Display omitted
Non-viral vectors are promising nucleic acid carriers which have been utilized in gene-based cancer immunotherapy. The aim of this study is to compare the transfection efficiency and ...cytotoxicity of three cationic non-viral vectors namely Polyethylenimine (PEI), Lipofectamine 2000 (LPF) and stable nucleic acid lipid particles (SNALPs) of different lipid compositions, for the delivery of plasmid DNA (pDNA) expressing immunostimulatory molecules, OX40L or 4-1BBL, to cancer cells in vitro. The results indicate that PEI and LPF are efficient vectors for pDNA delivery with high transfection efficiency obtained. However, pDNA-PEI and pDNA-LPF complexes up-regulated the expression of programmed death ligand-1 (PD-L1) and induced significant cytotoxicity in both B16F10 and CT26 cell lines. The up-regulation of PD-L1 expression induced by pDNA-PEI and pDNA-LPF complexes was independent of cancer cell line, nor was it linked to the presence of GpC motifs in the pDNA. In contrast, the use of biocompatible SNALPs (MC3 and KC2 types) resulted in lower pDNA transfection efficiency, however no significant up-regulation of PD-L1 or cytotoxicity was observed. A strong correlation was found between up-regulation of PD-L1 expression and cytotoxicity. Up-regulation of PD-L1 expression could be mitigated with RNAi, maintaining expression at basal levels. Due to the improved biocompatibility and the absence of PD-L1 up-regulation, SNALPs represent a viable non-viral nucleic acid vector for delivery of pDNA encoding immunostimulatory molecules. The results of this study suggest that PD-L1 expression should be monitored when selecting commercial transfection reagents as pDNA vectors for cancer immunotherapy in vitro.
Tumour−specific, immuno−based therapeutic interventions can be considered as safe and effective approaches for cancer therapy. Exploitation of nano−vaccinology to intensify the cancer vaccine potency ...may overcome the need for administration of high vaccine doses or additional adjuvants and therefore could be a more efficient approach. Carbon nanotube (CNT) can be described as carbon sheet(s) rolled up into a cylinder that is nanometers wide and nanometers to micrometers long. Stemming from the observed capacities of CNTs to enter various types of cells via diversified mechanisms utilising energy−dependent and/or passive routes of cell uptake, the use of CNTs for the delivery of therapeutic agents has drawn increasing interests over the last decade. Here we review the previous studies that demonstrated the possible benefits of these cylindrical nano−vectors as cancer vaccine delivery systems as well as the obstacles their clinical application is facing.
Display omitted
The facile and controlled fabrication of homogeneously grafted cationic polymers on carbon nanotubes (CNTs) remains poorly investigated, which further hinders the understanding of interactions ...between functionalized CNTs with different nucleic acids and the rational design of appropriate gene delivery vehicles. Herein, we describe the controlled grafting of cationic poly(2-dimethylaminoethylmethacrylate) brushes on CNTs via surface-initiated atom transfer radical polymerization integrated with mussel-inspired polydopamine chemistry. The binding of nucleic acids with different brush-CNT hybrids discloses the highly architectural-dependent behavior with dense short brush-coated CNTs displaying the highest binding among all the other hybrids, namely, dense long, sparse long, and sparse short brush-coated CNTs. Additionally, different chemistries of the brush coatings were shown to influence the biocompatibility, cellular uptake, and silencing efficiency in vitro. This platform provides great flexibility for the design of polymer brush-CNT hybrids with precise control over their structure–activity relationship for the rational design of nucleic acid delivery systems.
With a dismal survival rate, pancreatic cancer (PC) remains one of the most aggressive and devastating malignancies, predominantly due to the absence of a valid biomarker for diagnosis and limited ...therapeutic options for advanced diseases. Exosomes (Exo) as cell-derived vesicles, are widely used as natural nanocarriers for drug delivery. P21-activated kinase 4 (PAK4) is oncogenic when overexpressed, promoting cell survival, migration and anchorage-independent growth. Herein we validated PAK4 as a therapeutic target in an in vivo PC tumour mouse model using Exo-mediated RNAi following intra-tumoural administration. PC derived Exo were firstly isolated by ultracentrifugation on sucrose cushion and characterised for their surface marker expression, size, number, purity and morphology. SiRNA was encapsulated into Exo via electroporation and dual uptake of Exo and siRNA was investigated by flow cytometry and confocal microscopy. In vitro siPAK4 silencing in PC cells following uptake was assessed by flow cytometry, western blotting, and in vitro scratch assay. In vivo efficacy (tumour growth delay and mouse survival) of siPAK4 was evaluated in PC bearing NSG mouse model. Ex vivo tumours were examined using Haematoxylin and eosin (H&E) staining and immunohistochemistry. Results showed high quality PC-derived PANC-1 Exo were obtained. SiRNA was incorporated in Exo with 16.5% encapsulation efficiency. In vitro imaging confirmed Exo and siRNA co-localisation in cells. PAK4 knockdown was successful with 30 nM Exo-siPAK4 at 24 h post incubation in vitro. Intra-tumoural administration of Exo-siPAK4 (0.03 mg/kg siPAK4 and 6.1 × 1011 Exo, each dose, two doses) reduced PC tumour growth in vivo and enhanced mice survival (p < 0.001), with minimal toxicity observed compared to polyethylenimine (PEI) used as a commercial transfection reagent. H&E staining of tumours showed significant tissue apoptosis in siPAK4 treated groups. PAK4 knockdown prolongs survival of PC-bearing mice suggesting its potential as a new therapeutic target for PC. PANC-1 Exo demonstrated comparable efficacy but safer profile than PEI as in vivo RNAi transfection reagent.
Display omitted
Despite recent advances in treatment and vector control, malaria is still a leading cause of death, emphasizing the need for an effective vaccine. The malaria life cycle can be subdivided into three ...stages: the invasion and growth within liver hepatocytes (pre-erythrocytic stage), the blood stage (erythrocytic stage), and, finally, the sexual stage (occurring within the mosquito vector). Antigen (Ag)-specific CD8
T cells are effectively induced by heterologous prime-boost viral vector immunization and known to correlate with liver-stage protection. However, liver-stage malaria vaccines have struggled to generate and maintain the high numbers of
-specific circulating T cells necessary to confer sterile protection. We describe an alternative "prime and target" vaccination strategy aimed specifically at inducing high numbers of tissue-resident memory T cells present in the liver at the time of hepatic infection. This approach bypasses the need for very high numbers of circulating T cells and markedly increases the efficacy of subunit immunization against liver-stage malaria with clinically relevant Ags and clinically tested viral vectors in murine challenge models. Translation to clinical use has begun, with encouraging results from a pilot safety and feasibility trial of intravenous chimpanzee adenovirus vaccination in humans. This work highlights the value of a prime-target approach for immunization against malaria and suggests that this strategy may represent a more general approach for prophylaxis or immunotherapy of other liver infections and diseases.
Immune checkpoint blockade involves targeting immune regulatory molecules with antibodies. Preclinically, complex multiantibody regimes of both inhibitory and stimulatory targets are a promising ...candidate for the next generation of immunotherapy. However, in this setting, the antibody platform may be limited due to excessive toxicity caused by off target effects as a result of systemic administration. RNA can be used as an alternate to antibodies as it can both downregulate immunosuppressive checkpoints (siRNA) or induce expression of immunostimulatory checkpoints (mRNA). In this study, we demonstrate that the combination of both siRNA and mRNA in a single formulation can simultaneously knockdown and induce expression of immune checkpoint targets, thereby reprogramming the tumor microenvironment from immunosuppressive to immunostimulatory phenotype. To achieve this, RNA constructs were synthesized and formulated into stable nucleic acid lipid nanoparticles (SNALPs); the SNALPs produced were 140–150 nm in size with >80% loading efficiency. SNALPs could transfect macrophages and B16F10 cells in vitro resulting in 75% knockdown of inhibitory checkpoint (PDL1) expression and simultaneously express high levels of stimulatory checkpoint (OX40L) with minimal toxicity. Intratumoral treatment with the proposed formulation resulted in statistically reduced tumor growth, a greater density of CD4+ and CD8+ infiltrates in the tumor, and immune activation within tumor-draining lymph nodes. These data suggest that a single RNA-based formulation can successfully reprogram multiple immune checkpoint interactions on a cellular level. Such a candidate may be able to replace future immune checkpoint therapeutic regimes composed of both stimulatory- and inhibitory-receptor-targeting antibodies.
Apoptotic cells and cell fragments, especially those produced as a result of immunogenic cell death (ICD), are known to be a potential source of cancer vaccine immunogen. However, due to variation ...between tumours and between individuals, methods to generate such preparations may require extensive ex vivo personalisation. To address this, we have utilised the concept of in situ vaccination whereby an ICD inducing drug is injected locally to generate immunogenic apoptotic fragments/cells. These fragments are then adjuvanted by a co-administered cell reactive CpG adjuvant. We first evaluate means of labelling tumour cells with CpG adjuvant, we then go on to demonstrate in vitro that labelling is preserved following apoptosis and, furthermore, that the apoptotic body-adjuvant complexes are readily transferred to macrophages. In in vivo studies we observe synergistic tumour growth delays and elevated levels of CD4+ and CD8+ cells in tumours receiving adjuvant drug combination. CD4+/CD8+ cells are likewise elevated in the tumour draining lymph node and activated to a greater extent than individual treatments. This study represents the first steps toward the evaluation of rationally formulated drug-adjuvant combinations for in situ chemo-immunotherapy.
Display omitted
Immunotherapy is currently a standard of care in the treatment of many malignancies. However, predictable side effects caused by systemic administration of highly immunostimulatory molecules have ...been a serious concern within this field. Intratumoural expression or silencing of immunogenic and immunoinhibitory molecules using nucleic acid-based approaches such as plasmid DNA (pDNA) and small interfering RNA (siRNA), respectively, could represent a next generation of cancer immunotherapy. Here, we employed lipid nanoparticles (LNPs) to deliver either non-specific pDNA and siRNA, or constructs targeting two prominent immunotherapeutic targets OX40L and indoleamine 2,3-dioxygenase-1 (IDO), to tumours in vivo.
In the B16F10 mouse model, intratumoural delivery of LNP-formulated non-specific pDNA and siRNA led to strong local immune activation and tumour growth inhibition even at low doses due to the pDNA immunogenic nature. Replacement of these non-specific constructs by pOX40L and siIDO resulted in more prominent immune activation as evidenced by increased immune cell infiltration in tumours and tumour-draining lymph nodes. Consistently, pOX40L alone or in combination with siIDO could prolong overall survival, resulting in complete tumour regression and the formation of immunological memory in tumour rechallenge models. Our results suggest that intratumoural administration of LNP-formulated pDNA and siRNA offers a promising approach for cancer immunotherapy.
Display omitted
•I.t injection of non-specific LNPs led to strong local immune activation and tumour growth inhibition even at low doses.•pOX40L + siIDO LNPs induced more prominent immune activation than non-specific pDNA+siRNA LNPs.•pOX40L or pOX40L + siIDO LNPs prolonged survival, led to complete tumour regression and immunological memory generation.•I.t injection of LNP-formulated pDNA and siRNA offers a promising approach for cancer immunotherapy.
Therapeutic nucleic acids (TNAs) comprise an alternative to conventional drugs for cancer therapy. Recently, stable nucleic acid lipid particles (SNALPs) have been explored to deliver TNA efficiently ...and safely both in vitro and in vivo. Small interfering RNA (siRNA) and messenger RNA (mRNA) based drugs have been suggested for a wide range of pathologies, and their respective lipid nanoparticle (LNP) formulations have been optimised using a Design of Experiments (DoE) approach. However, it is uncertain as to whether data obtained from DoE using simple experimental outputs can be used to generate a general heuristic for delivery of diverse TNA both in vitro and in vivo. Using plasmid DNA (pDNA), for which limited DoE optimisation has been performed, and siRNA to represent the two extremities of the TNA spectrum in terms of size and biological requirements, we performed a comparative DoE for both molecules and assessed the predictive qualities of the model both in vitro and in vivo. By producing a minimum run of 24 SNALP formulations with different lipid compositions incorporating either pDNA or siRNA, DoE models were successfully established for predicting the effect of individual lipid composition on particle size, TNA encapsulation and transfection both in vitro and in vivo. The results showed that the particle size, and in vitro and in vivo transfection efficiency of both pDNA and siRNA SNALP formulations were affected by lipid compositions. The encapsulation efficiency of pDNA SNALPs but not siRNA SNALPs was affected by the lipid composition. Notably, the optimal lipid compositions of SNALPs for pDNA/siRNA delivery were not identical. Furthermore, in vitro transfection efficiency could not be used to predict promising LNP candidates in vivo. The DoE approach described in this study may provide a method for comprehensive optimisation of LNPs for various applications. The model and optimal formulation described in this study can serve as a foundation from which to develop other novel NA containing LNPs for multiple applications such as NA based vaccines, cancer immunotherapies and other TNA therapies.
Adenovirus vectors offer a platform technology for vaccine development. The value of the platform has been proven during the COVID-19 pandemic. Although good stability at 2-8 °C is an advantage of ...the platform, non-cold-chain distribution would have substantial advantages, in particular in low-income countries. We have previously reported a novel, potentially less expensive thermostabilisation approach using a combination of simple sugars and glass micro-fibrous matrix, achieving excellent recovery of adenovirus-vectored vaccines after storage at temperatures as high as 45 °C. This matrix is, however, prone to fragmentation and so not suitable for clinical translation. Here, we report an investigation of alternative fibrous matrices which might be suitable for clinical use. A number of commercially-available matrices permitted good protein recovery, quality of sugar glass and moisture content of the dried product but did not achieve the thermostabilisation performance of the original glass fibre matrix. We therefore further investigated physical and chemical characteristics of the glass fibre matrix and its components, finding that the polyvinyl alcohol present in the glass fibre matrix assists vaccine stability. This finding enabled us to identify a potentially biocompatible matrix with encouraging performance. We discuss remaining challenges for transfer of the technology into clinical use, including reliability of process performance.
PDF
