Conspectus The discovery of CRISPR/Cas has revolutionized the field of genome editing. CRIPSR/Cas components are part of the bacterial immune system and are able to induce double-strand DNA breaks in ...the genome, which are resolved by endogenous DNA repair mechanisms. The most relevant of these are the error-prone nonhomologous end joining and homology directed repair pathways. The former can lead to gene knockout by introduction of insertions and deletions at the cut site, while the latter can be used for gene correction based on a provided repair template. In this Account, we focus on the delivery aspects of CRISPR/Cas for therapeutic applications in vivo. Safe and effective delivery of the CRISPR/Cas components into the nucleus of affected cells is essential for therapeutic gene editing. These components can be delivered in several formats, such as pDNA, viral vectors, or ribonuclear complexes. In the ideal case, the delivery system should address the current limitations of CRISPR gene editing, which are (1) lack of targeting specific tissues or cells, (2) the inability to enter cells, (3) activation of the immune system, and (4) off-target events. To circumvent most of these problems, initial therapeutic applications of CRISPR/Cas were performed on cells ex vivo via classical methods (e.g., microinjection or electroporation) and novel methods (e.g., TRIAMF and iTOP). Ideal candidates for such methods are, for example, hematopoietic cells, but not all tissue types are suited for ex vivo manipulation. For direct in vivo application, however, delivery systems are needed that can target the CRISPR/Cas components to specific tissues or cells in the human body, without causing immune activation or causing high frequencies of off-target effects. Viral systems have been used as a first resort to transduce cells in vivo. These systems suffer from problems related to packaging constraints, immunogenicity, and longevity of Cas expression, which favors off-target events. Viral vectors are as such not the best choice for direct in vivo delivery of CRISPR/Cas. Synthetic vectors can deliver nucleic acids as well, without the innate disadvantages of viral vectors. They can be classed into lipid, polymeric, and inorganic particles, all of which have been reported in the literature. The advantage of synthetic systems is that they can deliver the CRISPR/Cas system also as a preformed ribonucleoprotein complex. The transient nature of this approach favors low frequencies of off-target events and minimizes the window of immune activation. Moreover, from a pharmaceutical perspective, synthetic delivery systems are much easier to scale up for clinical use compared to viral vectors and can be chemically functionalized with ligands to obtain target cell specificity. The first preclinical results with lipid nanoparticles delivering CRISPR/Cas either as mRNA or ribonucleoproteins are very promising. The goal is translating these CRISPR/Cas therapeutics to a clinical setting as well. Taken together, these current trends seem to favor the use of sgRNA/Cas ribonucleoprotein complexes delivered in vivo by synthetic particles.
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IJS, KILJ, NUK, PNG, UL, UM
Antibody titer and viable cell density (VCD) are two important parameters that need to be closely monitored during the process of cell line development and manufacturing of therapeutic antibodies. ...Typically, determination of each parameter requires 10–100 μL of supernatant sample, which is not suitable for small scale cultivation. In this study, we demonstrated that as low as 2 μL of culture supernatants were sufficient for the analysis using UV-Vis spectrum assisted with partial least squares (PLS) model. The results indicated that the optimal PLS models could be used to predict antibody titer and VCD with the linear relationship between reference values and predicted values at
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values ranging from 0.8 to > 0.9 in supernatant samples obtained from four different single clones and in polyclones that were cultured in various selection stringencies. Then, the percentage of cell viability and productivity were predicted from a set of samples of polyclones. The results indicated that while all predicted % cell viability were very similar to the actual value at RSEP value of 6.7 and
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of 0.8908, the predicted productivity from 14 of 18 samples were closed to the reference measurements at RSEP value of 22.4 and
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of 0.8522. These results indicated that UV-Vis combined with PLS has potential to be used for monitoring antibody titer, VCD, and % cell viability for both online and off-line therapeutic production process.
Graphical abstract
The process of multivariate analysis and partial least squares regression of UV-Vis spectrum for the determination of CHO cell densities and antibody titers obtained from small volume of cell culture supernatant samples.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Dendritic cells play a central role in antigen presentation and generation of cytotoxic T lymphocyte (CTL) response required for anticancer vaccination. The review focuses on use of particulate ...carriers like lipid and polymeric nanoparticles for targeting tumor antigens to the dendritic cells. The role of various physicochemical parameters of nanocarriers such as size, surface charge in passive targeting is detailed. Utilization of different ligands such as mannose, Fc receptor, CD11c/CD 18, DEC-205 and DC-SIGN on DC for active targeting is reviewed. Smart nanocarriers such as pH sensitive nanocarriers, pre forming liposomes, cell penetrating peptide containing systems and virosomes that can specifically increase the CTL response generating greater antitumor immunity have also been discussed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk ...analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications. From the Clinical Editor The authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Nano-sized extracelullar vesicles (EVs) released by various cell types play important roles in a plethora of (patho)physiological processes and are increasingly recognized as biomarkers for disease. ...In addition, engineered EV and EV-inspired liposomes hold great potential as drug delivery systems. Major technologies developed for high-throughput analysis of individual EV include nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (tRPS) and high-resolution flow cytometry (hFC). Currently, there is a need for comparative studies on the available technologies to improve standardization of vesicle analysis in diagnostic or therapeutic settings.
We investigated the possibilities, limitations and comparability of NTA, tRPS and hFC for analysis of tumor cell-derived EVs and synthetic mimics (i.e. differently sized liposomes). NTA and tRPS instrument settings were identified that significantly affected the quantification of these particles. Furthermore, we detailed the differences in absolute quantification of EVs and liposomes using the three technologies. This study increases our understanding of possibilities and pitfalls of NTA, tRPS and hFC, which will benefit standardized and large-scale clinical application of (engineered) EVs and EV-mimics in the future.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
In 2018 I was appointed full professor of Pharmaceutical Biotechnology & Delivery at the Pharmaceutics division of the department of Pharmaceutical Sciences at Utrecht University, The Netherlands. In ...this contribution to the Orations – New Horizons of the Journal of Controlled Release I will introduce my research group (see also www.uu.nl/pharmaceutics) and will highlight my current and future research projects. In coming years the focus of my research will be on the administration of biotherapeutics, aiming to control their fate from the site of injection to the site of action. I will discuss issues related to formulation of biotherapeutics into nanomedicines (NMs), intracellular delivery of nucleic acids as well as protein therapeutics, and targeted delivery of biotherapeutics beyond the liver. In addition, I will provide a forward view on how current developments in the drug delivery and gene therapy field may result in sustainable and cost-effective dosing regimens for biotherapeutics.
“The Nanomedicine Universe” according to Enrico Mastrobattista. Display omitted
•Highlights of research on intracellular delivery of protein and nucleic acid therapeutics using nanomedicine•Ideas on how to deliver biotherapeutics to tissues and organs other than liver and spleen•Future insights into sustainable dosing of biotherapeutics via genetic modification of endogenous cells•Importance of monitoring the effects of post-production handling of biopharmaceuticals on stability and potency
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Over the years, several chemical reactions have been developed that enable the covalent conjugation of synthetic molecules to natural proteins. The resulting bioconjugates have become important tools ...in the study of natural proteins. Furthermore, they form a new class of protein-based pharmaceuticals and biomaterials. However, classical bioconjugation reactions to natural amino acids suffer from poor site-specificity. To overcome this problem, a variety of uniquely reactive non-natural amino acids have recently been designed. These can be incorporated into proteins by specifically engineered bacterial strains. Such reactive non-natural amino acids create new possibilities for bio-orthogonal conjugation to proteins. This review first gives an overview of the various methods for site-specific introduction of non-natural amino acids into proteins. Both semisynthetic and entirely recombinant methods are addressed. Then, a detailed description is given of the reactive non-natural amino acids that have already been recombinantly introduced into proteins. The bio-orthogonal reactions that can be used for conjugation to these reactive non-natural amino acids are also discussed. These include the alkyne/azide ‘click’ reaction, carbonyl condensations, Michael-type additions, and Mizoroki-Heck substitutions.
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Wnt signalling proteins are essential for culture of human organ stem cells in organoids, but most Wnt protein formulations are poorly active in serum-free media. Here we show that purified Wnt3a ...protein is ineffective because it rapidly loses activity in culture media due to its hydrophobic nature, and its solubilization requires a detergent, CHAPS (3-(3-cholamidopropyl) dimethylammonio-1-propanesulfonate), that interferes with stem cell self-renewal. By stabilizing the Wnt3a protein using phospholipids and cholesterol as carriers, we address both problems: Wnt activity remains stable in serum-free media, while non-toxic carriers allow the use of high Wnt concentrations. Stabilized Wnt3a supports strongly increased self-renewal of organ and embryonic stem cells and the serum-free establishment of human organoids from healthy and diseased intestine and liver. Moreover, the lipophilicity of Wnt3a protein greatly facilitates its purification. Our findings remove a major obstacle impeding clinical applications of adult stem cells and offer advantages for all cell culture uses of Wnt3a protein.
The therapeutic potential of the CRISPR-Cas9 gene editing system in treating numerous genetic disorders is immense. To fully realize this potential, it is crucial to achieve safe and efficient ...delivery of CRISPR-Cas9 components into the nuclei of target cells. In this study, we investigated the applicability of the amphipathic cell-penetrating peptide LAH5, previously employed for DNA delivery, in the intracellular delivery of spCas9:sgRNA ribonucleoprotein (RNP) and the RNP/single-stranded homology-directed repair (HDR) template. Our findings reveal that the LAH5 peptide effectively formed nanocomplexes with both RNP and RNP/HDR cargo, and these nanocomplexes demonstrated successful cellular uptake and cargo delivery. The loading of all RNP/HDR components into LAH5 nanocomplexes was confirmed using an electrophoretic mobility shift assay. Functional screening of various ratios of peptide/RNP nanocomplexes was performed on fluorescent reporter cell lines to assess gene editing and HDR-mediated gene correction. Moreover, targeted gene editing of the CCR5 gene was successfully demonstrated across diverse cell lines. This LAH5-based delivery strategy represents a significant advancement toward the development of therapeutic delivery systems for CRISPR-Cas-based genetic engineering in in vitro and ex vivo applications.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric ...coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.
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