ConspectusThe 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...

Light triggered drug delivery systems offer attractive possibilities for sophisticated therapy, providing both temporal and spatial control of drug release. We have developed light triggered liposomes with clinically approved indocyanine green (ICG) as the light sensitizing compound. Amphiphilic ICG can be localized in different compartments of the liposomes, but the effect of its presence, on both triggered release and long term stability, has not been studied. In this work, we report that ICG localization has a significant effect on the properties of the liposomes. Polyethylene glycol (PEG) coating of the liposomes leads to binding and stabilization of the ICG molecules on the surface of the lipid bilayer. This formulation showed both good storage stability in buffer solution (at +4-37 °C) and adequate stability in serum and vitreous (at +37 °C). The combination of ICG within the lipid bilayer and PEG coating lead to poor stability at elevated temperatures of +22 °C and + 37 °C. The mechanisms of the increased instability due to ICG insertion in the lipid bilayer was elucidated with molecular dynamics simulations. Significant PEG insertion into the bilayer was induced in the presence of ICG in the lipid bilayer. Finally, feasibility of freeze-drying as a long term storage method for the ICG liposomes was demonstrated. Overall, this is the first detailed study on the interactions of lipid bilayer, light sensitizer (ICG) and PEG coating on the liposome stability. The localization of the light triggering agent significantly alters the structure of the liposomes and it is important to consider these aspects in triggered drug delivery system design.

The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse genetic disorders. However, for gene therapy applications, an efficient delivery vehicle is required, capable of delivering the CRISPR-Cas9 components into the cytosol of the intended target cell population. In this study, we optimized the formulation conditions of lipid nanoparticles (LNP) for delivery of ready-made CRISPR-Cas9 ribonucleic protein (RNP). The buffer composition during complexation and relative DOTAP concentrations were varied for LNP encapsulating in-house produced Cas9 RNP alone or Cas9 RNP with additional template DNA for gene correction. The LNP were characterized for size, surface charge, and plasma interaction through asymmetric flow field flow fractionation (AF4). Particles were functionally screened on fluorescent reporter cell lines for gene knock-out and gene correction. This revealed incompatibility of RNP with citrate buffer and PBS. We demonstrated that LNP for gene knock-out did not necessarily require DOTAP, while LNP for gene correction were only active with a low concentration of DOTAP. The AF4 studies additionally revealed that LNP interact with plasma, however, remain stable, whereby HDR template seems to favor stability of LNP. Under optimal formulation conditions, we achieved gene knock-out and gene correction efficiencies as high as 80% and 20%, respectively, at nanomolar concentrations of the CRISPR-Cas9 RNP.

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The aim of this study was to develop an injectable hydrogel delivery system for sustained ocular delivery of dexamethasone. To this end, a self-healing hydrogel consisting of a thermosensitive ABA triblock copolymer was designed. The drug was covalently linked to the polymer by copolymerization of methacrylated dexamethasone with N-isopropylacrylamide (NIPAM) and N-acryloxysuccinimide (NAS) through reversible addition–fragmentation chain transfer (RAFT) polymerization, using poly(ethylene glycol) (PEG) functionalized at both ends with a chain transfer agent (CTA). Hydrogel formation was achieved by mixing aqueous solutions of the formed thermosensitive polymer (with a cloud point of 23 °C) with cystamine at 37 °C, to result in covalent cross-linking due to the reaction of the N-hydroxysuccimide (NHS) functionality of the polymer and the primary amines of cystamine. Rheological analysis showed both thermogelation and covalent cross-linking at 37 °C, as well as the self-healing properties of the formed network, which was attributed to the presence of disulfide bonds in the cystamine cross-links, making the system injectable. The release of dexamethasone from the hydrogel occurred through ester hydrolysis following first-order kinetics in an aqueous medium at pH 7.4 over 430 days at 37 °C. Based on simulations, administration of 100 mg of hydrogel would be sufficient for maintaining therapeutic levels of dexamethasone in the vitreous for at least 500 days. Importantly, dexamethasone was released from the hydrogel in its native form as determined by LC–MS analysis. Cytocompatibility studies showed that at clinically relevant concentrations, both the polymer and the cross-linker were well tolerated by adult retinal pigment epithelium (ARPE-19) cells. Moreover, the hydrogel did not show any toxicity to ARPE-19 cells. The injectability of the hydrogel, together with the long-lasting release of dexamethasone and good cytocompatibility with a retinal cell line, makes this delivery system an attractive candidate for treatment of ocular inflammatory diseases.

The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse ge-netic disorders. However, for gene therapy applications an efficient delivery vehicle is required, capable of delivering the CRISPR-Cas9 components into the cytosol of the intended target cell population. Once there, the ribonucleoprotein complex (RNP) can be transported into the nucleus. Lipid nanoparticles (LNP) serve as promising candidates for delivery of CRISPR-Cas9 RNP. These delivery vehicles have been optimized for the delivery of nucleic acids, such as mRNA. Co-delivery of Cas9 encoding mRNA with the accompanying sgRNA leads to translation of the Cas9 protein and formation of the Cas9 RNP inside the cell. Only recently, direct delivery of the CRISPR-Cas9 RNP complexes has been explored, which requires adjustments to the LNP formulation. In this study, the importance of buffer composition and cationic charge during RNP and ssDNA en-trapment in LNP are demonstrated. After optimizing several formulation parameters, LNP were prepared that were colloidally stable in human plasma and efficiently deliver the SpCas9 RNP and ssDNA for HDR-correction in reporter cells. Under optimal formulation conditions, gene knock-out and gene correction efficiencies as high as 80% and 20%, respectively were achieved at nanomolar CRISPR-Cas9 RNP concentrations.