Enabling Precision Medicine with CRISPR-Cas Genome Editing Technology: A Translational Perspective.

Genome editing technologies, particularly CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR) associated nucleases), are redefining the boundaries of therapeutic gene therapy. CRISPR-Cas is a robust, straightforward, and programmable genome editing tool capable of mediating site-specific DNA modifications. The rapid advancements from discovery to clinical adaptation have expanded the therapeutic landscape to treat genetically defined diseases. Together with the technical developments in human DNA and RNA sequencing, CRISPR-directed gene therapy enables a new era to realize precision medicine where pathogenic mutations underlying monogenic disorders can potentially be corrected. Also, protective or therapeutic genomic alterations can be introduced as preventative or curative therapy. Despite its high therapeutic potential, CRISPR-Cas´ clinical translation is still in its infancy and is highly dependent on its efficiency, specificity in gene corrections, and cell-specific delivery. Therefore, this chapter focuses on the challenges and opportunities the CRISPR-Cas toolbox offers together with delivery vehicles to realize its use for therapeutic gene editing. Furthermore, we discuss the obstacles the CRISPR-Cas system faces for successful clinical translation and summarize its current clinical progress.

Extracellular vesicle-mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin-kexin type 9 (Pcsk9) in primary mouse hepatocytes.

The loss-of-function of the proprotein convertase subtilisin-kexin type 9 (Pcsk9) gene has been associated with significant reductions in plasma serum low-density lipoprotein cholesterol (LDL-C) levels. Both CRISPR/Cas9 and CRISPR-based editor-mediated Pcsk9 inactivation have successfully lowered plasma LDL-C and PCSK9 levels in preclinical models. Despite the promising preclinical results, these studies did not report how vehicle-mediated CRISPR delivery inactivating Pcsk9 affected low-density lipoprotein receptor recycling in vitro or ex vivo. Extracellular vesicles (EVs) have shown promise as a biocompatible delivery vehicle, and CRISPR/Cas9 ribonucleoprotein (RNP) has been demonstrated to mediate safe genome editing. Therefore, we investigated EV-mediated RNP targeting of the Pcsk9 gene ex vivo in primary mouse hepatocytes. We engineered EVs with the rapamycin-interacting heterodimer FK506-binding protein (FKBP12) to contain its binding partner, the T82L mutant FKBP12-rapamycin binding (FRB) domain, fused to the Cas9 protein. By integrating the vesicular stomatitis virus glycoprotein on the EV membrane, the engineered Cas9 EVs were used for intracellular CRISPR/Cas9 RNP delivery, achieving genome editing with an efficacy of ±28.1% in Cas9 stoplight reporter cells. Administration of Cas9 EVs in mouse hepatocytes successfully inactivated the Pcsk9 gene, leading to a reduction in Pcsk9 mRNA and increased uptake of the low-density lipoprotein receptor and LDL-C. These readouts can be used in future experiments to assess the efficacy of vehicle-mediated delivery of genome editing technologies targeting Pcsk9. The ex vivo data could be a step towards reducing animal testing and serve as a precursor to future in vivo studies for EV-mediated CRISPR/Cas9 RNP delivery targeting Pcsk9.

TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery.

Extracellular vesicles (EVs) have emerged as biocompatible drug delivery vehicles due to their native ability to deliver bioactive cargo to recipient cells. However, the application of EVs as a therapeutic delivery vehicle is hampered by effective methods for endogenously loading target proteins inside EVs and unloading proteins after delivery to recipient cells. Most EV-based engineered loading methods have a limited delivery efficiency owing to their inefficient endosomal escape or cargo release from the intraluminal attachment from the EV membrane. Here, we describe the 'Technology Of Protein delivery through Extracellular Vesicles' (TOP-EVs) as a tool for efficient intracellular delivery of target proteins mediated via EVs. The vesicular stomatitis virus glycoprotein and the rapamycin-heterodimerization of the FKBP12/T82L mutant FRB proteins were both important for the effective protein delivery through TOP-EVs. We showed that TOP-EVs could efficiently deliver Cre recombinase and CRISPR/Cas9 ribonucleoprotein complex in vitro. Moreover, our results demonstrated that the capacity of TOP-EVs to deliver intracellular proteins in recipient cells was not an artifact of plasmid contamination or direct plasmid loading into EVs. Finally, we showed that TOP-EVs could successfully mediate intracellular protein delivery in the liver in vivo. Taken together, TOP-EVs are a versatile platform for efficient intracellular protein delivery in vitro and in vivo, which can be applied to advance the development of protein-based therapeutics.

Extracellular vesicle-mediated protein delivery to the liver.

Extracellular vesicles (EVs) are nanoscale particles that facilitate intercellular communication. They are regarded as a promising natural drug delivery system for transporting and delivering bioactive macromolecules to target cells. Recently, researchers have engineered EVs with FKBP12/FRB heterodimerization domains that interact with rapamycin to load and deliver exogenous proteins for both in vitro and in vivo applications. In this study, we examined the tissue distribution of EVs using near-infrared fluorescent imaging. We evaluated the effectiveness of EV-mediated delivery of Cre recombinase specifically to hepatocytes in the livers of Ai9 Cre-loxP reporter mice. Intravenous injection resulted in more efficient Cre protein delivery to the liver than intraperitoneal injections. Depleting liver-resident macrophages with clodronate-encapsulated liposome pre-treatment did not enhance EV-mediated Cre delivery to hepatocytes. Moreover, we demonstrated that multiple intravenous injections of Cre-EVs facilitated functional Cre delivery to hepatocytes. To the best of our knowledge, this is the first study to simultaneously investigate the tissue distribution of FKBP12/FRB-engineered EVs and their subsequent intracellular protein delivery in Ai9 Cre-loxP reporter mice. These insights can inform preclinical research and contribute to developing next-generation EV-based platforms for delivering therapeutic proteins or genome editing technologies targeting the liver.

Biofabrication of Cell-Derived Nanovesicles: A Potential Alternative to Extracellular Vesicles for Regenerative Medicine.

Extracellular vesicles (EVs) are mediators of intercellular communication by transferring functional biomolecules from their originating cells to recipient cells. This intrinsic ability has gained EVs increased scientific interest in their use as a direct therapeutic in the field of regenerative medicine or as vehicles for drug delivery. EVs derived from stem cells or progenitor cells can act as paracrine mediators to promote repair and regeneration of damaged tissues. Despite substantial research efforts into EVs for various applications, their use remains limited by the lack of highly efficient and scalable production methods. Here, we present the biofabrication of cell-derived nanovesicles (NVs) as a scalable, efficient, and cost-effective production alternative to EVs. We demonstrate that NVs have a comparable size and morphology as EVs, but lack standard EV (surface) markers. Additionally, in vitro uptake experiments show that human fetal cardiac fibroblast, endothelial cells, and cardiomyocyte progenitor cells internalize NVs. We observed that cardiac progenitor cell-derived NVs and EVs are capable of activating mitogen-activated protein kinase 1/2 (MAPK1/2)-extracellular signal-regulated kinase, and that both NVs and EVs derived from A431 and HEK293 cells can functionally deliver Cre-recombinase mRNA or protein to other cells. These observations indicate that NVs may have similar functional properties as EVs. Therefore, NVs have the potential to be applied for therapeutic delivery and regenerative medicine purposes.