Kevin J. Kauffman

Delivery technologies for genome editing

With the recent development of CRISPR technology, it is becoming increasingly easy to engineer the genome. Genome-editing systems based on CRISPR, as well as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs), are becoming valuable tools for biomedical research, drug discovery and development, and even gene therapy. However, for each of these systems to effectively enter cells of interest and perform their function, efficient and safe delivery technologies are needed. This Review discusses the principles of biomacromolecule delivery and gene editing, examines recent advances and challenges in non-viral and viral delivery methods, and highlights the status of related clinical trials.

Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs.

Intracellular delivery of messenger RNA (mRNA) has the potential to induce protein production for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-containing lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Experiment (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formulations previously used for siRNA delivery. Key features of this optimized formulation were the incorporation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and increased ionizable lipid:mRNA weight ratios. Interestingly, the optimized lipid nanoparticle formulation did not improve siRNA delivery, indicating differences in optimized formulation parameter design spaces for siRNA and mRNA. We believe the general method described here can accelerate in vivo screening and optimization of nanoparticle formulations with large multidimensional design spaces.

Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery

Thousands of human diseases could be treated by selectively controlling the expression of specific proteins in vivo. A new series of alkenyl amino alcohol (AAA) ionizable lipid nanoparticles (LNPs) capable of delivering human mRNA with unprecedented levels of in vivo efficacy is demonstrated. This study highlights the importance of utilizing synthesis tools in tandem with biological inspiration to understand and improve nucleic acid delivery in vivo.

RNAi targeting multiple cell adhesion molecules reduces immune cell recruitment and vascular inflammation after myocardial infarction

Nanoparticles deliver siRNA for multigene silencing of endothelial cell adhesion molecules, which dampens leukocyte recruitment in atherosclerosis and myocardial infarction in mice. Knocking down adhesion, knocking out inflammation Cells that are central to inflammation lodge in damaged or fatty regions of the vessels (called plaques) by “feeling out” the vessel surface. Neutrophils and monocytes first “roll” along the wall, then firmly plant themselves at an ideal site, and lastly pass through the cells lining the blood vessel: the endothelial cells. This recruitment and transmigration process is mediated by surface receptors called cell adhesion molecules (CAMs). Sager et al. developed a nanomedicine approach to preventing such inflammatory cell adhesion and exacerbation of plaques, by transiently knocking down five different CAMs simultaneously. The authors delivered small interfering RNA (siRNA) targeting the CAMs inside nanoparticles that had been optimized to reach endothelial cells. The five siRNAs reduced leukocyte recruitment to atherosclerotic plaques in mice that were engineered to develop certain features of human atherosclerosis. In the same mice, the siRNAs also attenuated inflammation after myocardial infarction—the equivalent of a heart attack. Current therapies for atherosclerosis and cardiovascular disease do not target inflammatory cells, and this multipronged siRNA-based nanomedicine approach could complement existing options to prevent heart disease from worsening. Myocardial infarction (MI) leads to a systemic surge of vascular inflammation in mice and humans, resulting in secondary ischemic complications and high mortality. We show that, in ApoE−/− mice with coronary ligation, increased sympathetic tone up-regulates not only hematopoietic leukocyte production but also plaque endothelial expression of adhesion molecules. To counteract the resulting arterial leukocyte recruitment, we developed nanoparticle-based RNA interference (RNAi) that effectively silences five key adhesion molecules. Simultaneously encapsulating small interfering RNA (siRNA)–targeting intercellular cell adhesion molecules 1 and 2 (Icam1 and Icam2), vascular cell adhesion molecule 1 (Vcam1), and E- and P-selectins (Sele and Selp) into polymeric endothelial-avid nanoparticles reduced post-MI neutrophil and monocyte recruitment into atherosclerotic lesions and decreased matrix-degrading plaque protease activity. Five-gene combination RNAi also curtailed leukocyte recruitment to ischemic myocardium. Therefore, targeted multigene silencing may prevent complications after acute MI.

Materials for non-viral intracellular delivery of messenger RNA therapeutics.

Though therapeutics based on messenger RNA (mRNA) have broad potential in applications such as protein replacement therapy, cancer immunotherapy, and genomic engineering, their effective intracellular delivery remains a challenge. A chemically diverse suite of delivery materials with origins as materials for cellular transfection of DNA and small interfering RNAs (siRNAs) has recently been reported to have promise as non-viral delivery agents for mRNA. These materials include covalent conjugates, protamine complexes, nanoparticles based on lipids or polymers, and hybrid formulations. This review will highlight the use of delivery materials for mRNA, with a specific focus on their mechanisms of action, routes of administration, and dosages. Additionally, strategies in which these materials can be adapted and optimized to address challenges specific to mRNA delivery are also discussed. The technologies included have shown varying promise for therapeutic use, specifically having been used to deliver mRNA in vivo or exhibiting characteristics that could make in vivo use a possibility. In so doing, it is the intention of this review to provide a comprehensive look at the progress and possibilities in applying nucleic acid delivery technology specifically toward the emerging area of mRNA therapeutics.

Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing

Efficient genome editing with Cas9–sgRNA in vivo has required the use of viral delivery systems, which have limitations for clinical applications. Translational efforts to develop other RNA therapeutics have shown that judicious chemical modification of RNAs can improve therapeutic efficacy by reducing susceptibility to nuclease degradation. Guided by the structure of the Cas9–sgRNA complex, we identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and we develop an optimal set of chemical modifications for in vivo applications. Using lipid nanoparticle formulations of these enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9, we show that a single intravenous injection into mice induces >80% editing of Pcsk9 in the liver. Serum Pcsk9 is reduced to undetectable levels, and cholesterol levels are significantly lowered about 35% to 40% in animals. This strategy may enable non-viral, Cas9-based genome editing in the liver in clinical settings.

Polymer–Lipid Nanoparticles for Systemic Delivery of mRNA to the Lungs

Therapeutic nucleic acids hold great promise for the treatment of disease but require vectors for safe and effective delivery. Synthetic nanoparticle vectors composed of poly(β-amino esters) (PBAEs) and nucleic acids have previously demonstrated potential utility for local delivery applications. To expand this potential utility to include systemic delivery of mRNA, hybrid polymer-lipid nanoformulations for systemic delivery to the lungs were developed. Through coformulation of PBAEs with lipid-polyethylene glycol (PEG), mRNA formulations were developed with increased serum stability and increased in vitro potency. The formulations were capable of functional delivery of mRNA to the lungs after intravenous administration in mice. To our knowledge, this is the first report of the systemic administration of mRNA for delivery to the lungs using degradable polymer-lipid nanoparticles.

Cancer Nanotherapeutics in Clinical Trials

To be legally sold in the United States, all drugs must go through the FDA approval process. This chapter introduces the FDA approval process and describes the clinical trials required for a drug to gain approval. We then look at the different cancer nanotherapeutics and in vivo diagnostics that are currently in clinical trials or have already received approval. These nanotechnologies are catagorized and described based on the delivery vehicle: liposomes, polymer micelles, albumin-bound chemotherapeutics, polymer-bound chemotherapeutics, and inorganic particles.

Barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics

Significance The effectiveness of nucleic acid drugs is limited by inefficient delivery to target tissues and cells and by unwanted accumulation in off-target organs. Although thousands of chemically distinct nanoparticles can be synthesized, nanoparticles designed to deliver nucleic acids in vivo were first tested in cell culture, yielding poor predictions for delivery in vivo. To facilitate testing of many nanoparticles in vivo, we designed and optimized a high-throughput DNA barcoding system to simultaneously measure nucleic acid delivery mediated by dozens of distinct nanoparticles in a single mouse. This nano-barcoding system can be used to study hundreds, or even thousands, of nanoparticles directly in vivo and could dramatically accelerate the discovery and understanding of nanoparticle drug delivery systems. Nucleic acid therapeutics are limited by inefficient delivery to target tissues and cells and by an incomplete understanding of how nanoparticle structure affects biodistribution to off-target organs. Although thousands of nanoparticle formulations have been designed to deliver nucleic acids, most nanoparticles have been tested in cell culture contexts that do not recapitulate systemic in vivo delivery. To increase the number of nanoparticles that could be tested in vivo, we developed a method to simultaneously measure the biodistribution of many chemically distinct nanoparticles. We formulated nanoparticles to carry specific nucleic acid barcodes, administered the pool of particles, and quantified particle biodistribution by deep sequencing the barcodes. This method distinguished previously characterized lung- and liver- targeting nanoparticles and accurately reported relative quantities of nucleic acid delivered to tissues. Barcode sequences did not affect delivery, and no evidence of particle mixing was observed for tested particles. By measuring the biodistribution of 30 nanoparticles to eight tissues simultaneously, we identified chemical properties promoting delivery to some tissues relative to others. Finally, particles that distributed to the liver also silenced gene expression in hepatocytes when formulated with siRNA. This system can facilitate discovery of nanoparticles targeting specific tissues and cells and accelerate the study of relationships between chemical structure and delivery in vivo.

Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes

B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T-cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non-Hodgkins lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose-dependent and time-controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells. Here, the design, synthesis, and biological evaluation of a lipid nanoparticle (LNP) system that can encapsulate mRNA, navigate to the spleen, transfect B lymphocytes, and induce more than 60 pg of protein expression per million B cells within the spleen is described. Importantly, this LNP induces more than 85% of total protein production in the spleen, despite LNPs being observed transiently in the liver and other organs. These results demonstrate that LNP composition alone can be used to modulate the site of protein induction in vivo, highlighting the critical importance of designing and synthesizing new nanomaterials for nucleic acid delivery.