In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.

Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.

Amine headgroups in ionizable lipids drive immune responses to lipid nanoparticles by binding to the receptors TLR4 and CD1d.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery vehicle for RNA therapeutics, partly because of established lipid structure-activity relationships focused on formulation potency. Yet such knowledge has not extended to LNP immunogenicity. Here we show that the innate and adaptive immune responses elicited by LNPs are linked to their ionizable lipid chemistry. Specifically, we show that the amine headgroups in ionizable lipids drive LNP immunogenicity by binding to Toll-like receptor 4 and CD1d and by promoting lipid-raft formation. Immunogenic LNPs favour a type-1 T-helper-cell-biased immune response marked by increases in the immunoglobulins IgG2c and IgG1 and in the pro-inflammatory cytokines tumour necrosis factor, interferon γ and the interleukins IL-6 and IL-2. Notably, the inflammatory signals originating from these receptors inhibit the production of anti-poly(ethylene glycol) IgM antibodies, preventing the often-observed loss of efficacy in the LNP-mediated delivery of siRNA and mRNA. Moreover, we identified computational methods for the prediction of the structure-dependent innate and adaptive responses of LNPs. Our findings may help accelerate the discovery of well-tolerated ionizable lipids suitable for repeated dosing.

GalNAc-Lipid nanoparticles enable non-LDLR dependent hepatic delivery of a CRISPR base editing therapy.

Lipid nanoparticles have demonstrated utility in hepatic delivery of a range of therapeutic modalities and typically deliver their cargo via low-density lipoprotein receptor-mediated endocytosis. For patients lacking sufficient low-density lipoprotein receptor activity, such as those with homozygous familial hypercholesterolemia, an alternate strategy is needed. Here we show the use of structure-guided rational design in a series of mouse and non-human primate studies to optimize a GalNAc-Lipid nanoparticle that allows for low-density lipoprotein receptor independent delivery. In low-density lipoprotein receptor-deficient non-human primates administered a CRISPR base editing therapy targeting the ANGPTL3 gene, the introduction of an optimized GalNAc-based asialoglycoprotein receptor ligand to the nanoparticle surface increased liver editing from 5% to 61% with minimal editing in nontargeted tissues. Similar editing was noted in wild-type monkeys, with durable blood ANGPTL3 protein reduction up to 89% six months post dosing. These results suggest that GalNAc-Lipid nanoparticles may effectively deliver to both patients with intact low-density lipoprotein receptor activity as well as those afflicted by homozygous familial hypercholesterolemia.

Lipid nanoparticles silence tumor necrosis factor α to improve wound healing in diabetic mice.

Diabetes mellitus is a mounting concern in the United States, as are the mortality and morbidity that result from its complications. Of particular concern, diabetes patients frequently suffer from impaired wound healing and resultant nonhealing diabetic foot ulcers. These ulcers overproduce tumor necrosis factor α (TNFα), which reduces wound bed cell migration and proliferation while encouraging apoptosis. Herein, we describe the use of siRNA-loaded lipid nanoparticles (LNPs) as a potential wound treatment to combat an overzealous immune response and facilitate wound closure. LNPs were formulated with an ionizable, degradable lipidoid and siRNA specific for TNFα. Topical application of nanoparticles reduced TNFα mRNA expression in the wound by 40-55% in diabetic and nondiabetic mice. In diabetic mice, this TNFα knockdown accelerated wound healing compared to untreated controls. Together, these results serve as proof-of-concept that RNA interference therapy using LNPs can reduce the severity and duration of chronic diabetic wounds.

Recent advances in biomaterials for the treatment of diabetic foot ulcers.

Diabetes mellitus is one of the most challenging epidemics facing the world today, with over 300 million patients affected worldwide. A significant complication associated with diabetes is hyperglycemia, which impairs wound healing. The rise in the diabetic patient population in recent years has precipitated an increase in the incidence and prevalence of chronic diabetic wounds, most commonly the diabetic foot ulcer. Although foot ulcers are difficult to treat due to their complicated pathology, outcomes have improved with the development of increasingly sophisticated biomaterials that accelerate healing. In this review, we describe recently developed biomaterials that elicit healing through cell-material interactions and/or the sustained delivery of drugs. These tunable therapeutic systems increase angiogenesis, collagen deposition, cell proliferation, and growth factors concentrations, while decreasing inflammation and enzymatic degradation of the extracellular matrix. As the field of biomaterials for wound healing continues to mature, we expect to witness a broader range of clinical options that will speed healing times and improve patient quality of life.

Silencing TNFα with lipidoid nanoparticles downregulates both TNFα and MCP-1 in an in vitro co-culture model of diabetic foot ulcers.

Diabetes is one of the most formidable diseases facing the world today, with the number of patients growing every year. Poor glycemic control yields a host of complications, such as impaired wound healing. This often results in the formation of diabetic foot ulcers, which carry a poor prognosis because they are notoriously difficult to treat. Current therapies do not address the increased number of infiltrating macrophages to the wound bed that overproduce tumor necrosis factor α (TNFα), which increases fibroblast apoptosis and collagen dismantling and decreases angiogenesis. In this study, we investigated the potential of RNA interference therapy to reduce the inappropriately high levels of TNFα in the wound bed. Although TNFα is a challenging gene silencing target, our lipidoid nanoparticles potently silence TNFα mRNA and protein expression at siRNA doses of 5-100nM without inducing vehicle-related gene silencing or cell death. We also describe the creation of an in vitro macrophage-fibroblast co-culture model, which reflects the TNFα and monocyte chemotactant protein-1 (MCP-1/CCL2) cross-talk that exists in diabetic wounds. Because TNFα induces fibroblasts to produce MCP-1, we show that silencing TNFα results in a downregulation of MCP-1, which should inhibit the recruitment of additional macrophages to the wound. In co-culture experiments, a single lipidoid nanoparticle dose of 100nM siTNFα downregulated TNFα and MCP-1 by 64% and 32%, respectively. These data underscore the potential of lipidoid nanoparticle RNAi treatment to inhibit a positive feedback cycle that fuels the pathogenesis of diabetic foot ulcers. STATEMENT OF SIGNIFICANCE: Diabetic foot ulcers are a rapidly growing issue worldwide, with current ulcer treatments not as effective as desired. RNA interference therapy represents a largely untapped possible solution to impaired wound healing. We show that siRNA-loaded lipidoid nanoparticles silence the overexpression of tumor necrosis factor α (TNFα) in inflammatory macrophages which leads to a subsequent downregulation of fibroblast-produced macrophage chemotactant protein-1 (MCP-1). Both TNFα and MCP-1 are critical components of the inflammatory feedback loop that exists in chronic wounds. In contrast to the majority of wound drug delivery studies, our study utilizes macrophage/fibroblast co-culture experiments to recapitulate a multicellular wound environment in which cytokine signaling influences inflammation. Results underscore the therapeutic potential of siRNA nanoparticles directed against TNFα in inhibiting two key inflammatory targets in chronic wounds.