Abstract
Harnessing CRISPR-Cas9 technology for cancer therapeutics has been hampered by low editing efficiency in tumors and potential toxicity of existing delivery systems. Here, we describe a safe and efficient lipid nanoparticle (LNP) for the delivery of Cas9 mRNA and sgRNAs that use a novel amino-ionizable lipid. A single intracerebral injection of CRISPR-LNPs against PLK1 (sgPLK1-cLNPs) into aggressive orthotopic glioblastoma enabled up to ~70% gene editing in vivo, which caused tumor cell apoptosis, inhibited tumor growth by 50%, and improved survival by 30%. To reach disseminated tumors, cLNPs were also engineered for antibody-targeted delivery. Intraperitoneal injections of EGFR-targeted sgPLK1-cLNPs caused their selective uptake into disseminated ovarian tumors, enabled up to ~80% gene editing in vivo, inhibited tumor growth, and increased survival by 80%. The ability to disrupt gene expression in vivo in tumors opens new avenues for cancer treatment and research and potential applications for targeted gene editing of noncancerous tissues.
INTRODUCTION
In recent years, molecularly targeted inhibitors and immunotherapy have greatly improved cancer responses with reduced toxicity and adverse reactions. However, the high recurrence rate and the development of drug resistance for most types of cancers highlight the need for new therapeutic modalities. Most cancer drugs require repeated administration, which increases treatment-related toxicity and treatment cost and severely reduces patient quality of life. CRISPR-Cas9 gene editing has the potential to permanently disrupt tumor survival genes, which could overcome the repeated dosing limitations of traditional cancer therapies, improve treatment efficacy, and require fewer treatments (1, 2). The Cas9 nuclease is directed by a single-guide RNA (sgRNA) to modify a specific chromosomal DNA sequence by inducing a sequence-specific double-strand break (DSB) (3, 4). DSBs are predominantly resolved via the error-prone nonhomologous end-joining repair mechanism, which can induce insertions or deletions that result in gene disruption. However, the large size of Cas9 (160 kDa, 4300 bases) and sgRNA (~31 kDa, 130 bases) is an obstacle for conventional viral and nonviral delivery systems. Moreover, current delivery systems for nonliver tissues and tumors only result in relatively low gene editing percentages (5, 6). For an effective cancer therapy, substantially higher editing efficiencies would be needed.
Lipid nanoparticles (LNPs) are clinically approved nonviral nucleic acid delivery systems capable of delivering potentially such large payloads. Cationic ionizable lipids are the key component of LNPs that enables efficient nucleic acid encapsulation, cellular delivery, and endosomal release. However, LNP formulations that were optimized for small interfering RNA (siRNA) delivery do not efficiently deliver large nucleic acids (e.g., mRNAs and plasmids) (7, 8). Most in vivo studies of gene editing have relied on adeno-associated virus (AAV) to deliver CRISPR components locally to the retina or skeletal muscle or to the liver. Nevertheless, AAV applications are limited by its small carrying capacity, immune responses, hepatoxicity at high doses, and the lack of cellular targeting (9, 10). Several nonviral delivery vehicles for CRISPR components have been reported in recent years (5, 11). These systems were evaluated for liver-associated genetic diseases, demonstrated gene editing of up to 60% in mice and rat livers, and almost a complete reduction of target protein in the blood. However, formulations designed for other tissues were less efficient (i.e., up to ~15% in the lung and ~3% in melanoma) (5, 6). Therefore, the development of efficient and safe delivery systems for nonliver tissues remains an important missing link for therapeutic translation of CRISPR editing.
Here, we report the development of a targeted nonviral LNP delivery system for therapeutic genome editing and evaluate it in two aggressive and incurable cancer models.
RESULTS
Development and characterization of LNPs encapsulating Cas9 mRNA and sgRNA
To overcome the cargo limitation of currently available LNP formulations, LNPs were designed to coencapsulate Cas9 mRNA and sgRNA, using ionizable cationic lipids from a novel ionizable amino lipid library (Fig. 1A) (12). This library was constructed using a novel class of ionizable amino lipids based on hydrazine, hydroxylamine, and ethanolamine linkers with a linoleic fatty acid chain and amine head groups (12). Lipids 1, 6, 8, and 10 were the top hits of the screen and were chosen for further evaluation for CRISPR-Cas9 gene editing (Fig. 1B). Cas9 mRNA was chosen, instead of plasmid DNA, to reduce long-term exposure to the nuclease to minimize off-target gene modifications (13, 14). To enhance RNA stability and minimize immunogenicity, Cas9 mRNA was chemically modified with 5-methoxyuridine, and highly modified sgRNAs were used (IDT sgRNA XT) (15, 16). CRISPR-LNP (cLNP) formulations containing Cas9 mRNA and an sgRNA were compared to Cas9 mRNA and sgRNAs encapsulated with the clinically approved LNP formulation, used for siRNA therapeutics, based on DLin-MC3-DMA as the ionizable cationic lipid (MC3-cLNPs). cLNPs were uniform in size with a diameter of 71 to 80 nm, polydispersity index of 0.024 to 0.103, and ζ potential of −3 to 18.6 mV as measured by dynamic light scattering (Fig. 1C). The biophysical properties and transmission electron microscopy micrographs of L8-cLNPs were similar to those of MC3-cLNPs (Fig. 1C and fig. S1A). The encapsulation efficiency of Cas9 mRNA and sgRNA in L6, L8, L10, and MC3-LNPs was similarly high (>90%) but lower in L1-cLNPs (~65%) (Fig. 1D). Next, we evaluated the in vitro gene disruption efficiency of the cLNP formulations encapsulating a GFP sgRNA by measuring the loss of green fluorescent protein (GFP) fluorescence in human embryonic kidney (HEK) 293 cells stably expressing GFP (HEK293/GFP) (Fig. 1E) (17). L1-, L8-, and L10-cLNPs all disrupted GFP in a concentration-dependent manner, and L8 was the most efficient. GFP fluorescence was detected in only 4% of L8-cLNP–treated cells after incubation with cLNPs containing total RNA (1.0 μg/ml). Although Cy5.5-labeled MC3-cLNPs were taken up more efficiently than L8-cLNPs into HEK293/GFP cells as measured by flow cytometry (fig. S1B), MC3-cLNPs did not reduce GFP expression at any concentration (0.1 to 1.0 μg/ml total RNA, 0.7 to 7 nM total RNA). On the basis of these data, L8-cLNPs were chosen for further study….
