Lecithin nano-liposomal particle as a CRISPR/Cas9 complex delivery system for treating type 2 diabetes
Protein-based Cas9 in vivo gene editing therapeutics have practical limitations owing to their instability and low efficacy. To overcome these obstacles and improve stability, we designed a nanocarrier primarily consisting of lecithin that can efficiently target liver disease and encapsulate complexes of Cas9 with a single-stranded guide RNA (sgRNA) ribonucleoprotein (Cas9-RNP) through polymer fusion self-assembly.
In this study, we optimized an sgRNA sequence specifically for dipeptidyl peptidase-4 gene (DPP-4) to modulate the function of glucagon-like peptide 1. We then injected our nanocarrier Cas9-RNP complexes directly into type 2 diabetes mellitus (T2DM) db/db mice, which disrupted the expression of DPP-4 gene in T2DM mice with remarkable efficacy. The decline in DPP-4 enzyme activity was also accompanied by normalized blood glucose levels, insulin response, and reduced liver and kidney damage. These outcomes were found to be similar to those of sitagliptin, the current chemical DPP-4 inhibition therapy drug which requires recurrent doses.
Our results demonstrate that a nano-liposomal carrier system with therapeutic Cas9-RNP has great potential as a platform to improve genomic editing therapies for human liver diseases.
KeywordsCRISPR-Cas system Nanoliposome Type 2 diabetes mellitus Dipeptidyl peptidase-4 gene
clustered regularly interspaced short palindromic repeats
single-stranded guide RNA
type 2 diabetes mellitus
dipeptidyl peptidase-4 gene
lecithin-based liposomal carrier particle
nuclear localization signal
1,2-dioleoyl-sn-glycerol-3-[(N-(5-amino-1-carboxylpentyl)iminodiacetic acid)succinyl]-(nickel salt)
dynamic light scattering
PEI-fused Cas9-RNP complexes into the NL
rhodamine B isothiocyanate
confocal laser scanning microscopy
protospacer adjacent motif
hypoxia-inducible factor 2
oral glucose tolerance test
intraperitoneal insulin tolerance test
Clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein (Cas) nuclease complexes have been established as tools for human genome engineering therapy . They are expected to facilitate drastic changes in the treatment of intractable genetic diseases . However, conventional methods of transfecting cells with plasmid DNA to encode single-stranded guide RNA (sgRNA) and Cas nuclease exhibit critical limitations such as off-targeting, integration of foreign DNA, and toxicity due to the transfection agents [3, 4]. Direct delivery of protein-based nucleases such as Cas9 or Cpf1 with sgRNA complexes is another possible therapeutic strategy, but it faces low encapsulation efficiency, poor cell membrane permeability, and proteolytic instability, especially in vivo [5, 6]. As such, there is great need for a biocompatible vesicle carrier that can effectively encapsulate protein/sgRNA complexes. Liposomal particles are great candidate materials given their simple preparation method, easy surface modification, and high biocompatibility. For example, Wang et al.  have recently reported a bio-reducible lipid nanocarrier complex for protein-based Cas9 genome editing. It was produced through electrostatic interaction of cationic lipids and super-negatively charged complexes via protein–protein fusion.
In this study, type 2 diabetes mellitus (T2DM) was used as a target disease given its suitability for genetic therapy concerning liver. T2DM is a complex disease characterized by high glucose levels in the bloodstream, reduced glucose processing capacity in adipocytes, and insulin resistance in the body . Elevating glucagon-like peptide-1 (GLP-1), an important target hormone that stimulates insulin secretion, is one of recent therapeutic approaches . However, this hormone has a short half-life due to its extremely rapid degradation by enzyme dipeptidyl peptidase-4 (DPP-4) . To prevent GLP-1 degradation, various drugs inhibiting DPP-4 such as sitagliptin, vildagliptin, saxagliptin, and linagliptin have been developed for insulin-mediated glucose control of T2DM . Moreover, since DPP-4 inhibitors are widely used in clinical practice, they are also investigated as potential new therapeutics against the development of hepatic fibrosis and steatosis . However, small-molecule antidiabetic drugs must be administered daily. In addition, they are associated with adverse effects such as hepatic impairment. A therapeutic method capable of effectively down-regulating DPP-4 enzyme with low side effects, such as a CRISPR/Cas9-based solution, would be an appropriate remedy for T2DM.
We prepared recombinant Cas9 nuclease complexes with an sgRNA as a ribonucleoprotein (Cas9-RNP) designed to edit the DPP-4 gene. To deliver the Cas9-RNP complex, a lecithin-based liposomal nanocarrier particle (NL) was developed. To increase encapsulation efficiency, a cationic polymer was integrated with the Cas9-RNP complex to compensate for the NL’s negatively charged lipid structure. This is because loading efficiency is strongly dependent on electrostatic interactions . Moreover, in consideration of biodistribution, NL are suitable for targeting liver diseases due to the natural metabolism of lecithin in the liver. Effects of Cas9-RNP incorporated NL were demonstrated by observing glucose tolerance and insulin resistance in T2DM mice.
Lecithin, cholesterol, rhodamine-B-Isothiocyanate (RITC), dimethyl sulfoxide (DMSO), and isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from Sigma-Aldrich. DOGS-NTA-Ni was purchased from Avanti Polar Lipids. ICG-NHS was purchased from Goryo Chemicals. All chemicals were used directly without any purification.
Synthesis of various NL particles
To synthesize NL particles, 1 mM lecithin, 0.7 mM cholesterol, and 0.3 mM DOGS-NTA-Ni were dissolved in 10 mL chloroform at 1:0.7:0.3 molar ratio and homogeneously mixed. The solution was evaporated to remove organic solvent and formed a thin lipid film. Recombinant Cas9-RNP complex prepared in 2 ml PBS (250 nM Cas9) was added to the lipid film and shaken gently for re-dispersion. The resulting multi-lamellar vesicle solution was then subjected to three cycles of freeze-thawing and probe sonication at 4 °C. To prepare fluorescent NL, primary amine reactive 19 μM rhodamine-B-Isothiocyanate (RITC) or 3.2 μM ICG-NHS in dimethyl sulfoxide (DMSO) solution was mixed with Cas9-RNP reaction buffer solution (0.1 M NaHCO3, pH 8.3) for 1 h in the dark. The mixing solution was loaded onto a spin-column (Thermo-Fisher), spun at 10,000g for 5 min, and washed 3 times with 100 μL PBS buffer solution. The labeled Cas9-RNP on the membrane was collected and dissolved in 200 μL PBS.
Synthesis of recombinant Cas9 protein and sgRNA complexation
Escherichia coli BL21 competent cells (RBC Bioscience) were transformed with a pET28-(a) vector encoding SpCas9-NLS fused to a 6×His-tag with a C-terminal NLS followed by a His-tag (Addgene). Cells were incubated at 28 °C overnight. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was then added at 0.5 mM to induce Cas9 protein expression. These cells were collected by centrifugation and lysed in lysis buffer [20 mM Tris–HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole, 1× protease inhibitor cocktail, 1 mg/mL lysozyme] by sonication. Soluble lysate was obtained by centrifugation at 20,000g for 20 min at 4 °C. The soluble fraction was incubated with a column containing Ni–NTA agarose resin (Qiagen) at 4 °C to capture His-tagged Cas9 protein. Column-bound Cas9 protein was eluted with elution buffer [20 mM Tris–HCl (pH 8.0), 300 mM NaCl, 250 mM imidazole and 1 × protease inhibitor cocktail] and dialyzed in a storage buffer [50 mM Tris–HCl (pH 8.0), 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 20% glycerol]. Purification fractions of Cas9 protein were quantified using a bicinchoninic acid (BCA) assay kit (Pierce Biotechnology) and analyzed by SDS-PAGE and Coomassie staining. To prepare Cas9 and sgRNA complex with PEI, 75 μg Cas9 in 0.5 mL PBS buffer was homogenized with sgRNA solution (26 μg sgRNA with 0.5 mL PBS). Then 160 μg PEI (2 kDa) was incubated with the protein solution for 10 min.
In vitro transcription of sgRNA
DNA fragment templates for sgRNA transcription were generated using an oligo containing T7 promoter and 20-bp sgRNA-specific target sequence overlapping with a second reverse complement oligo containing the constant region of the sgRNA backbone. All sgRNAs were transcribed using Guide-it™ sgRNA Screening kit (Clontech). In vitro transcribed RNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v), saturated with 10 nM Tris (pH 8.0) and 1 mM EDTA, precipitated with 2-propanol, and then re-dissolved in water. sgRNA concentration was quantified using a NanoDrop spectrophotometer and visualized after agarose gel electrophoresis.
Intracellular delivery of NL particles
SNU398 cells were seeded into 60 mm2 dishes before the experiment. The prepared NL solution (250 nM Cas9) was mixed with FBS-free medium and added to cells. Cells were incubated at 37 °C for 2 h. After the 2 h incubation, excess nano-liposomes were removed, and treated cells were incubated in FBS-containing medium. To study the uptake mechanism of NL@Cas9-RNP, SNU398 cells were pretreated with 200 μM genistein, 30 μM chlorpromazine, 50 μM nocodazole, and 0.01% sodium azide or 5 μM cytochalasin B for 30 min at 37 °C or 4 °C followed by treatment with the NL solution (250 nM Cas9).
In vitro cell analysis after treatment of NL@Cas9-RNP
For confocal laser scanning microscopy (CLSM) imaging of intracellular uptake of NL particles, SNU398 cells were seeded at density of 5000 cells/well into 4-well chamber slides (SPL) before the experiment. After treatment with RITC-labeled NL@Cas9-RNP for 2 h, treated cells were fixed with 4% paraformaldehyde and incubated in 1% BSA blocking buffer. Cells were then washed twice with PBS and incubated with anti-Cas9 (Abcam). After primary antibody incubation, cells were washed with PBS-T and incubated with CFL488-coupled secondary antibodies at room temperature for 1 h in the dark. At the end of incubation, cells were washed with TBS-T three times and counterstained with DAPI (Fluoroshield with DAPI mounting media). CLSM images were captured with a Leica TCS-SP5 laser scanning confocal microscope equipped with an oil objective.
DNA sequencing and efficiency analysis
Genomic DNA was isolated from cells (SNU398) or mouse liver tissue using genomic isolation kit (Bioneer) according to the manufacturer’s instruction. PCR amplicons including nuclease target sites were generated using primers listed in Additional file 1: Figures S7a and S10b. To analyze DNA sequences, amplified PCR products corresponding to genomic modification were purified using PCR purification kit (Macherey–Nagel) and cloned into T-Blunt vector using PCR cloning kit (SolGent). Cloned products were sequenced by dye-terminator sequencing method (Bioneer). To analyze genomic editing efficiency, PCR products were purified with a PCR purification kit (Macherey–Nagel) and quantified. Purified PCR products were subsequently combined, denatured, and then annealed through thermos-cycling for 5 min (95 to 85 °C at 2 °C/s; 85 to 20 °C at 0.2 °C/s). Annealed DNA was incubated with T7 endonuclease I (T7EI, New England Biolabs) for 30 min at 37 °C and analyzed by 2% agarose gel electrophoresis.
In vivo study for T2DM therapy
C57BL/6 wild-type mice and homozygous leptin receptor deficient db/db mice were purchased from Charles River Laboratories Japan, Inc. Mice were housed in a temperature- and humidity-controlled facility with a 12-h light/dark cycle for adaptation. To deliver particle under optimized conditions, 200 μL NL@Cas9-RNP (50 nM Cas9) was incubated at room temperature for 30 min before each injection. Sitagliptin (50 mM) was given daily to its treatment group by oral administration. Five mice were used for each treatment group. An EMD Millipore Multiplex MAP kit was used to assess concentrations of GLP-1 at the end of the 28-day treatment.
In vivo monitoring of NL@Cas9-RNP
Optical imaging was performed post injection of ICG-labeled NL@Cas9-RNP using a homemade high-quality in vivo imaging system connected to an EMCCD camera (DU897-EX, iXon Ultra; Andor Technology). Fluorescence images of mice were acquired using an 808-nm (0–15 W) diode laser (OCLA) as an excitation light source and an emission filter (840 ± 12 nm, Semrock). Prior to imaging experiments, mice were anesthetized with 4% isoflurane/O2 (v/v) and maintained in 1–2% (v/v) isoflurane/O2 atmosphere throughout the experiment.
In vivo therapeutic effect
Oral glucose tolerance test (OGTT) was performed at the end of the study. We carried out OGTT by taking baseline blood sample (200-μL) from the tail under local anesthesia and then gavaging with 50% glucose solution (Gibco) at 12 mL/kg to deliver 6 g of glucose per kg of body weight. Blood samples were taken at 30, 60, 120, and 180 min after the glucose meal and analyzed for blood glucose using Accu-Chek (Roche). For insulin tolerance test (ITT), regular human insulin (0.75 U/kg, Eli Lilly) was intraperitoneally injected and then blood samples were taken at 15, 30, 45, and 60 min post injection.
At 28 days after treatment, mice were euthanized and then liver and pancreas were harvested. For immunochemistry staining, tissues were fixed with 4% paraformaldehyde at 4 °C overnight. Paraffin-embedded tissue sections were then cut into 5 μm thick sections and dried at 60 °C for 1 h. These sections were deparaffinized in xylene and rehydrated through graded ethanol solutions followed by rehydration with water. Antigen detection was performed using anti-DPP-4 (Origene) and anti-insulin (Abcam). After rinsing with tap water, sections were stained with hematoxylin and cover-slipped using a mounting medium.
Results and discussion
Screening sgRNA sequences for DPP-4 gene targeting
To ensure high gene targeting efficiency, we designed sgRNA sequences and delivered Cas9/sgRNA plasmids into SNU398 human liver carcinoma cells. We cloned Streptococcus pyogenes Cas9 (SpCas9) vectors co-expressing one of the sgRNA options that recognize the DPP-4 gene and grafted a nuclear localization signal (NLS) sequence as well to enter the cell nucleus (Additional file 1: Figure S1a) . Cells transfected with Cas9/sgRNA plasmids were differentiated based on mRNA expression, protein level, and enzyme activity using quantitative real-time PCR, western blotting, and DPP-4 enzyme activity assay, respectively (Additional file 1: Figure S1b–d). A further validation study showed that sgRNA2 targeting DPP-4 gene worked better than sgRNA1 in decreasing mRNA, protein expression, and enzyme activity.
Preparation of recombinant Cas9 protein nuclease and complexing with sgRNA
Recombinant Cas9 protein was prepared following a standard procedure previously reported . Recombinant Cas9 protein containing NLS was purified from Escherichia coli and complexed with in vitro-transcribed sgRNA for targeting the DPP-4 gene (Additional file 1: Table S1 and Figure S2a, b). We then monitored cleavage of the DPP-4 gene caused by the prepared Cas9-RNP complex using an in vitro cleavage assay. Fragments (500 bp) of DPP-4 genomic DNA were allowed to react with Cas9 protein alone or Cas9-RNP complex for 1 h. The cleavage was then determined based on agarose gel electrophoresis (Additional file 1: Figure S2c). Cas9 protein alone did not affect the genomic fragment. However, the Cas9-RNP complex showed clear gene editing activity, indicating target DPP-4 gene recognition by the sgRNA complexed with Cas9 protein.
Preparation of NL and encapsulation of the Cas9-RNP complex
Gene editing in human cells by NL@Cas9-RNP
Off-target cleavage effects of NL@Cas9-RNP in cells
To evaluate gene editing specificity, we assessed the capability of NL@Cas9-RNP to reduce genome-wide off-target effects at different positions depending on various target sequences of chromosomes (Additional file 1: Figure S7a). In vitro genomic editing efficiency was found to be ~ 31% by T7 endonuclease I (T7EI) assay. We exploited seven different genomic off-target sites with 3- to 5-bases mismatched sequences of chromosome (Additional file 1: Figure S7b). Results showed that NL@Cas9-RNP was highly specific to DPP-4 gene sequence as an on-target site. These results demonstrated that our protein-based gene editing system could effectively recognize target sequences with high specificity, thus substantially improving sgRNAs targeting to homopolymeric sequences.
In vivo gene editing by NL@Cas9/gDPP-4 delivery in a T2DM mouse model
Blood analysis for glucose regulation and organ function test
Protein delivery for genome editing has undergone much recent development for therapeutic applications given its advantages such as low off-target effects and non-integration over plasmid delivery. Delivery of Cas9 in protein form has been previously attempted by other groups using methods such as local delivery, lipid nanoparticles, DNA nanoclew, and gold nanoparticle [21, 22, 23, 24]. We implemented a nanocarrier system, mainly composed of lecithin, that could effectively deliver Cas9-RNP complexes into hepatocytes via accumulation in the liver. Here were report the synthesis and application of NL containing therapeutic Cas9-RNP complexes for genome editing in vitro and in vivo. The encapsulation of Cas9-RNP complexes was enhanced by increasing the loading efficiency through fusion of a positively charged polymer. Spontaneous electrostatic interaction between charge-compensated complexes and negatively charged lipids resulted in self-assembly of NL spheres with uniform size distribution. Unlike unprotected protein therapeutic strategies that have low delivery efficacy due to enzymatic breakdown, our genome platform has exemplary biocompatibility with low cytotoxicity and high solution stability, making it optimal for genetic and chronic human disease therapies. As proof of concept, T2DM was selected as disease of focus. We designed a therapeutic strategy whereby the DPP-4 gene was disrupted by NL@Cas9-RNP delivery. We optimized an sgRNA sequence to target the DPP-4 gene. NL@Cas9-RNP successfully edited this gene with very low cytotoxicity. In db/db mice, NL@Cas9-RNP particle was able to decrease levels of DPP-4. Such disruption induced up-regulation of GLP-1 concentration in the blood. The enhanced efficacy of the gene disruption effect might be attributed to extended retention of NL in the liver given that hepatocytes can effectively uptake lecithin-based NL particles. Down-regulation of DPP-4 by NL@Cas9-RNP treatment also reconciled glucose tolerance and insulin resistance in a manner similar to sitagliptin. Interestingly, blood analysis showed that unlike sitagliptin, NL@Cas9-RNP treatment maintained normal liver and kidney function probably because of lessened drug dispensation. NL@Cas9-RNP can be applied as an effective treatment option for mitigating T2DM and its development. Moreover, our therapeutic strategy using NL@Cas9-RNP to target DPP-4 can be used for other diseases as well given that DPP-4 is known to exert substantial pleiotropic effects for a variety of diseases such as obesity, chronic renal disease, cardiovascular disease, and inflammation. Our results suggest that NL@Cas9-RNP system possesses several advantages: (1) highly efficient encapsulation of the Cas9-RNP complex, (2) effective and stable delivery in vivo, and (3) effective therapy concerning liver disease. More studies are warranted to characterize and optimize the pharmacokinetics, efficacy, and safety of such DPP-4 gene editing strategy using NL@Cas9-RNP in animal models.
EC, JR and HL carried out experiments, analyzed data and wrote the manuscript. SH, HP and KH conducted in vivo treatment and imaging analysis. HP, HK and TJ supervised entire project and involved in the designing of all experiments and revised the manuscript. All authors read and approved the final manuscript.
We thank Emmanuel A. Hui for reviewing the manuscript and Dr. Young Seok Cho (Seoul St. Mary’s Hospital) for performing T2DM therapy.
The authors declare that they have no competing interests relevant to this study.
Availability of data and materials
The authors declare that the data supporting findings of this study are available within the article and its Additional file 1.
Ethics approval and consent to participate
All experiments were performed in accordance with Ajou University Safety and Ethics guidelines. Animal experiments were carried out according to the animal experiment procedure approved by Institutional Animal Care and Use Committee (IACUC) of the Laboratory Animal Research Center of Ajou University (Permission No. 2016-0033).
This work was supported by the GRRC program of Gyeonggi province (GRRC 2016B02, Photonics-Medical Convergence Technology Research Center), and Ajou University research fund.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 19.Williams KH, De Vieira Ribeiro AJ, Prakoso E, Veillard AS, Shackel NA, Brooks B, Bu Y, Cavanagh E, Raleigh J, McLennan SV, McCaughan GW, Keane FM, Zekry A, Gorrell MD, Twigg SM. Circulating dipeptidyl peptidase-4 activity correlates with measures of hepatocyte apoptosis and fibrosis in non-alcoholic fatty liver disease in type 2 diabetes mellitus and obesity: a dual cohort cross-sectional study. J Diabetes. 2015;7:809–19.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.