Table 1:
Summary of select NP formulations
| NP formulation | NP traits | CRISPR/Cas9 cargo | In vitro results (cells used) | In vivo results |
|---|---|---|---|---|
| Gold nanoclusters/Tat complexed with cargo (core): cationic lipid shell (75) | • ~70 nm d. (SEM) • ~103 nm d. (DLS) •+35 mV ZP |
Cas9 protein + pDNA encoding Plk1-targeting sgRNA – co-loaded into NP | •TE: 55.7% (A375) •EE: 26.6% (A375) Treatment: 2 μg pDNA or Cas9 protein/1×106 cells/ml |
•IT delivery of NPs significantly reduced tumor growth (>70%) in a melanoma model (A375) in athymic mice Dosing: 10 μg pDNA or Cas9 protein/mouse (7 doses over 15 days) |
| Lipid NPs: cholesterol, C14-PEG 2000, DOPE and ionizable lipid cKK-E12 formulated with cargo (77) | ~70 nm d. (DLS) (165) *1 | Pcsk9-targeting chemically modified sgRNAs + Cas9 mRNA | •IV delivery: > 80% EE (hepatocytes) •Low frequency of off-target indels •No liver toxicity or cytokine induction Dosing: 1.2 mg/kg Cas9 mRNA and 0.5 mg/kg of each sgRNA/mouse |
|
| Lipid NPs: bioreducible lipid (3-O14B), cholesterol, DOPE, and C16-PEG2000−ceramide formulated with cargo (78) | • ~292 nm d. (DLS) •−9 mV ZP |
sgRNA/Cas9 RNP targeting GFP | EE: approx. 70% (HEK293-GFP) Treatment: 25 nM RNP (6 μg/ml lipid) |
|
| Lipidoid NPs (optimal formulation: 76-O17Se) + cargo (81) | • ~314 nm d. (DLS) •−13.2 mV ZP |
sgRNA/Cas9 RNP targeting GFP | EE: approx. 50.2% (HEK293-GFP) Treatment: 25 nM RNP (3.3 mg/L lipidoids) |
|
| Cationic liposomes embedded with PSMA-specific RNA aptamer (linked to DSPE-PEG) plus cargo (86) | • ~150 nm (DLS) •+40 mV ZP |
Cas9 protein + pDNA encoding Plk1-targeting sgRNA | > 90% down-regulation of Plk1 mRNA (LNCap cells) compared to 80% down regulation by liposomes without aptamer and Lipofectamine 2000 | IV delivery (day 0) reduced LNCap tumor volumes by 1.66-fold versus 2.56-fold increase for PBS control (over days 13 – 27) in athymic mice Dosing: 40 μg liposomes-sgRNA + 40 μg Cas9 |
| PEGylated NPs comprising cationic alpha-poly-glutamate-based polypeptides (P-HNPs) plus cargo (29) |
• ~100 nm (DLS) •+20 mV ZP |
•In vitro: Cas9 pDNA + sgRNA targeting AAVS1 and/or HPRT1 • In vivo: Cas9 pDNA + sgRNA targeting Plk1 Note: pDNA and sgRNA were independently loaded into P-HNPs |
EE: 40 – 50% (HEK293T) Treatment: 1 μg/ml pDNA and 0.5 μg/ml each sgRNA |
IT delivery: 1) >71% tumor suppression 2) Improved survival (60% versus 0% (untreated/ controls)) 3) EE: 35% (mice with HeLa tumors) Dosing: P-HNPs containing 20 μg pDNA (delivered days 7,9.11,13 and 15 PTC) + P-HNPs containing 20 μg sgRNA (delivered on days 8,10,12,14, and 16 PTC) |
| Lipid NPs: biodegradable ionizable lipid (LP01), cholesterol, DSPC and PEG2k-DMG formulated with RNA cargo (N:P ratio of 4.5:1) (90) | • ~105 nm d. (DLS) | Chemically modified sgRNA (targeting TTR) and Cas9 mRNA (wt ratio of 1:1 for mRNA:sgRNA) | •IV delivery : ~ 70% EE (liver): > 97% reduction in serum TTR levels (CD-1 mice) Dosing: 3 mg/kg NPs •IV delivery: ~ 65% EE (Rats) Dosing: 5 mg/kg NPs |
|
| ZAL NPs: “ZA3-EP10” ZAL:cholesterol:PEG lipid + cargo (88) |
• ~170 nm d. (DLS) • ~−6 mV ZP |
•In vitro: sgRNA ( ~100 nt) targeting luciferase (Luc)
•In vivo: sgRNA targeting LoxP + Cas9 mRNA (wt ratio of 1:4) |
EE: 95% (HeLa-Luc-Cas9) Treatment: 7 nM sgRNA |
•EE: 1.5 −3.5% (hepatocytes) •Confocal microscopy of sectioned organs demonstrated tdTO positive cells in liver, lung and kidney (mice) •IV delivery resulted in preferential accumulation in lungs of mice Dosing: 5 mg/kg total RNA |
| Self-assembling NPs with an inorganic core (inc. protamine sulfate + cargo) and a chitosan shell with targeting moieties (58) | • ~300 nm d. •−9 mV ZP |
pDNA encoding both Cas9 and sgRNA targeting CDK11p110 gene | > 90% reduction in CDK11p110 expression by MCF-7 cells (Western blotting) Treatment: 1 μg/ml pDNA |
|
| Cationic arginine functionalized gold NPs (10 nm) + cargo (149) | 475 (± 60) nm (“nanoassemblies”) | sgRNA/Cas9glut (+NLS) RNP targeting AAVS1 gene (or PTEN gene) (1:1 molar ratio of sgRNA:Cas9) | •UE: 80–90% (HeLa) •EE: 30% (Hela/HEK-293T/RAW) Treatment: 125 nM NPs/62 nM RNP |
|
| ZIF-8 plus cargo (Loading capacity: 1.2 wt %) (153) |
• ~100 nm d. (DLS) •+5 mV ZP |
sgRNA/Cas9 RNP targeting EGFP (1:1 molar ratio of sgRNA:Cas9) | Reduced EGFP fluorescence by 37% (CHO-EGFP) Treatment: 240 nM RNP |
|
| GO-PEG-PEI NPs plus cargo (154) | • ~220 nm (DLS) •+18.5 mV |
sgRNA/Cas9 RNP targeting EGFP (1:1 molar ratio of sgRNA:Cas9) | EE: 39% (AGS-EGFP) Treatment: 30 μg/ml GO-PEG-PEI/; 100 nM RNP |
|
| PEI-coated self-assembling DNA nanoclews complexed with cargo (148) | • ~56 nm d. (DLS) •+18 mV ZP |
sgRNA/Cas9 RNP targeting EGFP | EE: 36% (U2OS-EGFP) Treatment: 8 μg/ml DNA nanoclews; 100 nM RNP |
IT delivery: ~ 25% loss of EGFP+ve cells at the site of injection (mice) Dosing: 5 μM RNP |
| PT24/Cas9 RNP NPs (32) (Ratio PT24:Cas9 RNP = 62.5:1) |
• ~270 nm (DLS) •+4 mV ZP |
sgRNA/Cas9 RNP targeting HPRT1 | EE: 30–40% (HeLa) Treatment: 160 nM RNP |
|
| Fluorinated G5DP NPs complexed with cargo (N:P ratio of 5:1) (117) | • ~150 nm (DLS) •+30 mV ZP |
pDNAs encoding sgRNA/Cas9d-VP64 targeting MASPIN | Five-fold increase in MASPIN expression over control in MCF-7 cells Treatment: 1 μg pDNAs/well |
EE = editing efficiency; TE = transfection efficiency; UE = uptake efficiency; DLS = dynamic light scattering; SEM = scanning electron microscopy; IT = intratumoral; IV = intravenous; DSPC = distearoylphosphatidyl choline; ZAL = zwitterionic amino lipid; NLS nuclear localization sequence; ZP = zeta potential; d. = diameter; nt = nucleotides; tdTO = tdTomato; wt = weight; G5DP NPs = Nanoparticles made from G5 dendronized polymers; Plk1 = polo-like kinase 1; DSPE-PEG = poly(ethylene glycol)-grafted 1,2-distearoyl-sn-glycero- 3-phosphatidylethanolamine; PTC = post tumor challenge; ZIF-8 = zeolitic imidazolate framework-8; GO = graphene oxide
*1
size of NPs here is when no cargo was present. Authors did not provide data on size of NPs + cargo.