Shaping the future from the small scale: dry powder inhalation of CRISPR-Cas9 lipid nanoparticles for the treatment of lung diseases

Figures & data

Figure 1. Schematic structure and mechanism of action of CRISPR-Cas9. 1. sgRNA representation. 2. sgRNA + Cas9 protein illustration: the sgRNA-Cas9 exploits the PI domain to recognize and match with PAM sequences in the DNA, (3) triggering the strand separation of the target DNA duplex and promoting sgRNA-DNA hybrid formation, which enhances the DNA DSB. 4. Consequently, the cell pathways for genome repair NHEJ and HDR are enabled and exploited for gene deletions or insertions.

Figure 2. Illustration of LNP formulations for CRISPR-Cas9 and siRNA delivery. The main components of LNP formulations are cationic/ionizable lipids, helper lipids, PEG-lipids, and cholesterol. Cationic/ionizable lipids enable the efficient loading of CRISPR-Cas9 and/or siRNA into LNPs.

Table 1. Summary of studies developing CRISPR-Cas9-loaded LNPs, the main delivery target, and the administration route.

CRISPR-Cas9 form	Target gene	Target organ	Administration route	Ref.
Cas9 and sgRNA	EGFP	Brain	Brain injection	[99]
Cas9 and sgRNA	SV40 polyA	Brain	Brain injection	[109]
Cas9 and sgRNA	Pten	Lung	Intravenous injection	[109]
Cas9 and sgRNA	SV40 polyA; p53; Pten; Eml4; Alk; RB1	Lung	Intravenous injection	[109]
Cas9 and sgRNA	Eml4; Alk rearrangement	Lung	Intravenous injection	[109]
Cas9 and sgRNA	DMD	Tibialis anterior muscle	Intravenous injection	[109]
Cas9 and sgRNA	Pcsk9	Liver	Intravenous injection	[109]
Plasmid DNA encoding for Cas9 and sgRNA	EGFP	embryonic mesenchymal cells	In vitro	[101]
Plasmid DNA encoding for Cas9 and sgRNA	Plk1	Implanted A375 tumor	Intratumoral injection	[102]
Plasmid DNA encoding for Cas9 and sgRNA	PLK1	Xenograft tumor	Intratumoral injection	[93]
Plasmid DNA encoding for Cas9 and sgRNA	Pcsk9	Liver	Intravenous injection	[94]
Cas9 mRNA and sgRNA	DMD1	Hindlimb muscle	Intravenous injection	[54]
Cas9 mRNA and sgRNA	Pten; Pcsk9; Plk1; PLK1	Multiple Organs	Intravenous injection	[105]
Cas9 mRNA and sgRNA	PCSK9	Liver	Intravenous injection	[95]
Cas9 mRNA and sgRNA	Pah; TTR	Liver	Intravenous injection	[96]
Cas9 mRNA and sgRNA	Ttr; TTR	Liver	Intravenous injection	[97,104]
Cas9 mRNA and sgRNA	Angptl3	Liver	Intravenous injection	[98]

Figure 3. Schematic representation of NEM preparation and pulmonary delivery. (a) Nucleic acid-loaded nanoparticles are mixed with an entrapping matrix excipient to ensure the preparation of dry powders by spray drying. (b) NEMs are loaded into a dry powder inhaler and, due to ideal aerodynamic properties, the formulation can penetrate the lungs and release RNA-based nanoparticles at the target site.

Table 2. Examples of successful inhalable nucleic acid-based NEMs.

Formulation to be dried	Key excipients	Drying method	Main findings	Ref.
siRNA-dendrimer nanocomplexes	Trehalose, inulin, mannitol	Spray drying (SD)	After SD: successful reconstitution of nanocomplexes, siRNA integrity and function were preserved.	[148]
siRNA-PLGA nanoparticles	Trehalose, lactose and mannitol	SD	SD parameters were optimized and mannitol as excipient achieved the lowest residual moisture. siRNA integrity was preserved after SD, but gene silencing was not reported.	[149]
siRNA-lipid PLGA nanoparticles	Mannitol	SD	SD did not affect the physicochemical characteristics of nanoparticles; both siRNA and gene silencing activity were preserved in dry powder.	[150]
siRNA solid lipid nanoparticles	Mannitol	Thin-film freeze-drying, SD, and conventional shelf freeze-drying	Thin-film freeze-drying improved physicochemical and aerosol properties compared to SD or conventional shelf freeze-drying.	[151]
Peptide-DNA nanoparticles	Mannitol	SD	Mannitol concentration, inlet temperature and spray rate had a significant effect on DNA recovery, dry powder size, and peptide-DNA-NEM redispersion.	[152]
DNA-PEI polyplexes and DNA-lipopolyplexes	Poly(vinyl alcohol)	SD	NEMs derived from DNA-polyplexes and lipopolyplexes presented appropriate aerodynamic properties, even without the use of stabilizers. Both NEMs increased transfection efficacy compared to the fresh counterparts and ensured successful in vivo transgene expression.	[153]
miRNA-lipid–polymer hybrid nanoparticles (LPN)	-	Nebulization	Aerosolized miRNA-LPN had similar physicochemical properties as before nebulization and suitable aerodynamic properties to ensure lung deposition. After nebulization, the miRNA-loaded LPNs retained their ability to inhibit IL-8 secretion.	[154]
siRNA-loaded human serum albumin	Mannitol	SD	siRNA powder had appropriate aerodynamic properties and up to 78% siRNA was preserved upon SD.	[155]
DNA-PEI polyplexes	Mannitol and Trehalose	SD	NEMs derived from DNA-polyplexes and presented appropriate aerodynamic properties, and redispersability was optimized by the excipient content. NEMs increased transfection efficacy compared to the fresh counterparts and ensured successful in vitro transgene expression. Aerodynamic properties were assessed based on DNA content.	[122]
siRNA-PEG-PCL-PEI and Transferrin-PEI polyplexes	Mannitol and Trehalose	SD	SD temperatures and excipient content were optimized to ensure appropriate aerodynamic properties, siRNA integrity and unaffected polyplex gene silencing efficacy after SD and redispersion for both PEG-PCL-PEI and Transferrin-PEI polyplexes in vitro and ex vivo in primary T cells. Aerodynamic properties were assessed based on siRNA content.	[123]
siRNA-lipid nanoparticles	Lactose	SD	SD temperatures were optimized for siRNA-LNPs for efficient redispersion, appropriate aerodynamic properties and unaffected gene silencing efficacy in vitro and ex vivo in human lung tissue.	[160]

Table

COPD	Chronic obstructive pulmonary disease
CF	Cystic fibrosis
CFTR	Cystic fibrosis transmembrane conductance regulator
TNF-α	Tumor necrosis factor-alpha
CRISPR-Cas9	Clustered regularly interspaced short palindromic repeats
DSs	Delivery systems
LNP	Lipid nanoparticles
sgRNA	Single guide RNA
REC	Recognition
BH	Bridge Helix
NUC	Nulclease
PI	Protospacer adjacent motif interacting
PAM	Protospacer adjacent motif
DSB	Double-strand break
NHEJ	Non-Homologous End Joining
HDR	Homology Direct Repair
HITI	Homology-independent targeted integration
AAV	Adeno-associated viruses
PEI	Polyethyleneimine
PEG	Polyethylene glycol
DLin-MC3-DMA	Dilinoleylmethyl-4-dimethylaminobutyrate
ApoE	Apolipoprotein E
NEM	Nano-embedded microparticles
SD	Spray drying
SORT	Selective Organ Targeting

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