Liposomes for Gene Editing

LIPOSOMES FOR GENE EDITING Delivery of CRISPR/Cas9 An expert in Lipid-based Drug Delivery Services

Creative Biolabs| Lipid-based Drug Delivery

Creative Biolabs| Lipid-based Drug Delivery

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Background

CRISPR/Cas9

Liposomal Delivery of CRISPR/Cas9

Liposome Surface Modifications

Recent Studies on Liposomal Delivery of CRISPR/Cas9

Creative Biolabs| Lipid-based Drug Delivery

Background In recent years, new gene-editing techniques have been developed and applied to various diseases treatment. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) appears to be the most suitable for preclinical and clinical applications compared to other gene-editing technologies due to its high gene editing efficiency and simultaneous multiple loci editing.

However, several barriers limited the direct use of CRISPR in gene therapy. First, CRISPR/Cas9 could be digested by endonuclease in the serum. Second, CRISPR/Cas9 is anionic, which reduces cell uptake efficiency due to electrostatic repulsion. Therefore, it is necessary to select carriers that meet the following key features for efficient CRISPR delivery: (1) Prolonged blood circulation time; (2) Efficient delivery into target tissues; (3) High cellular uptake and efficient endosomal escape.

Among the existing delivery carriers, liposomes with characteristic lipid bilayers that resemble the cell plasma membrane are the most widely studied gene carriers due to their excellent biodegradability and biocompatibility. In the liposomal CRISPR delivery system, many parameters including size, surface charge, PEGylation, and targeting ligands can be adjusted as need. Moving forward, liposomes will deliver their promise for further applications providing their serum stability, higher endosomal escape, and transfection efficiency.

Creative Biolabs| Lipid-based Drug Delivery

CRISPR/Cas9 •

Fuse crRNA and tracrRNA into single-guide RNA (sgRNA) to improve the stability

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Base pairing between the 5‘ -terminal 20 nt of sgRNA and target DNA

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Cas9 recognizes the PAM sequence at the 3’ – terminal of the target DNA

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Double-strand break (DSB) after CRISPR/Cas9 cleaves the target DNA

Creative Biolabs| Lipid-based Drug Delivery

Variants of Cas9

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dCas9 only retained the ability to be guided into the genome by gRNA, and the cleavage enzyme activity was completely lost

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The Cas9nickase is a mutant form of the Cas9 nuclease where one of its cleavage domains has been disabled to minimize the off-target effects of wild type Cas9

Creative Biolabs| Lipid-based Drug Delivery

Liposomal Delivery of CRISPR/Cas9 The liposome is an ideal drug delivery carrier with characteristic lipid bilayers that resemble the cell plasma membrane and high biosecurity.

They can incorporate a variety of drug candidates including anti-cancer formulations, nucleic acid and antigens. Liposomes are the most widely used carriers for delivering CRISPR/Cas9 due to their capacity to extend blood circulation time and prevent the enzymatic digestion in the serum.

Besides, cationic liposomes can effectively avoid the electrostatic repulsion of negatively charged cell membranes and improve cellular uptake and endosomal escape. Different structures of liposomes such as lipoplexes, stable nucleic-acid-lipid particles (SNALPs), lipopolyplexes, and membrane/core nanoparticles (MCNPs) can provide encapsulation of CRISPR/Cas9 plasmid, Cas9 mRNA with sgRNA or Cas9–gRNA ribonucleoprotein. The flexibility of liposomes also allows the many parameters to be adjusted as needed, including the degree of PEGylation, targeting ligands, and surface charge.

Creative Biolabs| Lipid-based Drug Delivery

Liposome Surface Modifications

Modify targeting ligands on liposomes to enhance cellular uptake further increases the gene-editing efficiency

Adjust surface charge of liposomes to control cell uptake, endosomal escape, blood circulation and tissue accumulation

PEGylation to increase structural stability and circulation time of liposomes

Creative Biolabs| Lipid-based Drug Delivery

Recent Studies on Liposomal Delivery of CRISPR/Cas9 Name

Lipid

Form of CRISPR

Ligands

Disease model

Liposome-templated hydrogel nanoparticles (LHNPs)

DOTAP

Cas9 protein/Minicircle DNA

iRGD, mHph3

Cancer

Liposome-coated mesoporous silica nanoparticle (lipoMSN)

DOTAP

Cas9/gRNA RNPs

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Cardiovascular disease

Lipofectamine 2000

Cationic liposome

Cas9/gRNA RNPs

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Genetic deafness

Lecithin-based liposomal nanocarrier particle (NL)

Lecithin

Cas9/gRNA RNPs

PEI

Type 2 diabetes mellitus

Creative Biolabs| Lipid-based Drug Delivery

Creative Biolabs’ Services of Liposome

Liposome Design • • • •

Formulation Structure Function Cargo

Liposome Production • • • • • • •

Encapsulation Combination of Nanoparticles & Materials Surface Charge Adjustment PEGylation Targeting Ligands Modification Controlled Release Other Surface Modifications

Liposome Analysis and Characterization • • • •

Basic characterization Analysis Formulation Stability Monitoring Functional Verification & Analysis Other custom Services

Creative Biolabs| Lipid-based Drug Delivery

Creative Biolabs’ Services of Liposome Our liposomes custom services can achieve prolonged blood circulation time, high cellular uptake, efficient endosomal escape and functional modification for liposomal delivery of CRISPR/Cas9. Different structures and ligands are optional according to requirements. Our services include liposome structure design, liposome encapsulation, liposome ligands modification and characterization. We can also provide strategies to combine liposomes with nanoparticles or other materials.

Features of our Liposomes Custom Services: •

High biocompatibility, low toxicity

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High cellular uptake and endosomal escape efficiency

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Encapsulation of different form of CRISPR/Cas9

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Multi-functionalization

As a liposome contract manufacturer, we are making high-end liposomal products for customers.

Creative Biolabs can customize the design & production of a liposomal product for you completely in line with your requirements. Our ground- breaking research, objectified working processes, and state-of-the-art equipment all mean that we can develop distinctive liposomal formulations and products. Our liposomes custom services offer genuine phospholipid bilayered vesicles containing the active substance. We can formulate liposomes for any active substance. Besides, our unique liposome products are subject to stringent quality control, and we can guarantee that our liposomes retain their quality and efficacy during storage. For more details about our services, please contact us directly.

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