Application of Nanoparticles in CRISPR/Cas9 Gene Therapy

At present, genetic diseases are a large category of diseases that affect human health. So far, we can only treat a small part of them, and most of the treatment methods are “treating the symptoms but not the root cause”. Therefore, gene therapy that can “cure the root cause” is highly anticipated. Gene therapy is a treatment method that uses modern molecular biology methods to repair disease-causing genes to achieve relief and cure of diseases. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system won the Nobel Prize for related discoveries just a few years later, showing its vigorous vitality in gene editing.

In gene therapy, safe and efficient delivery is undoubtedly an important link that restricts its further application. Today, the delivery systems used for CRISPR/Cas9 are mainly divided into two categories: viral vectors and non-viral vectors. Adeno-associated virus (AAV) is the representative of viral vectors. Although viral vectors show high transfection efficiency, their clinical application is still limited by immune rejection and packaging capacity. Because 40%–80% of adults have been infected with wild-type AAV, and the packaging capacity of AAV is <4.7kb. Therefore, more and more researchers are now turning their attention to the development of non-viral vectors in the hope of achieving a breakthrough.

Nanoparticles in non-viral vectors have become a research hotspot for delivering CRISPR/Cas9 systems. The reasons are mainly as follows. First, they can be designed to be taken up by specific types of cells or tissues, providing effective targeting capabilities. Second, they can protect the cargo to be delivered (such as nucleic acids and protein substances) from degradation. Third, nanoparticles can also transport large-sized “cargoes”, such as CRISPR/Cas9 plasmids and nucleoprotein complexes. Fourth, nanoparticles have high biosafety and are not mutagenic compared to viral vectors. Fifth, nanoparticles are easy to prepare on a large scale, which is conducive to clinical translational applications.

Overview of Nanoparticles

Nanoparticles are defined as particles that have at least one dimension in the nanoscale range (1-100nm) in three-dimensional space or that are used as basic units. Because nanoparticles are composed of tiny units equivalent to molecular or even atomic sizes, they have some special physical or chemical properties. Nanoparticles are roughly divided into organic nanoparticles and inorganic nanoparticles. Among organic nanoparticles, the nanoparticles used to deliver CRISPR/Cas9 systems mainly include nanomicelles, lipid-based nanoparticles, and polymer nanoparticles. Inorganic nanoparticles that are widely used include mesoporous silica, magnetic nanoparticles (superparamagnetic iron oxide), nanogold, quantum dots QD (Cd, Se, Te), and lanthanide ions (Gd3+, Eu3+). Using the characteristics of nanoparticles in photoelectric, thermal, and magnetic aspects, they play an important role in the encapsulation delivery, imaging diagnosis, and modification of gene therapy.

CRISPR and Gene Therapy

Based on the classification of CRISPR’s cas loci, CRISPR systems can be divided into 6 different types (Ⅰ–Ⅵ), and each system uses a unique set of Cas proteins and CRISPR-related RNA (crRNA) for CRISPR interference. Among them, the type II Cas9 system is widely used, mainly because of its high efficiency and simple design and application. The iterative updates of the CRISPR/Cas9 system have also made gene therapy more mature and perfect. CRISPR-based gene therapy is showing vigorous vitality. From the development of more effective gene editing tools to the treatment of cancer and genetic diseases, CRISPR has begun to gradually move from a basic research tool to clinical application.

Biological Applications of Nanoparticles in Delivering CRISPR/Cas9 Systems

There are three main forms of delivery of CRISPR/Cas9 systems. They are plasmid DNA (pDNA), mRNA, and Cas9 protein complexes (RNPs). Different delivery forms have their own advantages and disadvantages and are suitable for different purposes. The advantages of pDNA are: (1) simple and convenient operation; (2) circular DNA is more stable. The disadvantages are: (1) large overall size (>10kb), more difficult delivery and expression; (2) nuclear transcription is required, which reduces editing efficiency; (3) plasmid form prolongs the duration of Cas9 protein, which may lead to higher off-target effects and stronger immune responses. This strategy is mainly used in in vitro cell experiments. The advantages of mRNA are: (1) no need to enter the nucleus, which can exercise editing function faster; (2) transient expression of Cas9 mRNA, which may help reduce the occurrence of off-target editing and avoid the risk of insertion mutations. The disadvantages are: (1) short expression time may also lead to low efficiency; (2) mRNA is unstable and prone to degradation. Therefore, this strategy is mainly used for genome editing of fertilized eggs, early embryos and cultured cells. Based on the form of RNPs, its advantages are: (1) RNPs can produce gene editing effects faster than pDNA and mRNA; (2) they can be rapidly degraded in cells, which can reduce off-target effects to a certain extent; (3) the complex form of protein and sgRNA can protect sgRNA; (4) the delivery of RNPs can avoid the risk of integration into the genome. Its disadvantages are: (1) the protein form may induce immune response; (2) the overall size is large (Cas9 protein is about 160 kDa), which makes effective delivery more difficult; (3) it is necessary to obtain Cas9 protein with guaranteed purity and activity in large quantities, which has a high investment cost. The most widely used lipid-based nanoparticles, polymer nanoparticles, gold nanoparticles and biofilm nanoparticles.

• Lipid-based Nanoparticles (LNPs)

Because LNPs have controllable composition, cell membrane structure, high carrying capacity and low toxicity, they have become one of the most widely used non-viral gene carriers for delivering CRISPR/Cas9. The classic liposome structure is a phospholipid bilayer, which can form vesicles that encapsulate lipophilic components in the bilayer, or encapsulate hydrophilic components in the water core. Due to the special design of the components, lipid-based nanoparticles can also form monolayer, bilayer and vesicle-like structures. Unlike classic liposomes, the surface of these lipid-based nanoparticles can still be modified with other components to add additional functions.

LNPs-mediated pDNA Delivery

Current commercial lipid systems, such as Lipofectamine2000, still have low transfection efficiency for CRISPR/Cas9. There are two main reasons for this: too large size and incomplete encapsulation. To overcome these obstacles, some researchers have proposed a “core-shell” strategy. First, Cas9-sgPLK-1 plasmid/chondroitin sulfate (CS)/fish protein was used as the core (to make the whole highly concentrated, small and stable), and then cationic lipids (DOTAP/DOPE/cholesterol) were encapsulated (to improve the mechanical strength of the lipid layer) as the shell, and finally DSPE-PEG was used to further modify the whole (to increase stability, solubility and half-life) to form a nanocomplex referred to as PLNP/DNA. The overall size was about 156.5 nm and the zate potential was 23.2 mV. In the in vitro transfection experiment, the transfection efficiency in A375, PC3 and MCF-7 cell lines was 47.4%, 36.2% and 37.8%, respectively, which was much higher than the transfection efficiency of Lipofectamine2000 as a control. After PLNP/Cas9-sgPLK-1 was administered to tumor-bearing mice (injected with A375 cell line), the volume of the tumor was significantly reduced, and about 3% of effective editing was found at the genomic level. The pDNA format is convenient to use, but further studies on its in vivo delivery are relatively limited.

LNPs-mediated mRNA Delivery

Ionizable LNPs (iLNPs) in liposomes have been widely used in the study of mRNA delivery in recent years, mainly because they are neutral in physiological environments, but can ionize in the acidic environment of endosomes, causing overall collapse, solution to release the properties of the goods. In CRISPR/Cas9-mediated gene therapy, one-time administration and long-lasting effectiveness has always been the goal pursued by scientists. Researchers developed and reported a type of LNPs called LNP-INT01, which delivered Cas9 mRNA and sgRNA to mouse livers. In a single dose, it was able to significantly edit 70% of the mouse transthyretin gene (TTR) in the liver. In order to prevent the carrier components from causing toxic side effects due to bioaccumulation, the researchers designed the connecting region of LP01, the main component of LNP-INT01, into an unstable ester bond to facilitate degradation. From this study, it can be seen that iLNPs show excellent potential in mediating gene editing in vivo, especially in the liver.

LNPs-mediated RNP Delivery

The rapid development of CRISPR/Cas9 has also promoted the development of commercial transfection reagents. Researchers have reported that a new transfection reagent, Lipofectamine CRISPRMAX, can effectively transfect in mammalian cell lines. Co-delivery of donor donors with Cas9 RNPs into a stable cell line carrying a disrupted EmGFP expression cassette mediated efficient replacement of up to 17%. Formulation utilizing RNPs may be a safe and effective format for in vivo delivery of gene therapy.

• Polymer Nanoparticles

Currently, the main polymer nanoparticles used for gene delivery include polyethyleneimine (PEI), poly(amidoamine), PAMAM, and chitosan. These polymers interact with nucleic acids to form compact nanosized polymers. This charge-neutralized dense polymer core is conducive to maintaining the stability of nucleic acids. The polymer/nucleic acid complex can enter cells through the clathrin-mediated endocytosis mechanism to complete delivery.

Polyethyleneimine (PEI)

PEI is a cationic polymer in which the -NH- groups give it a high density of positive charges, which can bind to negatively charged nucleic acids through electrostatic interactions and mediate effective transfection. Branched and linear forms of PEI show high transfection ability both in vivo and in vitro. Studies have evaluated the ability of branched PEI (molecular weight at 25kDa, BPEI-25k) to deliver CRISPR/Cas9 plasmids. The results showed that the CRISPR plasmid targeting the Slc26a4 gene was successfully delivered to Neuro2a cells and mediated about 22.9% effective editing. The limitation of further application of PEI lies mainly in its cytotoxicity. The existing strategy is to reduce the toxicity of PEI by combining it with biodegradable molecules (such as heparin, polycaprolactone PCL, dextran, chitosan, pullulan and folic acid, etc.).

Poly(amidoamine, PAMAM)

PAMAM is a dendritic macromolecule, and its size is expressed by generation (G). The periphery of the PAMAM polymer has a high density of primary amine groups, which can form a strong complexation with nucleic acids. The high density of tertiary amines inside helps the escape of the nanocomplex.

Chitosan (CS)

Chitosan is a natural polysaccharide with abundant sources, non-toxicity, and biodegradability. It is obtained by N-deacetylation of chitin. In acidic and neutral environments, due to the presence of amino groups, it carries a higher density of positive charges as a whole, so that it can form NPs with nucleic acids. As a gene carrier, chitosan has the advantages of low immunogenicity, good biocompatibility, and low cytotoxicity. In addition, chitosan can be degraded into common amino sugar-N-acetylglucosamine, which is excreted from the body through the metabolic pathway of glycoproteins and has good biodegradability.