Nucleic acid therapy can achieve lasting and even curative effects through gene augmentation, gene suppression, and genome editing. However, it is difficult for naked nucleic acid molecules to enter cells. As a result, the key to nucleic acid therapy is the introduction of nucleic acid molecules into cells. Cationic polymers are non-viral nucleic acid delivery systems with positively charged groups on their molecules that concentrate nucleic acid molecules to form nanoparticles, which help nucleic acids cross barriers to express proteins in cells or inhibit target gene expression. Cationic polymers are easy to synthesize, modify, and structurally control, making them a promising class of nucleic acid delivery systems.
Cationic polymers are a type of non-viral vector (Figure 1
). A common feature of their structures is the presence of many positively charged groups in the molecule, which are protonated into positively charged polymers. Cationic polymers can bind nucleic acids through electrostatic interactions and condense them into small nanoparticles 
. Positive charges improve interactions with negatively charged cell membranes, and aid polyplexes in escaping endosomes before lysosomal degradation occurs. 
. Subsequently, polyplexes escape from the endosome by the proton sponge effect that is mediated by positively charged groups on the cationic polymer 
. Furthermore, nucleic acids must dissociate from cationic polymers before finally performing their function 
Figure 1. Cationic polymers are used as nucleic acid delivery vehicles.
Gene therapy is the primary application of cationic polymers as transfection agents. By carrying plasmid DNA, mRNA, and siRNA, cationic polymers achieve therapeutically relevant functions such as gene augmentation, gene suppression, and genome editing.
2. Gene Augmentation
The most straightforward and perhaps simplest strategy for gene therapy involves the addition of a new protein-coding gene. For monogenic recessive diseases in which the disease-causing mutated gene is not functional, the therapeutic gene to be delivered will be the normal wild type of the gene. Delivery of the correct copy of the gene is expected to restore the production of the defective or missing protein, and thus restore the disease phenotype.
A non-viral vector for oral insulin gene delivery was created by Lin et al. 
. A copolymer of highly substituted branched chain polyethylenes (CS-g-bPEI) on chitosan makes up the vector, which helps the plasmid go through the intestinal epithelium, and prevents it from being destroyed by stomach acid. Such CS-g-bPEI/insulin plasmid DNA nanoparticles (NPs) enable systemic transgene expression for a number of days. In diabetic mice, a single dosage of oral NPs (600 micrograms of plasmatic insulin (pINS)) provided the animals with more than 10 days of protection from hyperglycemia. Similar hypoglycemia results were generated using a repeated dosage three times at 10-day intervals, with no liver injury noted.
The large-scale use of mRNA vaccines in the context of the current epidemic has ignited a passionate interest in nucleic acid drugs. Theoretically, mRNA is capable of expressing any kind of protein; thus, it can be useful in the treatment of other diseases, in addition to preventing infectious diseases as a vaccine. Examples include the treatment of tumors, rare diseases, metabolic diseases, etc. The current mRNA delivery technology is based on a lipid nanoparticle platform, and the patents for this technology are in the hands of a few companies. Moreover, mRNA vaccines composed of lipid nanoparticles should be stored and transported at ultralow temperatures, severely limiting the use of vaccines in areas with high temperatures or limited conditions. Therefore, cationic polymers are uniquely suited as an alternative to liposomal nanoparticles.
Liu et al. delivered mRNA to the spleen and lymph nodes in vivo via alkylated dioxophosphoryl oxides to cationic phospholipidated polymers (ZPPs) 
. This modular post-modification approach readily produced tunable amphiphilic species for antiserum purposes and simultaneously introduced alkyl chains to enhance endosomal escape, thereby transforming defective cationic polymers into effective amphiphilic mRNA carriers without the need for elaborate syntheses of functional monomers. ZPPs mediated 39,500-fold higher protein expression in vitro than their cationic parent, and selectively delivered effective mRNA into the spleen and lymph nodes after intravenous administration in vivo.
Nanoparticles often show significant adjuvant effects in vaccine delivery. Cationic polymers, including PEI, polylysine, cationic dextran, and cationic gelatin, have been reported to exhibit a strong stimulation of Th1 responses characterized by the induction of CD4(+) T-cell proliferation and the secretion of Th1-related cytokines 
. In addition, cationic polymers strongly inhibit LPS-induced TNF-α secretion from macrophages. The stimulatory ability of a cationic polymer is related to its degree of cationicity, and neutralization of a cationic polymer with an anionic polymer can completely abolish the stimulatory effect. The molecular weight of the polymer also affects its stimulatory ability, with larger molecules implying greater stimulatory ability.
In addition to mRNA vaccines, DNA vaccines are also good options. With the aid of polyglucose, spermine (PG) conjugates, and fourth-generation polyamide dendrimers (PAMAM G4), researchers concentrated on ways to administer synthetic T-cell immunogens as DNA vaccines 
. They improved the PG and PAMAM G4 complexes’ size, motility, and surface charge before testing the vaccine designs’ immunogenicity in BALB/c mice. As a result of being packaged in both the PG and the PAMAM G4 envelopes, the DNA vaccine’s immunogenicity increased, according to the findings. The strongest T-cell responses were seen in mice that were administered DNA vaccination complexes coated with PG, and these responses were noticeably higher than those shown in the group of animals that were prescribed the naked DNA combination and the DNA combination coated with PAMAM 4G.
3. Gene Suppression
Small interfering RNA (siRNA) is an initiator of RNA interference that stimulates the silencing of target mRNAs complementary to it, which is important for gene regulation and disease treatment. After entering the cytoplasm, siRNA is either loaded directly onto RISC or enters the Dicer-mediated interference process 
. The activated RISC localizes to the homologous mRNA transcript by base-pairing, and cleaves the mRNA at a position 12 nt from the 3’ end of the siRNA to achieve gene silencing. Chemically synthesized siRNAs have the advantages of easy operation, high transfection efficiency, and low toxic effects on cells or tissues; moreover, they can be prepared on a large scale. These advantages are especially suitable for screening effective fragments of siRNAs under the uncertainty of gene target sites.
Huang et al. combined siRNA and chemotherapeutic agents in the same nanoparticle to achieve in situ-activated ROS and CPT-based cascade therapy 
. They synthesized the copolymer mPEG-P (Asp-co-TkCPT) (PTkCPT) by covalently linking the hydrophobic chemotherapeutic drug CPT to the side chain of poly(ethylene glycol)-block poly(aspartic acid) (PEG-PAsp) via the dithione bond (Tk) of ROS-labile. PTkCPT was then self-assembled into a micelle containing a large number of -COOH groups in the micelle interlayer, and was used as a nanotemplate for CaP mineralization. In addition, Arf6 siRNA was loaded in the CaP layer. When PTkCPT/siRNA nanocapsules were endocytosed into cancer cells, the acidity of lysosomes may cause degradation of the CaP layer, and thus promote the release of siRNA. Arf6 siRNA blocked the Arf6 signaling pathway and promoted mitochondrial aggregation and subsequent ROS surge. ROS not only directly killed cancer cells, but also triggered chemotherapy by breaking the dithione bond in combination with CPT. Through this in situ cascade process, the nanoparticles significantly inhibited tumor growth in mice, with minimal side effects.
shRNA technologies are DNA-based. Most shRNAs are transcribed through plasmid vectors, and shRNA-based therapies are, therefore, dependent on the delivery of plasmid DNA. The transcription of shRNA is typically driven by the U6 promoter, which drives high levels of constitutive expression, or by the weaker H1 promoter. After transcription, the shRNA sequence is recognized by an endogenous enzyme, Dicer, which processes the shRNA into siRNA duplexes. As with exogenously synthesized siRNA oligonucleotides, this endogenously produced siRNA binds to the target mRNA and is incorporated into the RISC complex for degradation of the target mRNA. A major advantage of shRNAs over siRNA systems is that shRNAs can be designed to be inducible. Another advantage of shRNAs is that their expressing sequences can be integrated into the genome and cause persistent silencing of the target gene.
Kim et al. designed biodegradable (methoxy)poly(ethylene glycol)-b-(polycaprolactone-poly(lactic acid)) copolymers (MP) that derive spermine groups with cationic properties (MP-NH2
) at the pendant position of the MP chain 
can act as a gene carrier for St3-shRNA by forming electrostatic complexes with cationic spermine. This increases the stability of the complexes due to the protective effect of PEG in the biological environment, and can exhibit a sol-gel phase transition near body temperature, resulting in an intra-tumor injected MP-NH2
hydrogel library for the storage of St3-shRNAb. These complexes are not affected by external biomolecules, such as serum, DNase, and heparin, for a relatively long period of time (≥72 h). Intra-tumoral injections and St3-shRNA/MP-NH2
complex-loaded hydrogels showed prolonged and effective antitumor effects due to Stat3 knockdown.
4. Genome Editing
The field of genome editing has undergone a revolution, thanks to the discovery of CRISPR/Cas. The CRIPSR/Cas component of the bacterial immune system causes genome-wide double-stranded DNA breaks, and facilitates gene editing by internal DNA repair processes. It has been noted that the cationic polymer polyethylenediamine-cyclodextrin (PC) facilitates the effective delivery of plasmids encoding Cas9 and sgRNA 
. When large plasmids are delivered by PC, they can coalesce and enclose them at high N/P ratios; this effectively edits two genomic loci: hemoglobin subunit beta (19.1%) and rhomboid 5 homolog 1 (RHBDF1 (7.0%)). Researchers developed macrophage-specific promoter-driven Cas9 expression plasmids (pM458 and pM330), and encapsulated them in cationic lipid-assisted PEG-b-PLGA nanoparticles to solve the problem of being unable to execute precise gene editing in target tissues and cells (CLAN) 
. Due to the CD68 promoter’s unique production of Cas9, the Ntn1 gene was not disrupted in other cells. This method offers additional options for precise CRISPR/Cas9 in vivo gene editing. In vitro and in vivo, the CRISPR/Cas9 plasmid-encapsulated nanoparticles were able to exclusively express Cas9 in macrophages and their progenitor monocytes. More significantly, the resulting CLAN pM330/sgNtn1 successfully disrupted in vivo the expression of Ntn1 in macrophages and their Ntn1 gene in precursor monocytes, thereby reducing the expression of netrin-1 (encoded by Ntn1) and subsequently improving type 2 diabetes (T2D) symptoms.
Scientists have discovered that a related CRISPR system uses an enzyme called Cas13 that recognizes and cuts RNA instead of DNA 
. Among its other functions, RNA acts as a messenger, passing instructions between DNA and cellular machinery to make proteins. The ability to edit RNA before it is translated into proteins could open up new therapeutic options for human diseases. Recently, researchers have used PBAE-based polymers to deliver Cas13a mRNA and guide RNA into the respiratory tracts of mice and hamsters for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) treatment by nebulization 
. In the study, mRNA-encoding Cas13a was used to attenuate influenza A virus and SARS-CoV-2 infections in mice and hamsters, respectively. The researchers designed CRISPR RNAs (crRNAs) that targeted highly conserved regions of influenza viruses PB1 and PB2, and CRISPR RNAs targeting the replicase and nucleocapsid genes of SARS-CoV-2. Polymer-formulated Cas13a mRNA and validated guide RNA were delivered into the respiratory tract via a nebulizer. Results showed that in mice, Cas13a effectively degraded influenza RNA in lung tissue upon post-infection delivery, while in hamsters, Cas13a delivery reduced SARS-CoV-2 replication and alleviated symptoms.