Non-viral vectors for gene delivery have attracted attention in the past decades becasue of their potential for limited immunogenicity, the ability to accomodate and deliver alrge size genetic materials and the potential for modification of theri surface structures.

Noevertheless, inherent to nonviral vectors are also important challenges. First, nonviral systems face aprpeciable plasmid loss during their mitotic segration in dividing cells. Second, silencing of transgene expression may occur due to epigentic events. Their, there is variable efficiency of plasmid delivery into various cells, both by physical and chemical methods. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

The major categories of non-viral vectors include cationic lipids and cationic polymers. 

Cationic lipid derived vectors:

Cationic lipid dervied vectors represent the most extensively investigated systems for non-viral gene delviery. Cationic polymer non-viral vectors have gained increasing attention becasue of flexibility in their synthesis and structural modifictions for specific biomedical applications. Both cationic lipid and cationic polymer systems deliver genes by forming condensed complxes with negatively charge deoxyribonucleic acid (DNA) through electrostatic interactions. Polyplexes formed between cationic polyemrs and DNA are relatively more stable than lipoplexes formed between cationic lipds and DNA. (Zhou “Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery” Nat Mater: 11(1) 82-90 (2012). 

Lipids and Lipid-based nanoparticles:

Companies: Beam Therapeutics

Polymers, liposomes, or other nanoscale structures can be modified to enhance their biocompatility, reduce cytotoxicity (e.g., PEG), and augment their potential to target specific tisseus or cell types. Polyemr and liposme ehcists increasingly collaborate with biologists to optimize the capablity of syntehtic nanostructures for improving nonviral gene delivery. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Concurrent advances in the development of syntehtic materials that encapsulate RNA, such as polymers, lipids and lipid nanoparticles (LNPs) ahve invigorated research into non-viral based delivery systems leading to FDA approval of subutaneously administered N-acetylgalactosamine (GalNAc)-siRNA conjugates that target hepatocytes, intravenously adminsitered LNP based siRNA drugs that target hepatocytes and emergency use authorizaton (EUA) and FDA approval for intramuscularly administered LNP based mRNA COVID vaccines. Tehse approvals suggest that improved delivery to non-liver tissues (also known as extrahepatic tissues) as well as local deliveyr to the CNS, eye and ear oculd result in new drugs. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022).

LNPs:

Lipid Nanoparticles (LNPs) are a key class of drug delivery system that includes nanoparticles approved by the FDA for liver siRNA delivery and form fRNA vaccine delivery. On the basis of the size of the hydrophoilic head group relative to the size of the hydrophobi tail or tails, lipids form distinct structures including micelles, liposomes and LNPs. FDA approved LNPs contain variations of four basic components: a cationic or ionizalbe lipid cholesterol, a helper lipid, and a poly (ethylene glycol) (PEG) lipid. In addition to the RNA payload, the Alnylam, Moderna and Pfizer/BioNTech/Acuitas LNPs include four components: the cationic or ionizable lipids DLin-MC3-DMA (Alnylam), SM-102 (Moerna) or ALC-0315 (Pfizer/BioNTech/Acuitas), cholesterol, the PEG-lipids PEG-2000-C-DMG (Alnylam), PEG-2000-DMG (Moderna) or ALC-0159 (Pfizer/BioNTech/Acuitas) adn DSPC. (Dahlman, “Drug delivery systems for RNA therapeutics” Nature Reviews Genetics, 23, May 2022)

LNPs are a key class of drug delivery system that includes nanoparticles approved by teh FDA for liver siRNA delivery and for mRNA vaccine delivery. On the basis of the size of the hydrophilic head group relative to the size of the hydrophobic tail or tails, lipids form distinct structures including micelles, liposomes and LNPs. FDA aprpoved LNPs contain variations of four basis components: a cationic or ionizable lipid, cholesterol, a helper lipid and poly(ehtylene glycol) (PeG)-lipd. Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)

Moderna discloses lipids and compounds useful for the nanopartcile composition for delivery of RNA therapeutics. (see US 9,533,047, US 9,867,888, US 10,195,156, 10,933,127  and US 10,799,463).

In addition to the RNA payload, the Alnylam, Moderna and Pfizer/tioNTech/Acuias LNPs comprise four componetns: the cationic or ionizable lipdis DLin-MC3-DMA (Alnylam), SM-102 (Moderna) or ALC-0315 (Pfizer), cholesterol, the PEG-lipids PEG-2000-C-DMG (Alnylam), PEG-2000-DMG (Moderna) or ALC-0159 )Pfizer). Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)

Liposomes and synthetic polymers are chemical means that exploit the nanoscale size and cotrollable surface properties of organic and polyemric molecuels for gene delivery. These chemical carreirs are intended ot match or exceed the performance of viral vectors with fewer immunogenic complciaitons. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–PEGylated siRNA-loaded lipid nanopartciles: have addressed the greatest challenge in implementing siRNA therapeutics, which is their delivery. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–In vivo Gene therapies:

In vivo gene therapeis can be delivered via the nonviral vehicle lipid nanoparticle (LNP). In fact, gene editing tools can be delivered to patients through intravenous infusion of LNPs targeted to hematopietic stem cells (HSCs), elimianting the need for transplantation altogtther. 

Virus-Like Particles:

Virus-like particles represent another class of nonviral gene dleivery vectors, and these typically consist of self assembled viral protein nanostructures. 

Polymers and Polymer-based nanoparticles:

Many non-viral RNA delivery systems utilize polymers and polymeric nanoparticles. Chemists can vary polymer traits including charge, degradability and molecualr weight, all of which influence how polymers delivery RNA into cells. One frequently used polymer is poly(lactic-co-glycolic acid) (PLGA). PLGA drug delivery systems have been approved by the FDA for the delivery of small molecule drugs but not for the devliery of nucleic acids. At neutral pH, PLGA does not have the positive charge required to complex the anionic RNA phosphodiester backbone. Thus, to utilze PLGA as an RNA deliveyr system, scientists have added cationic chemical groups such as chitosan to delivery siRNA in mice. (Dahlman, “Drug delivery systems for RNA therapeutics” Nature Reviews Genetics, 23, May 2022)

Episomal vectors/Episomes/Exosomes/Plasmids: 

Most plasmids (natural or aritifical) used in trasnfections, remain in the nucleus only transiently. Nonviral episomes have to pass thorugh a number of critical stages, starting with gaining cellular entry, until they can modulate sustained, long term maintenance within cells. The fate of pEPI-based, established, and onintegrating episomal vectors is characterized by a sequence of fundamental stages inside the host cell: establishment, replication, mitotic stability, and plasmid segregation in daughter cells at mitosis. These are ocmplex processes mediated by specific chromatin structures and DNA features, and studies during the recent years have greatly explanded how episomes can function as gene transfer vectors in a gene therapy context. Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Exosomes:

Exosomes are small secreted vesicles that are produced by all cells, are present at high concentrations in interstitial and other body fluids, and have been present throughout the life of every person and the evolution of every animal. They are in fact the only biologically nomral nanovesicle in existence. In contrast to LNPs, which have been shown to elicit cellular toxicity, exosomes were shown to have no adverse effects at any dose tested. Moreover, mRNA-loaded exomsomes were characterize by efficient mRNA encapsulation, high mRNA content, consistent and consistent size. (Gould, “Exosome-medaited mRNA delivery in vivo is safe adn can be used to induce SARS-CoV-2 immunity” J. Biiological Chemistry, 2021). 

Nonviral and nonintegrating episomal vectors are reemerging as a valid, alternative technology to integrating viral vectors for gene therapy, due to their more favorable saftey profile, significantly lower risk for insertional mutagenesis, and a lesser potential for innate immune reacitons, in addition to their low production cost. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Selaru (WO 2016/123556) discloses extracellular vesicles (Evs) derived from a cancer assocaited fibroblast (CAF) which includes a payload such as a polynucleotide such as miR-195, miR-126 or miR-192, identified as being dwon-regulated in the CAF. The extracellular vesicel selectively target a cancer cell. 

Episomes:

The term episome was propsoed by Fancois Jacob and Elie Wollman in 1958 to describe extrachromosomal genetic material that may replicate autonomously or become integrated into the chromosome. Although the term episome is now somewhat interchangeable with the term plasmid, episomes are larger in size and are retained for a longer period of tiem following transfection of host cells. Episome integration is a rare event and can occur through canonical sequence independent, nonhomolgous end joining or microhomology medaited end joining. Nevertheless. episomal integration must be inhibited if episomes are to become safe vihciles for gene therapy applciations. Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Episomal vectors or “episomes” are free, circular, extrachromosomal DNA molecuels of viral and nonviral origin. Viral epiosmes refer to viruses whose natural life cycle includes a stage of remaining as free, viral DNA within the cell nucleus, retaining the ability to encode proteins without integrating into the host cell’s genome. Examples include Adenoviruses (see outline). Non-viral episomes are effectively plasmids, usually of a larger size relative to conventional plasmids, and can exist independent of the genomic DNA for longer periods than covnentional plasmids. Both types of episomes are used as vectors of gene transfer in gene therpay. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Successful episome based gene expression hinges on the ability to safely and precisely deliver episome vectors to complex biological environments. Although bare nucleic acids can be delivered in vivo by direct introduction of DNA or RNA into cells, rpaid clearance and loss of expression limit the effectiveness of this approach. Several carreir systems can be utilized to deliver episomes and these include liposomes, syntetic polyemrs, or physical means of gene delivery (electroporation and sonoporation, among others). (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Episomes have also been successfully delivered in several tissues using lysin-PEG and polycationic comb polymers with nuclear loalizing sequences, among others. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–Types of episomal vectors:

The most commonly used episomal vector ahs been pEPI-1, which originated form pGFP-C1 with the addition of the S/MARs anchoring elements, which keep the chromosomal DNA tethered into the nuclear matrix or scaffold. pEPI-1 was the first vecotr reproted as nonintegrative in the long term following trasnfection and the maintenance of key vecotr egiones. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

Sleeping Beauty (SB) Transporson System:

The sleeping Beauty (SB) transposon system has emerged as a promising alternative to viral vectors. The SB system shares with viral vectors the need to integrate into the recipient cell’s genome to be functional. However, ocntrary to viral vectors, it does not show integration preference for sites within highly transcribed gehome domains. And since the vast majority of the genome does not contain highly transcribed gehnome domains, random integraiton of SB outside these domains reduces its risk for insertional mutagenesis. (Mulia “Advances in the development and the applications of nonviral, episomal vectors for gene therapy” Human Gene Therapy, 32 (19-20), 2020).

–Modified mRNA: Modern disclsoes a modified mRNA which overcome probleems with respect to the modulation of intracellular translation and processing of nuceic acids encoding polypetpides (US 9,950,068).