GeneTherapy An emerging therapeutic approach

GeneTherapy

An emerging therapeutic approach

Gene therapies are promising treatment options for diseases that are considered untreatable. These therapies are designed by either of three methods, gene silencing, gene replacement, and gene editing to achieve the final therapy outcome. Although there are various methods to transfer the therapeutic gene into the target cells, viral vectors such as adeno-associated viruses (AAVs) seem to be the most preferred ones.

Sravan Kumar Pasumarthi1, Amit Khurana2*, Ralf Weiskirchen2*

1 The Business Research Company (TBRC ), India 2 Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH Aachen University Hospital

Gene therapy is a powerful therapeutic approach which relies on the modification or over-expression or suppression of gene expression. The conventional therapeutic approaches like use of small molecule inhibitors and biologics can't provide mitigation of genetic anomalies. Inherited genetic disorders involving mutation or deletion of genes may lead to defective metabolism, cell cycle, receptor-ligand interactions, protein synthesis and others. Hence, the unmet medical need in the area of genetic abnormalities is fulfilled by gene therapy. The past decade has seen great interest in the area of gene therapy with the approval of gene therapy based medicine Onpattro in 2018. In this editorial we provide an overview of the widely used methods for gene transfer, challenges in gene therapy and the applications of gene therapy in medicine.

Methods of Gene Transfer

Gene therapy relies on the process of transferring engineered genes to the targeted cells. Gene transfer is a process that aims to induce the synthesis of desired protein by inserting copies of manipulated genes into the living cells. Various methods are employed to transfer the genes into the target cells, which can be listed as physical methods, chemical methods, and biological methods. Several factors influence the choice of employing one of these methods for gene transfer, which include ease of use, efficiency, cost, reproducibility, toxicity, and mechanism of delivery. Figure 1 summarises the commonly used methods of gene transfer.

Physical methods help genes to enter the cell by exerting physical force on the cell membrane, thereby increasing the permeability of the cell. This method carries a risk of injury to cells due to the sheer physical force applied to it. Physical gene transfer methods include electroporation, biolistic, microinjection, laser, elevated temperature, ultrasound, and hydrodynamic applications.

Chemical methods help the genetic material to enter the cell by forming complexes. The chemical agents used in this method include calcium phosphate and diethylaminoethyl (DEAE)-dextran. These chemical compounds are mixed with DNA to form fine co-precipitates (complexes) which enter the cell through phagocytosis. Although this method is comparatively inexpensive, low transfection efficiency is considered a major disadvantage.

Biological methods used for gene transfer employ either viral transfection vector carriers or non-viral transfection vector carriers. Viral vectors can be adeno-associated viruses or lentivirus, whereas non-viral vectors include lipoplex and polyplex. Adenoviruses and lentivirus work differently, for instance, adenoviruses do not integrate within the host cell genome and have an episomal expression, whereas lentiviruses integrate inside the host genome which results in the formation of a stable and permanent expression of the gene and its products.

Although biological methods are highly effective there remain noticeable limitations such as large-scale virus production, immunogenicity, toxicity, and insertion of mutagenesis (in the case of integrating viruses).

In addition to the three methods discussed, there are novel delivery systems for gene transfer, which include cationic lipid-mediated transfection, liposomes, polymers, virus-like particles, erythrocyte ghost, exosomes, and other nanoparticles. Although some of these methods are considered better compared to the chemical methods in terms of efficiency, they cannot replace viral vectors. For instance, the transfection systems based on cationic lipid molecules are simple to synthesise and reliable but show disadvantages such as heterogeneity and structural instability, toxicity, and inactivation in blood with a lack of targeted delivery. Similarly, liposomes are also better than chemical methods but are not relatively efficacious as viral vectors. the cell, as this environment limits the direct delivery of macromolecules inside the target cell. One of the main barriers to successful gene delivery is the release of DNA from the endosome before its degradation at the lysosomal level. Employing liposomes, cationic polymers polyethylenimine (PEI), and fusogenic lipids (such as DOPE, and cholesterol) are helpful for endosomal escape. For instance, Lipoplexes are employed to help the DNA escape from endosome through the fusion of liposome with the endosomal membrane.

Although the DNA is protected from circulation and extracellular matrix, there remains a challenge to transferring the genes from exogenous genetic material from the cell membrane to the nucleus. Most of the methods for enhancing nuclear import include exploitation of the cellular protein nuclear import machinery.

Barriers to Gene Delivery

Major barriers to gene delivery include barriers to ease of formulation and largescale production of vectors coupled with the stability and storage of these products. In addition to these, there are three main barriers such as degradation of free plasmid DNA in the circulation, targeted delivery and intracellular trafficking, and efficient processing of gene delivery systems within the cell.

Free plasmid DNA is vulnerable to degradation within the circulation, cationic lips such as DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and DOPE (dio-leoylphosphatidylethanolamine) are employed to prevent DNA degradation. These cationic lips are used to encapsulate the plasmid and protect it from the enzymatic environment outside the cell.

Once the plasmid DNAs are protected from the circulatory environment, the next phase is to protect them from the extracellular matrix surrounding

Figure 1: Methods of gene transfer. Different methods of gene transfer can be grouped into physical, chemical and biological methods. The figure is reproduced with permission from N. Sayed, P. Allawadhi, A. Khurana, V. Singh, U. Navik, S.K. Pasumarthi, I. Khurana, A.K. Banothu, R. Weiskirchen, K.K. Bharani, Gene therapy: Comprehensive overview and therapeutic applications, Life sciences (2022) 120375