Tools For Gene Transfer Into The Cells In Stem Cell Research And Genome Engineering

Nowadays most gene therapies based on viral vectors to transfer nucleic acids into the cells, but there is remarkable interest in developing chemical-based methods, as polymer-based vectors, because of their low cost, tunability and immunocompatibility. The full potential of polymer-based transfer systems has not been realized, however, because most of the polymeric transfection reagents are too inefficient and too toxic to use in the medicine fields. From this point of view, development in carbohydrate-based cationic polymers, termed glycopolymers, for enhanced non-viral gene transfer has been demonstrated. As omnipresent elements of biological systems, carbohydrates are a very rich group of compounds that are able to be harnessed to develop the biocompatibility of the non-native polymers, as linear polyamines used for stimulating transfection. A new class of carbohydrate-based polymers that called Polyglycoamidoamines (PGAAs) was developed by Reineke et al. by step-growth polymerization of linear monosaccharides with linear ethyleneamines. These glycopolymers were displayed to be either biocompatible or efficient transfection reagents. Systematic changes of the structural elements of the PGAA system showed structure-activity relationships significant to its function, including its capacity degrading in situ. Study is now underway to convert the application of glycopolymers for safe and efficient transfer of nucleic acid cargo for GT and gene editing uses.

The possibilities available for genetic alteration of the central nervous system cells (CNS) have greatly improved in the last decade. An available panoply of viral and nonviral vectors supply multifunctional platforms to transport expression cassettes into plenty of structures and nuclei. These cassettes are able to replace abnormal genes, change a given pathway disturbed by diseases, or generate proteins that can be selectively activated by medications or stimulate neurons. This study observes the use of canine adenovirus type 2 (CAV-2) vectors for gene delivery into neurons in the brain and peripheral nervous system.

Genome engineering has concentrated on inducible pluripotent stem cells that can grow into all 3 germ layers to avoid the ethical issues of embryonic stem cells. Methylation patterns can be detected in these cells, thereby, it is possible to perform research into pluripotency markers. CRISPR system that recently developed has allowed broad use of genome engineering techniques. The CRISPR-Cas9 system can be applied for gene knockout or knock-in genome manipulations through exchange of a targeted genetic sequence with an interest of donor sequence. The Cas9 nuclease can initiate 2 types of genome engineering, homologous and nonhomologous DNA repair. Viral and nonviral transfer methods can realize delivery of the CRISPR-Cas9 and target donor vectors in human pluripotent stem cells. Nonviral delivery method has lipid-mediated transfection and electroporation. Now it is the most common and effective delivery method for human pluripotent stem cells in vitro. CRISPR might be combined with human pluripotnt stem cells to investigate genetic factors of lineage choice, differentiation, and stem cell death.

Inherited retinal degeneration (IRD). Based on the way of inheritance of the dystrophy, retinal GT has 2 main strategies. AR, XL, and AD IRDs with haploinsufficiency can be treated by transferring a functional copy of the gene using both viral and nonviral vectors. Various types of viral vectors and nonviral vectors are applied to transfer plasmid DNA both in vitro and in vivo.

Currently, latest methods of genome alteration known as genome editing, which use so called 'programmable' nucleases available to apply. Introduction of the CRISP-Cas system which Cas9 plays a major role. This system relies on the components of the bacterial and archaeal mechanism responsible for adaptive immunity against phage infections and delivery of foreign genetic inofrmation. There are different types of CRISPR-Cas systems between prokaryotes but only components of CRISPR type II are used in genome engineering. CRISPR-Cas type II uses small RNA molecules (crRNA and tracrRNA) to accurately direct the effector nuclease - Cas9 - to a particular site in the genome, i.e. to the sequence complementary to crRNA. The CRISPR-Cas-based methods successfully have been applied for production of animal and cell models of many diseases, e.g. specific types of cancer.

Gene therapy offers a promising cancer treatment characterizing high effectiveness and limited side effects, but it is blocked by a lack of safe and effective gene transfer vectors. Lipid-based nonviral gene vectors, cationic polymers have many advantages and have been investigated for cancer gene transfer, but their low gene-expression efficiencies limit their clinical applications. Big efforts have been performed to developing new carrier molecules and generating functional vectors aimed at developing gene expression, but the overall effectiveness is still at the same level. Cancer gene-delivery cascade and the barriers, the needed nanoproperties and the current strategies for overcoming these barriers, and outlines PEGylation, surface charge, size, and stability dilemmas in vector nanoproperties to efficiently accomplish the cancer gene-tansfer cascade have been analysed. Stability, surface, and size transitions (3S Transitions) are offered to resolve those problems and strategies to find out these transitions.

In the future, the genome editing by programmable nucleases can find widespread application in medicine e.g. in the therapies of specific diseases of genetic origin and in the therapy of HIV-infected patients. 

09 March 2021
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