Gene Therapy: Transfection And Molecular Basis PDF

Gene Therapy: Transfection and its Molecular Basis • Gene Therapy involves altering the genes inside the body's cells to treat disease. Loss or gain of function can be achieved through the production of good protein transcripts or the use of siRNA. • Transfection is the introduction of nucleic acids...

Gene Therapy: Transfection and its Molecular Basis • Gene Therapy involves altering the genes inside the body's cells to treat disease. Loss or gain of function can be achieved through the production of good protein transcripts or the use of siRNA. • Transfection is the introduction of nucleic acids into animal cells, while transduction refers to virus-mediated DNA transfer. Non-viral DNA transfer into bacteria, non-animal eukaryotic cells, and plant cells is known as transformation. • Understanding mutations and polymorphisms can help understand the biological aetiology of disease in cells rather than test tubes. • Studying protein expression in eukaryotic cells allows for proper folding and post-translational modifications, while RNA transfection allows for knock-down and knock-in experiments. • Transfection targets can include DNA, RNA, and protein, with transient and stable transfection options available. • Transient transfection does not integrate the DNA into the genome and produces a high level of expression for a few days, while stable transfection results in integration and long-term expression. Transfection Methods and Considerations for Protein Expression • High copy number of transfected genetic material leads to high protein expression, but cells are typically harvested within 24-96 hours of transfection, and it's generally unsuitable for studies using vectors with inducible promoters. • Stable transfection via DNA vectors results in permanent genetic alteration, but only single or low copy numbers yield lower protein expression. Selective screening and 2-3 weeks of selection are required for the isolation of stably transfected colonies. Suitable for studies with inducible promoters. • Homologous recombination is a rare event, which can be employed to direct error-free repair of double-strand DNA breaks. It enables knock-in and knock-out. Homologous recombination sequences of the targeted locus are surrounded by wild-type. • Transfection methods include cationic lipid, electroporation, in vivo, co-transfection, RNAi/siRNA, transient, stable, calcium phosphate, CRISPR, WHDNA, and mRNA transfection. The process involves attachment to cell, endocytosis into cell, escape from endosome, and nuclear entry. • Inserting the target sequence is done through PCR, cleanup, and mixing with plasmid and restriction enzymes. Applying selective pressure confirms that it's there. • Cloning vectors, including plasmids, cosmids, and phages, are small pieces of DNA employed to introduce foreign genes of interest into the host cell. They consist of origin of replication, unique restriction sites, reporter gene, and antibiotic resistance site. • Expression vectors are employed to express a foreign gene, consisting of a promoter to drive target gene expression, plasmid, and antibiotic resistance gene. Understanding Expression Vectors and Different Methods of DNA Transfection • An expression vector is a plasmid used to introduce a gene of interest into host organisms and analyze its protein product via expression. • It consists of regulatory elements like enhancers, promoters, termination sequences, transcription initiation site, and translation initiation sites. • A plasmid must possess these elements to be an expression vector. • Electroporation is a physical method of DNA transfection with high cell death and low efficiency but prevents DNA degradation. • Calcium Phosphate transfection is a buffered solution mixed with Plasmid DNA and adheres to the cell surface - a cheap method with high efficiency. • Liposomes or transfection complexes are used as a chemical method of DNA transfection. Various DNA Transfection Methods • In May 2023, DNA can be released through Cationic Liposomes AVA and Liposomes Micelles, which can work with various cell types and has no immune response. However, it may not be applicable to some cells and can degrade DNA. • Magnetic nanoparticle transfection is a quick and easy method that can have high transfection rates and minimal effect on cell health. However, it is relatively new and requires adherent cells. • The use of viral vectors, specifically retroviruses, adenoviruses, lentiviruses, and adeno-associated viruses, can infect and insert genetic material into both dividing and non-dividing cells. However, each type of virus has its own pitfalls, such as low immunogenicity or requiring co-infection with a helper virus. Gene Therapy and Transfection • Random insertion site highly immunogenic virus requires helper virus to grow; gene expression is high during this. Potential cytopathic effects are present as well. • RNA transfection has many advantages over DNA transfection. However, the mRNA carrying plasmid needs to be designed carefully. • Small inhibitory RNAs (siRNA) act like endogenous miRNAs, represses gene expression. Delivery could be lipid/polymer-mediated or virus-mediated. • miRNA and siRNA are used for RNA interference (RNAI), which is mediated by RdRP. Amplification of siRNA results in degradation of target mRNA. • Gene therapy treatment for Severe Combined Immunodeficiency (SCID) involves removing blood stem cells and transplanting the working copy of the ADA gene via viral transduction. However, it can lead to cancer (25%). • A common-place treatment involves injecting viruses carrying genes to halt cell death in Age-related Macular Degeneration patients. • There are ethical issues in gene therapy, such as deciding whether disabilities are diseases and whether searching for a cure demeans the lives of people who have disabilities. Gene Therapy and Human Genetic Revolution • A study published in Gene Therapy showed that miR-29a downregulates the peripheral myelin protein 22 gene in cellular models of Charcot-Marie-Tooth disease. • A news article on the NHS website reported that gene therapy has successfully halted the bubble boy immune disease. • An overview of the genetic revolution in human gene therapy was discussed in an article published on the Journal of the Association of Physicians of India website. • The article "What to do next?" poses questions for further research, including understanding the differences and techniques of transfection, transduction, and transformation methods in gene therapy. It also raises ethical considerations regarding gene alteration.