Non-viral gene therapy is the delivery of genetic material to treat genetic disease through the use of methods that do not use viruses to introduce genetic material into the body, tissues or cells. Gene therapy includes the replacement of function of an abnormal or non-functional gene with a functioning variant, the supplementation of an additional capability such as morphogen-induced tissue regeneration and also gene editing therapies such those based on CRISPR.
Gene replacement therapy is delivered as a therapeutic gene expression cassette most commonly by a viral vector. Along with the therapeutic gene, the gene expression cassette typically has a promoter sequence to drive gene expression and a termination signal to end gene transcription. As a delivery system, viruses have been favored for effective uptake and chromosomal integration for long lasting transgene expression. However undesirable viral effects such as adverse immune response and insertional mutagenesis can cause unwanted clinical outcomes. Non-viral vectors for gene therapy include nanoparticles, liposomes and cell-penetrating peptides. Other non-viral approaches include naked DNA or carrier-free delivery as circular plasmids or other forms of DNA. Transposon systems such as the sleeping beauty transposon system allow for integration of the therapeutic gene into the genome.
Carrier-free nucleic acid therapeutics are suitable for external or easily accessible tissues such as ocular, epidermal, pancreatic, pulmonary and colonic tissues. As of 2019 there were numerous examples of carrier-free nucleic acid therapies that are in clinical trials, however none of these involve the introduction of a replacement gene. Rather these are other types of nucleic acid therapies such as antisense oligonucleotide (ASO), siRNA and PEG-DNA aptamer therapies. ASOs and siRNAs are short synthetic oligonucleotides that interfere with translation of messenger RNA into protein. Intramuscular injection of naked DNA plasmid has been carried out in clinical trials. Clinical trials have used introduction of naked PCR product.
Naked DNA can be introduced into cells by electroporation and by using a “gene gun” that used high pressure gas to shoot DNA coated gold particles into the cell. To improve delivery of DNA into the cell, it can be protected from damage by being complexed with lipids in an organized structure like a micelle or liposome, forming a lipoplex. Cationic lipids are positively charged and naturally complex with negatively charged DNA. They also interact with the cell membrane so that endocytosis of the lipoplex results in the introduced DNA being released into the cytoplasm.
The cargo for CRISPR/Cas9 gene editing takes the following three approaches. 1) a DNA plasmid that encodes both the Cas9 protein and the guide RNA, 2) mRNA for Cas9 translation alongside a separate guide RNA or 3) Cas9 protein with guide RNA (ribonucleoprotein complex). Delivery of CRISPR gene editing cargo may be physical, viral or non-viral. Physical methods include microinjection and electroporation. Non-viral vector systems include systems such as lipid nanoparticles, cell-penetrating peptides (CPPs), DNA ‘nanoclews’, and gold nanoparticles. Editas Medicine is developing a CRISPR gene editing therapy that uses ribonucleoprotein (RNP) delivery of CRISPR cargo to blood cells ex vivo to treat non-malignant hematologic diseases.
