Junk DNA in Birds Inspires New Technique in Inserting Codes Into Genomes, Unlocks Safe and Efficient Gene Therapy

The approval of CRISpR-Cas9 therapy for sickle cell disease demonstrates the potential of gene editing tools in curing hereditary diseases. However, inserting entire genes into human genomes is still impossible, and defective or deleterious genes cannot be substituted.

This challenge persists even for other genetic diseases, where merely blocking or editing a gene using CRISPR may not be sufficient in addressing the consequences of genetic mutation. Experts believe a corrective gene must be added to the DNA to resolve the problem completely. This is where a novel method, Precise RNA-mediated Insertion of Transgenes (PRINT), comes in.

What is PRINT Technique?

PRINT refers to inserting new DNA into a cell using delivery methods similar to those used to deliver CRISPR-Cas9 into cells for genome editing. It leverages the ability of some retrotransposons to insert entire genes into the genomes efficiently without affecting other genome functions. It can complement CRISPR technology's ability to deactivate genes, make point mutations, and insert short segments of DNA.

Retrotransposons, also called retroelements, are pieces of DNA that when transcribed to RNA, code for enzymes that copy RNA back into DNA in the genome. This is a self-serving cycle that clutters the genome with retrotransposon DNA. About 40% of the human genome comprises this "selfish" new DNA, although most genes are disabled, also known as junk DNA.

For PRINT, a single piece of delivered RNA encodes a common retroelement protein called R2 protein. It has multiple active parts, like nickase and reverse transcriptase. The other RNA is the template for the transgene DNA to be inserted into gene expression control elements.

Utilizing Birds' Junk DNA

Many hereditary diseases are due to several mutations in the same gene, all of which disable the function of the genes. Any CRISPR-based gene editing therapy must be customized to an individual's specific mutation. In using PRINT for gene supplementation, the correct gene can be delivered to every person with the disease, enabling the body to produce the normal protein, regardless of the original mutation.

Many academic laboratories and startups invest in transposons and retrotransposons in inserting genes for gene therapy. One famous retrotransposon is Long Interspersed Element-1 (LINE-1), which has duplicated itself in humans and some hitchhiker genes to cover 30% of the genome.

At the University of California Berkeley, Professor Kathleen Collins and her colleagues developed the PRINT technique, which paved the way for safer gene therapy. The details of their study are discussed in the paper "Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci."

Collins noted that the LINE-1 retrotransposon protein would be difficult to engineer for safe and efficient insertion into the human genome. However, previous research suggests that inserting genes into the genome's repetitive, ribosomal RNA encoding region might work better.

Since R2 is not found in humans, the research team screened retroelements from different animal genomes. They tested insects, horseshoe crabs, and other multicellular eukaryotes to find an efficient way to insert long DNA lengths into rDNA regions.

Collins and her team discovered that the ideal candidates for this purpose are birds like zebra finches and white-throated sparrows. Experiments confirmed that R2 derived from birds worked well when they tried to create a message and delivered it to human cells. Many cells successfully incorporated the new gene into their DNA, and everything functioned normally.