Within the genome of the white-throated sparrow and the zebra finch lies the foundation of an innovative new approach to gene therapy published in Nature Biotechnology. This technique, dubbed Precise RNA-mediated INsertion of Transgenes (PRINT), utilizes a retrotransposon derived from these avian sources to seamlessly integrate genes into the human genome, offering a potential solution for a myriad of hereditary diseases. Developed in the laboratory of Professor Kathleen Collins at the University of California, Berkeley, PRINT represents an exciting new frontier in the treatment of genetic diseases. 

While the approval of CRISPR-Cas9 therapy for sickle cell disease marks a remarkable milestone in gene therapy, the ability to insert entire genes into the human genome remains out of reach. PRINT, however, offers hope in the endeavor to replace defective genes. Retrotransposons, often called “selfish” DNA, code for enzymes that copy RNA back into genomic DNA, consequently cluttering the genome with retrotransposon DNA. PRINT exploits this feature to randomly insert transgenes to substitute for defective native copies. Unlike traditional methods that risk random gene insertions, such as human virus vectors, these genes are inserted into genetic “safe harbors,” sites where the insertion won't disrupt essential genes or lead to cancer.

PRINT utilizes a common retroelement protein called R2 protein, which has multiple active parts, including a nickase—an enzyme that binds and nicks double-stranded DNA—and reverse transcriptase, the enzyme that generates the DNA copy of RNA. This is delivered into the cell as RNA, alongside another RNA comprising the template for the transgene DNA to be inserted, plus gene expression control elements—an entire autonomous transgene cassette that R2 protein inserts into the genome.

"A CRISPR-Cas9-based approach can fix a mutant nucleotide or insert a little patch of DNA— -sequence fixing. Or you can just knock out a gene function by site-specific mutagenesis," said Collins. "We're not knocking out a gene function. We're not fixing an endogenous gene mutation. We're taking a complementary approach, which is to put into the genome an autonomously expressed gene that makes an active protein—to add back a functional gene as a deficit bypass. It's transgene supplementation instead of mutation reversal. To fix loss-of-function diseases that arise from a panoply of individual mutations of the same gene, this is great."

Many genetic disorders, like cystic fibrosis and hemophilia, are the result of a multitude of mutations within a single gene. Conventional CRISPR-Cas9-based therapies necessitate bespoke modifications tailored to each patient. However, gene supplementation via PRINT could bypass the need for such intricacy, instead delivering the correct gene in its entirety to each patient, enabling their bodies to synthesize the requisite proteins regardless of the original mutation.

The efficacy of PRINT was demonstrated through meticulous laboratory experiments, where researchers successfully inserted functional genes into human cells. Notably, using the R2 protein ensures robust and efficient gene insertion without compromising cellular integrity. The R2 protein inserts the transgene into an area of the genome containing hundreds of identical copies of ribosomal RNA—the rDNA. The amount of redundant copies means that when the insertion disrupts one or a few ribosomal RNA genes, the loss of the genes is inconsequential.

Beyond providing a secure genetic harbor, the rDNA offers a conducive environment for gene insertion, nestled within the nucleolus. Characterized by stringent regulation and rapid DNA repair mechanisms, insertion to this locus mitigates oncogenic risks associated with gene insertion.

"The nucleolus is a giant ribosome biogenesis center," Collins explained. "But it's also a really privileged DNA repair environment with low oncogenic risk from gene insertion. It's brilliant that these successful retroelements—I'm anthropomorphizing them—have gone into the ribosomal DNA. It's multicopy, conserved, and a safe harbor in the sense that you can disrupt one of these copies, and the cell doesn't care."

While the potential of PRINT is clear, uncertainties persist regarding the intricate dynamics of R2 and rDNA transcription. It is still unknown the extent of disruption that rDNA is resilient against, and cell-specific dynamics may make certain cell types more vulnerable to these insertions. Collins and her team remain undaunted, navigating these uncharted waters with a blend of inquiry and innovation, aiming to optimize PRINT's efficacy in diverse cellular contexts.

Despite the lingering uncertainties, "it works," asserts Collins. "It's just that we have to understand a little bit more about the biology of our rDNA in order to really take advantage of it." 

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