Treating disease is often a matter of restoring the function of a specific protein, process, or pathway. But when a disease manifests in cellular chaos too widespread to correct or in drug targets that are lost altogether, researchers may resort to remedying its genetic origin. These gene therapies can deactivate aberrant genes with strategic breakages, replace mutation-ridden regions of genes, and even edit individual nucleotide bases.    

While gene editing technologies such as CRISPR-Cas9 go straight for the DNA source, the often overlooked intermediate in the DNA-to-protein pipeline is attracting attention as an alternate editing canvas. Robert Bell, a gene therapy scientist at Ascidian Therapeutics, is one of a group of researchers developing platforms to rewrite genes at the RNA level. Gregory Davis, a genome engineer at Sangamo Therapeutics, is among scientists advancing DNA editing systems. These two researchers lay out the advantages, disadvantages, and unique applications of each approach, revealing that together, they may broaden the scope of genes researchers can edit and diseases gene therapy can treat.

A ReNewAble target

While a powerful tool, gene editing can cause serious problems if things don’t go according to plan. For example, a 2020 study in Cell showed that a CRISPR-Cas9 system designed to cleave and rejoin DNA strands to correct a mutation created the break in the wrong location or failed to repair the break about half of the time, leading to the loss of entire chromosomes (1). “Once you've made those changes in the DNA, there's no going back,” Bell said. “There's inherent risk to all technology, but I think in particular, when you start to manipulate the genetic code in a way that you can't then change, it opens up other potential unknowns.” 

As researchers come to appreciate the risk of off-target activity and other unintended effects of gene editing, RNA provides an attractive, more forgiving target. Rather than being cemented in the cell’s blueprint forever, genetic changes introduced into short-lived RNA as it's continuously transcribed from DNA could be halted.        

The team at Ascidian Therapeutics developed an RNA editing platform inspired by its namesake, the ascidian, a sea squirt that re-engineers its RNA as it develops from a larva to an adult. The company’s technology targets pre-mRNA. The cell’s spliceosome complex modifies pre-mRNA to remove noncoding intron regions and join together coding exon regions to yield the final mRNA sequence. Mutations in exons can translate to dysfunctional proteins. 

Using high throughput synthetic and computational biology methods, the researchers design a chain of wildtype RNA exons attached to a synthetic RNA strand featuring domains that bind to a specific site in the pre-mRNA and engage with the spliceosome. When the pre-mRNA is spliced, the wildtype exons replace the mutated ones, producing mRNA that codes for a functional protein. 

To restore the working protein at the DNA level, researchers can replace the defective gene, but the stand in may not express the protein precisely as the cell intended (2). “It's very difficult to try to mimic exactly what all the cells in the body want in terms of the [right] level of protein and at the right time,” Bell said. Disrupting finely tuned endogenous gene expression patterns risks detrimental outcomes. For example, even modest overexpression of MECP2, a gene therapy target for neurodevelopmental disorders, yields neurotoxic effects in mice (3).  

Editing RNA ensures that the cell maintains its natural transcriptional regulation of protein production. “There's control of the transcriptome through a lot of complicated mechanisms that result in very different RNA expression in different cell types,” Bell said. “Ascidian Therapeutics’ RNA exon editing technology will only edit when the RNA is present in that cell, so the cell controls the fate of what editing occurs.”   

While the need for sustained activity presents a practical challenge for editing renewed RNA, Ascidian Therapeutics delivers its technology via a stable DNA construct that is transcribed in the nucleus to generate the RNA editor. This method could provide an indefinite source of the editor in nondividing cells, enabling the possibility of a one-time treatment. 

Ascidian Therapeutics’ lead program targets the ABCA4 gene, where mutations are responsible for progressive forms of blindness such as Stargardt disease. Researchers have identified more than 900 individual ABCA4 mutations across different patients, making single base editing approaches a poor fit. While full gene replacement would take care of any and all mutations, the ABCA4 gene is too large to fit inside the adeno-associated virus (AAV) vector, the gold standard gene delivery vehicle (2). By packaging only the necessary wildtype RNA exons rather than the entire gene, Ascidian Therapeutics developed an AAV-compatible editor that corrects about 60 percent of disease-linked ABCA4 mutations (4). The exon editor makes these changes in more than 25 percent of ABCA4 mRNA in cultured human cells, while other scientists have shown that 10 to 25 percent restoration of ABCA4 expression produces a therapeutic benefit in mice (4). The team also tested their technology in a primate model and observed efficient mRNA editing in retinal cells one month and three months after a single subretinal injection (5).

“We're just at the tip of the iceberg for what RNA exon editing can accomplish to address many of the underlying medical needs that exist in today's society,” Bell said. “I see a long-term vision of us re-engineering and rewriting RNA and the transcriptome for human health.”

Two and done

The fact that DNA constantly churns out RNA provides several upsides to editing at the DNA level, according to Davis. DNA editors need to act only on the two chromosomes in each cell. “That gives us the advantage of possibly lower dosing and more effective drug targeting. With RNA editing, you've got a lot more targets to take care of,” he said. 

Extending the lifespan of the RNA editor to reach all of those targets creates similar risks regarding lasting off-target effects, Davis said. “DNA editing would be more permanent and heritable, but if you want this RNA editor to be effective for the patient, it's going to be hanging around and being active. And if it has a wrong activity going along with that, you’re in the same situation,” he said. 

The need for longevity in RNA editing also heightens concerns about immunogenicity from nucleic acid-modifying enzymes. Ascidian Therapeutics' technology harnesses the existing splicing machinery in the cell and RNA single base editing platforms rely on endogenous adenosine deaminase acting on RNA. However, other RNA editing systems require foreign enzymes. “If you need to have a constant source, you need to have an enzyme that has no immunogenicty, essentially,” Davis said. However, a recent study in Nature Medicine suggests that an RNA-cleaving Cas13 nuclease could trigger an adaptive immune response in humans, raising questions about its safety when repeatedly administered (6).

For their DNA editing approach, researchers at Sangamo Therapeutics turn to zinc fingers, a class of zinc-stabilized human proteins that bind DNA. By iteratively screening and engineering a library of zinc fingers, the team identifies the protein with maximum affinity and selectivity for a specific DNA site. They can then attach the DNA-directing zinc finger to different enzymes for versatile editing applications. For example, the Sangamo Therapeutics team created a zinc finger cytidine deaminase that rewrites nucleotide bases to generate stop codons that knock out genes implicated in cancer and autoimmune disorders (7). They also developed zinc finger nucleases to shut off a gene involved in the HIV's infectious mechanism and replace a gene that encodes a faulty enzyme in the rare metabolic disease mucopolysaccharidosis type II (8,9).

Because introns are removed from RNA, pursuing DNA expands the range of potential editing targets, Davis said. For example, while some scientists are seeking strategies to directly correct the mutation that causes defective adult hemoglobin in sickle cell disease, the Sangamo Therapeutics team is exploring an indirect, intron-targeting approach. The researchers developed a zinc finger therapeutic that causes cells to make fetal hemoglobin again, providing an additional oxygen carrier that can relieve symptoms of sickle cell disease. Their technology acts on a DNA-regulating enhancer sequence located in an intron region in erythroid red blood cell precursors. The sequence promotes the expression of the BCL11A gene, which halts the production of fetal hemoglobin at birth. Sangamo Therapeutics’ zinc finger nuclease creates a break in this sequence, introducing erroneous insertions and deletions when it tries to repair itself. By deactivating the enhancer sequence in this way, the editor shuts off BCL11A expression in erythroids, thereby restoring the production of fetal hemoglobin. In a preliminary trial in patients with sickle cell disease, researchers collected, edited, and reinfused participants’ stem cells, resulting in increased fetal hemoglobin levels within weeks (10). 

“It would not be possible to do this at the RNA level because that intronic sequence would be spliced out,” Davis said. “So that's a good example of where going the DNA route gives you an option that you did not have at the RNA level.”

References

1. Zuccaro, M.V. et al. Allele-specific chromosome removal after Cas9 cleavage in human embryos. Cell  183, 1650-1664 (2020). 
2. Wang, D., Tai, P.W.L., & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov  18, 358-378 (2019).
3. Collins, A.L. et al. Mild overexpression of MeCP2 causes a progressive neurological disorder in mice. Hum Mol Genet  13, 2679-2689 (2004). 
4. Gray, J.M. et al. ABCA4 pre-mRNA exon editing in vitro and in vivo. At <https://www.ascidian-tx.com/publications-and-presentations.html>.
5. Krumbach, R. et al. Evaluation of ABCA4 RNA exon editing and replacement in non-human primate. At <https://www.ascidian-tx.com/publications-and-presentations.html>.
6. Tang, X.Z.E., Tan, S.X., Hoon, S., & Yeo, G.W. Pre-existing adaptive immunity to the RNA-editing enzyme Cas13d in humans. Nat Med  28, 1372-1376 (2022). 
7. Fauser, F. et al. A compact zinc finger architecture for highly efficient base editing in human cells. At <https://www.sangamo.com/wp-content/uploads/2022/05/560-ASGCT-Base-Editing-FREIDER-FAUSER-.pdf>.
8. Tebas, P. et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med  370, 901-910 (2014). 
9. Conway, A. et al. Non-viral delivery of zinc finger nuclease mRNA enables highly efficient in vivo genome editing of multiple therapeutic gene targets. Mol Ther  27, 866-877 (2019).
10. Alavi, A. et al. Preliminary safety and efficacy results from PRECIZN-1: An ongoing phase 1/2 study on zinc finger nuclease-modified autologous CD34+ HSPCs for sickle cell disease (SCD). At <https://www.sangamo.com/wp-content/uploads/2021/12/2930_PRECIZN-1_Preliminary-Safety-and-Efficacy-Results-FINAL.pdf>.