A novel gene editing technique has been successfully implemented to preserve retinal cells and recover visual function in mice suffering from an inherited retinal disease. The findings spell hope for millions worldwide affected by these currently untreatable blinding conditions.
Blindness is one of the most feared disabilities. Unfortunately, for millions of people worldwide who have lost or are losing their eyesight due to degenerative disorders that affect their eyes' retinas - the part of the eye that contains the light sensitive cells (photoreceptors) - treatment options are virtually non-existent.
Now, a team of researchers from the Gavin Herbert Eye Institute at the University of California, Irvine, was able to recover some lost vision and preserve remaining photoreceptors in mice suffering from photoreceptor loss caused by inherited retinal degeneration (IRD) by correcting errors in the DNA sequence of a mutated gene in their eyes. The 'repaired' gene, RPE65, is integral to the maintenance of these cells. Errors in its sequence lead to a condition known as Leber Congenital Amaurosis (LCA) which is characterised by rapid photoreceptor degeneration and is the leading cause of inherited blindness in children. Their results, published in the journal Nature Communications, pave the way for such treatments to one day also help human patients suffering from these debilitating conditions.
"LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function, but also protects photoreceptors from further degeneration", says Dr. Choi, lead author of the study.
In humans, retinal degenerative diseases, such as retinitis pigmentosa or age-related macula degeneration (AMD), are one of the leading causes of blindness, with AMD affecting ~1 in 12 individuals aged 45 to 85, and inherited retinal diseases affecting ~1 in 2000 individuals globally. While AMD is largely influenced by age, lifestyle and environment, in the inherited forms of these conditions, mutations in genes involved in retinal metabolism cause cell death and thus a progressive loss of photoreceptors in the retina. Depending on the affected gene(s) and their role in the retinal metabolism machinery, the course of disease can range from years to decades. However, in the vast majority of cases, and due to the current total lack of treatment options, cell loss inevitably results in blindness.
Encouragingly, research into treatments for inherited diseases has gained renewed momentum over the past two decades, thanks to several technological breakthroughs: gene therapy (i.e., the delivery of intact gene copies to cells containing mutated, defective versions of that gene) and gene editing (i.e., the correction of errors in the DNA sequence of a defective gene).
Gene therapy has been employed successfully in treating various inherited diseases, including inherited retinal diseases. Most notably, a gene therapy providing intact RPE65 to treat LCA was approved by the U.S. Food and Drug Administration in 2018 and has since been commercialised as Luxturna. But it remains unclear how long-lasting the positive effects of gene therapy treatments truly are; the supplied genetic material remains exogenous (not part of the cell's genome) and therefore may be prone to degradation.
Treatments via gene editing, on the other hand, promise to be a permanent cure as the corrections are carried out on the genomic DNA itself. Research into gene editing as a therapeutic tool has skyrocketed since the mainstream adoption of the Nobel prize winning CRISPR-Cas9 gene editing system in 2012. Yet, while no doubt an incredibly powerful tool with seemingly limitless untapped potential, major caveats of current gene editing approaches have posed significant obstacles on its path towards human clinical trials. At present, these methods suffer from low editing efficiency (i.e., only a small fraction of all expressed genes are in fact corrected) and insufficient editing accuracy (i.e., 'off-target' edits in regions of the genome that were not intended). The latter poses a serious health risk as off-target edits, at worst, could lead to cancerous cell growth.
In their experiments, Choi et al. used optimized gene editing tools, known as base editors, to 'repair' defective RPE65 genes in the retinal cells of LCA-mice. Base editors allow the direct conversion of specific DNA base pairs (i.e., the letters in a DNA sequence) into other base pairs at precise locations within the genome. But, unlike the most widely used gene editing systems, such as CRISPR-Cas9, base editors can perform said conversions without breaking the DNA double strand. This increases precision and consequently reduces the risk of potentially harmful off-target edits. Interestingly, Choi et al. were able to boost editing efficiency and accuracy compared to earlier approaches even further by screening base editor variants and different associated single guide RNAs (short RNA sequences that recognize specific base motifs at the target site in the genome) in cultured cells prior to application in mice.
Once the best-performing base-editor and guide RNA had been identified, constructs were packaged into lentiviruses and injected into the subretinal space. Using viruses such as lentivirus as carriers for the gene editing 'payload' exploits the viruses' innate ability to pass through the cell membranes for delivery of the gene editing construct. Moreover, these viruses elicit no or only a mild immune reaction.
Treatment success was assessed by comparing the amount of expressed RPE65 that contained the corrected sequence with that of uncorrected versions of the gene. This was done via single-cell RNA-sequencing, a novel sequencing technology with single-cell resolution. The analysis revealed that nearly 40% of all expressed RPE65 were of the 'repaired' version, meaning the editing method used was more efficient than previous ones.
Once sufficient editing efficiency had been confirmed, the team proceeded to determine whether correction of the RPE65-sequence did indeed improve visual function and preserve photoreceptors. To do this they used a method called electroretinography (ERG), which allows researchers to determine the electrical output of retinal photoreceptors by measuring differences between the electrical potential of the eye upon light stimulation and a reference potential. Only intact and healthy photoreceptors produce a measurable ERG signal. In addition to this, photoreceptor structure and abundance were determined via microscopy.
As expected, they found that in untreated LCA-mice, photoreceptors produced no measurable ERG signals. In gene-edited LCA-mice, on the other hand, photoreceptors produced electrical signals up to 1/3 the strength of those seen in healthy mice at the same age. Furthermore, photoreceptors in treated mice largely retained their structural integrity and abundance when compared to untreated LCA-mice.
Although this approach was tailored to exclusively treat IRD due to RPE65-mediated LCA, the results are nevertheless very encouraging for the development of similar strategies to address IRDs caused by mutations in different retinal genes.
On the whole, these outcomes raise hope for the prospect of modern medicine to some day soon be able to offer help to patients who suffer from these debilitating conditions.
The original research was published on 5 April 2022 in the journal Nature Communications and can be found here.
Comments