Researchers at the National Eye Institute (NEI), part of the National Institutes of Health, have developed a gene therapy that rescues cilia defects in retinal cells affected by a type of Leber congenital amaurosis (LCA), a disease that causes blindness in early childhood. Using patient-derived retina organoids—retinas-in-a-dish—the researchers discovered that a type of LCA caused by mutations in the NPHP5 (also called IQCB1) gene leads to severe defects in the primary cilium, a structure found in nearly all cells of the body. Their in vitro findings shed light on the function of NPHP5 protein in the primary cilium, but also led to a potential treatment for this blinding condition. The team developed an adeno-associated virus (AAV)-mediated IQCB1/NPHP5 gene augmentation therapy that ameliorated disease phenotype in NPHP5-LC retinal organoids.

“It’s so sad to see little kids going blind from early onset LCA,” said study lead investigator, Anand Swaroop, PhD, senior investigator at the NEI Neurobiology Neurodegeneration and Repair Laboratory. “NPHP5 deficiency causes early blindness in its milder form, and in more severe forms, many patients also exhibit kidney disease along with retinal degeneration. We’ve designed a gene therapy approach that could help prevent blindness in children with this disease and one that, with additional research, could perhaps even help treat other effects of the disease.”

Swaroop and colleagues reported on their development in Stem Cell Reports, in a paper titled, “In vitro modeling and rescue of ciliopathy associated with IQCB1/NPHP5 mutations using patient-derived cells.”

LCA is a rare genetic disease that leads to degeneration of the light-sensing retina at the back of the eye. Defects in at least 25 different genes can cause LCA. While there is an available gene therapy treatment for one form of LCA, all other forms of the disease have no treatment.

The type of LCA caused by mutations in NPHP5 is relatively rare. It causes blindness in all cases, and in many, it can also lead to failure of the kidneys, a condition called senior-Løken syndrome (SLSN). “Mutations in the gene encoding IQ calmodulin-binding motif containing B1 (IQCB1/NPHP5) are the most common cause of renal-retinal SLSN, and are also detected in patients with non-syndromic LCA, the authors commented. “Notably, patients with SLSN exhibit variable onset of kidney dysfunction, whereas visual function defects are observed in early childhood and have a significant impact on patients’ quality of life, making photoreceptors an important target for therapy development.”

For their study, the Swaroop team, including Kamil Kruczek, PhD, Zepeng Qu, PhD, and Emily Welby, PhD, collected stem cell samples from two patients with NPHP5 deficiency, at the NIH Clinical Center. These stem cell samples were used to generate retinal organoids, cultured tissue clusters that possess many of the structural and functional features of native retina. “For in vitro disease modeling, we obtained dermal fibroblasts from patients with NPHP5-LCA that were reprogrammed into induced pluripotent stem cells (iPSCs) and differentiated into retinal pigment epithelium (RPE) and retinal organoids,” the team explained.

Patient-derived retinal organoids are a particularly valuable model because they closely mimic the genotype and retinal disease presentation exhibited by patients, and also provide a “human-like” tissue environment for testing therapeutic interventions, including gene therapies. As in the patients themselves, the retinal organoids developed by the researchers showed defects in the photoreceptors, including loss of the portion of the photoreceptor called outer segments. The photoreceptor outer segment is a special type of primary cilium, an ancient structure found in nearly all animal cells.

In a healthy retina, photoreceptor outer segments contain light-sensing opsins. When the outer segment is exposed to light, the photoreceptor initiates a nerve signal that travels to the brain and mediates vision.

In a normal healthy eye, NPHP5 protein is believed to sit at a gate-like structure at the base of the primary cilium that helps filter proteins that enter the cilium. Previous studies in mice have shown that NPHP5 is involved in the cilium, but researchers don’t yet know the exact role of NPHP5 in the photoreceptor cilium, nor is it understood exactly how mutations affect the protein’s function.

Treating patient-derived retinal organoids with AAV-NPHP5 restores rhodopsin localization in photoreceptor outer segments. Top: Retinoids stained for DNA (blue), NPHP5 (red), and rhodopsin (green). Below: Close-up view of organoid photoreceptor layer stained green for rhodopsin. Adapted from Kruczek et al., 2022. [Anand Swaroop, PhD, and Kamil Kruczek, PhD, National Eye Institute]
In the present study, researchers found reduced levels of NPHP5 protein within the patient-derived retinal organoid cells, as well as reduced levels of another protein called CEP-290, which interacts with NPHP5 and forms the primary cilium gate. Mutations in the gene for CEP-290 constitute the most common cause of LCA.

In addition, photoreceptor outer segments in the retinal organoids were completely missing and the opsin protein that should have been localized to the outer segments was instead found elsewhere in the photoreceptor cell body. “Reduced CEP290 protein, as well as abnormal cilia morphology, provided useful mechanistic insights into NPHP5-LCA pathogenesis,” the team pointed out.

When the researchers introduced an AAV vector containing a functional version of NPHP5 as a gene therapy vehicle, the retinal organoids showed a significant restoration of opsin protein concentrated in the proper location in outer segments. The findings also suggested that functional NPHP5 may have stabilized the primary cilium gate. “The patient-specific retinal organoids, established in this study, should help in further dissection of NPHP5 functions in photoreceptor cilia,” the investigators noted.

Gene therapy using AAV vectors has emerged as an effective treatment approach for many inherited retinopathies, the authors commented, and interestingly, prior studies have demonstrated improved visual function in Nphp5 gene knockout mice, and in a spontaneous dog model carrying NPHP5 mutations, using an AAV vector to deliver the complete IQCB1/NPHP5 coding region. “The animal models provided essential in vivo proof of concept, whereas our work complements these studies with a human-specific system and evaluated the rescue of photoreceptor phenotype in patient-derived tissue in vitro,” the team concluded. “We could also rescue photoreceptor OS defects in retinal organoids by AAV-mediated gene therapy, highlighting the utility of this model system in developing future treatments for ciliopathies … Our studies thus establish a human disease model and a path for treatment of NPHP5-LCA.”