Treating a juvenile Batten disease mouse model with antisense oligonucleotides to correct its underlying disease-causing mutation led to less waste buildup in the brain, improved motor skills, and longer survival, a study reports.
These results are a key step toward finding treatments to address the genetic cause of the condition.
The study, “Therapeutic efficacy of antisense oligonucleotides in mouse models of CLN3 Batten disease,” was published in the journal Nature Medicine.
Juvenile Batten disease is an inherited neurodegenerative disorder that usually develops between the ages of 5 and 10. It affects the function of structures within cells called lysosomes, which are responsible for breaking down and recycling damaged or unused proteins in a process known as autophagy.
The condition is also referred to as CLN3 disease because it is caused by mutations in both copies of the CLN3 gene (one from each parent), which carries the instructions for a protein known as battenin. The most common mutation leads to a shortened, inactive form of the battenin protein called CLN3Δex7/8.
Notably, the missing portion of the battenin protein is the lysosomal targeting sequence, which normally directs the protein to the lysosomes for proper functioning. However, rare CLN3 mutations that leave the lysosomal targeting sequence intact also lead to less severe disease.
These observations prompted researchers at Rosalind Franklin University (RFU) in Illinois, along with scientists at the University of South Dakota and Ionis Pharmaceuticals, to create a version of battenin called CLN3Δex5/7/8 that restored the lysosomal targeting sequence, which may provide therapeutic value to those with Batten disease.
Researchers first showed that the production of the disease-associated form of the battenin protein (CLN3Δex7/8) in cells led to an increase in the number of autophagic vesicles (that fuse with lysosomes) and in the size of lysosomes, whereas normal battenin and the new form of battenin (CLN3Δex5/7/8) did not.
Based on these findings, antisense oligonucleotides, or ASOs, were designed to alter the production of battenin to generate the new form of CLN3Δex5/7/8 for further testing in cells and a mouse model.
ASOs are short stretches of nucleotides — the building blocks of DNA and RNA — that bind to messenger RNA (mRNA), the molecule that carries the protein instructions from DNA. Binding of ASOs to mRNA can alter protein production in a directed way.
Two ASOs were identified — ASO-20 and ASO-28 — which increased the production of the CLN3Δex5/7/8 mRNA in a dose-dependent manner and were more stable than the disease-causing CLN3Δex7/8 mRNA, which is rapidly degraded.
Furthermore, treating cells isolated from patients with the CLN3Δex7/8 mutation with ASO-28 partially restored the autophagic process.
A series of ASOs were designed to bind to mouse mRNA and test their efficacy in a live model. One ASO in particular, ASO-26, was the most active, also in a dose-dependent manner.
In mice with the same Cln3Δex7/8 mutation in one (heterozygous) or both (homozygous) of their Cln genes, ASO-26 was delivered directly into the central nervous system (CNS; consisting of the brain and spinal cord) by injection one or two days after birth. An ASO without a target was injected as a control.
After three weeks, an examination of tissue found the ASO was distributed throughout the brain and was stable for up to 14 months, demonstrating “long-term maintenance of ASOs in the CNS,” the researchers wrote.
Patients with CLN3 disease accumulate a protein called SCMAS in their brains. While homozygous mutant mice showed increased levels of SCMAS, ASO treatment lowered SCMAS accumulation in various parts of the brain, including the cortex, thalamus, striatum, and to a lesser extent, the hippocampus.
Motor tests revealed that heterozygous mutant mice performed significantly better than homozygous mutant mice. Treatment of ASO-26 in heterozygous mutant mice resulted in a significant improvement in performance compared with controls. Moreover, the treatment of homozygous mutant mice improved their performance to heterozygous mice levels, which indicated a “complete rescue of ability in these tasks,” according to the researchers.
Finally, as Cln mutations do not lead to early mortality in mice, to test for survival, homozygous mutant mice were crossed with mice carrying a mutant gene associated with familial Alzheimer’s disease, which resulted in a 50% survival rate of about 18 days.
Treating these mice with ASO-26 significantly improved survival, which was similar to the survival levels observed in heterozygous Batten mice.
“When rodents were treated with the therapeutic CLN3 corrector, their lifespan was extended, motor skills improved and waste buildup in the brain was lessened,” said Michelle Hastings, PhD, professor at RFU, in a press release. “It was a surprising demonstration that partial correction of the defective CLN3 gene can lead to meaningful improvements in an animal model with this disease.”
Future work will focus on finding a clinical candidate, which involves careful screening for activity and toxicity in animal models, the scientists noted.
“Our work tested a novel approach to therapeutically target the expression of the most common cause of the disease using ASOs — directed to the mutated form of the gene,” said lead author Jessica Centa, a graduate student at RFU. “These results are a critical step toward our long-term goal of developing a treatment for CLN3 Batten disease.”
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