Zinc-finger Protein Therapy Shows Potential in Angelman Mouse Model
Potentially safer way of activating silenced paternal UBE3A gene to treat disease
A novel treatment approach based on zinc-finger DNA-binding proteins, designed to activate the silenced paternal UBE3A gene, successfully eased the signs and symptoms of Angelman syndrome in a mouse model.
The strategy also demonstrated efficacy in brain cells derived from an Angelman patient, showing that it can be translated to human cells, and it may be safer than other approaches being examined, the researchers noted.
The study, “Transcriptional reprogramming restores UBE3A brain-wide and rescues behavioral phenotypes in an Angelman Syndrome mouse model,” was published in the journal Molecular Therapy.
Treatment goal is production of the functional UBE3A enzyme
The UBE3A gene carries instructions for ubiquitin protein ligase E3A, an enzyme that targets other proteins to be degraded and recycled, primarily within cells of the central nervous system (the brain and spinal cord).
Typically, one copy of the UBE3A gene, inherited from the mother, is active, while the other copy, inherited from the father, is selectively silenced by a molecule called UBE3A antisense transcript (UBE3A-ATS).
Deletion or malfunction in the maternal copy of the UBE3A gene causes Angelman syndrome.
One potential treatment approach is to activate the paternal UBE3A gene copy by blocking the action of UBE3A-ATS, allowing the production of functional UBE3A enzyme.
Antisense oligonucleotides (ASOs), short strings of genetic material (DNA or RNA) designed to suppress UBE3A-ATS production, are being tested in ongoing clinical trials as a treatment for Angelman.
Zinc-finger protein therapeutics — called ZFPs — is another approach to blocking UBE3A-ATS. These proteins are involved in several biological processes, including the regulation of gene activity, and can be designed to target a gene of choice selectively. As such, they might be a safer approach, the researchers noted, adding that ZFPs have undergone clinical assessment for other indications.
A team led by researchers at the University of California, Davis, designed ZFPs to suppress the production of UBE3A-ATS, with the goal of activating paternal UBE3A as a potential Angelman treatment.
First, the team tested their human ZFP candidate, called hATF-555, in patient-derived nerve cells with a maternal UBE3A gene mutation. In these cells, which only carried a functional paternal gene, there was an 11-fold increase in UBE3A-ATS accompanied by a 1.3 times decrease in UBE3A enzyme activity.
Delivered by a modified and harmless lentivirus, hATF-555 successfully reduced UBE3A-ATS by 1.25 times in nerve cells while partly activating paternal UBE3A activity by 1.33 times.
No off-target treatment effects or inflammatory immune activity seen in mice
To evaluate this approach further, the researchers treated an adult mouse model of Angelman syndrome with a ZFP that blocked mouse Ube3a-ATS, called ATF-S1K. Here, the treatment was injected into the tail vein, with targeted delivery to the mouse brain using a modified and harmless AAV virus.
Five weeks after treatment, Ube3a-ATS gene activity levels were 2.2 times lower in brain tissue, and the level of UBE3A enzyme increased to 26% of unaffected control animals in a dose-dependent manner. The average number of UBE3A-positive cells in the brains of treated Angelman mice was similar to controls.
Notably, there was no further increase in UBE3A-positive cells when control mice were treated with ATF-S1K. Researchers also did not detect any off-target effects on overall gene activity or inflammatory immune activation, “suggesting that the ATF-S1K treatment is not only specific but also well tolerated in vivo [in living animals],” they wrote.
Mice were then subjected to an open field test to assess motor abilities and willingness to explore. Without treatment, Angelman mice are less active than control mice. With ATF-S1K, Angelman mice showed motor improvements in horizontal directions, vertical rearing, and exploration.
On a rotarod test for motor coordination as well as balance, the Angelman mice performed significantly worse than controls. Treatment, on average, led to slight improvements.
Angelman mice also had longer walking strides at a lower stride frequency in the hindlimb and forelimb than controls, similar to deficits observed in patients, the researchers noted. AAV-S1K treatment improved motor abilities in the Angelman group, most notably for hindlimb function. No improvements were seen in other gait measures, including stride length or swing duration.
“We demonstrate that a single injection of an AAV delivering ATF-S1K (AAV-S1K) into the tail vein rescues molecular and behavioral phenotypes in young adult [Angelman syndrome] mice,” the team wrote. “The specificity and tolerability of a single AAV-S1K treatment in mice support the use of [ZFPs] for the treatment of [Angelman syndrome] and other genetic disorders.”
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