Project Summary
Alternating Hemiplegia of Childhood (AHC) is a devastating neurological disorder that is characterized by
bouts of paralysis and is often accompanied by developmental abnormalities. Mutations within the ATP1A3
gene, which encodes a neuronal isoform of the Na+/K+ ATPase, are the most common cause of AHC. AHC is
rare, and at present there is no cure. Two factors significantly contribute to the lack of progress in the
development of treatments. First, as with most rare genetic disorders, there are insufficient resources and
limited financial motivation in the private sector. Second, the mechanisms by which the mutations cause the
disease are not understood. AHC mutations are dominant recessive, and it is unclear how mutant Na+/K+
ATPases interfere with wild type versions to create physiological deficits that are higher than expected. For any
genetic disorder, the most direct treatment would be to correct the underlying mutation. In theory, this could be
accomplished by editing the gene or the messenger RNA that it encodes. For neural disorders, gene editing is
not practical because the most advanced systems using CRISPR technology don’t work well in neurons. In
addition, they are difficult to deliver in vivo because they are based on bacterial components which will likely
generate immunological complications. Recently, new systems for editing mRNAs, called site-directed RNA
editing (SDRE), offer distinct advantages for the treatment of genetic diseases. First, they can operate in
neurons, and they are based on enzymes that occur naturally in humans. Another advantage is that they are
relatively simple, being composed of a small oligonucleotide guide RNA coupled to a human RNA editing
enzyme. Because genetic information is encoded the same way between different RNAs in different cells, it
can be edited in much the same way wherever it is expressed. This make SDRE a semi-generic approach for
different genetic disorders. In this work, SDRE components will be optimized to efficiently and selectively
correct the most frequent mutation that underlies AHC (ATP1A3 D801N). Top guide RNAs will be identified
from pools of billions of randomized candidates through an iterative selection procedure. These will then be
tested in cells in combination with different versions of engineered RNA editing enzymes. These reagents will
then be packaged into virus particles so that they can be efficiently delivered to cells. Simultaneously, the
mechanisms by which the ATP1A3 D801N mutation alters Na+/K+ ATPase function will be studied, both in
enzymes that contain the mutation and in wild type enzymes. These experiments will provide a better
understanding of the physiological basis of AHC and help provide estimates of the proportion of mutants that
must be corrected to offset functional deficits. Taken together, the development of SDRE reagents coupled
with a clear understanding of the aberrant physiology caused by AHC mutations will allow us to begin to
develop the first therapeutics for this condition.
