Novel gene based therapy for nemaline myopathy

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PROJECT SUMMARY/ABSTRACT Nemaline myopathy (NM) is a childhood-onset skeletal muscle disease that is characterized by severe disabilities, including (in many cases) wheelchair and feeding tube dependence. Mutations in more than a dozen genes can cause NM. Most of these genes encode components of the thin filament (a principle part of the sarcomere), and mutations associated in these genes alter the structure and/or function of the thin filament, resulting in impaired muscle contraction and generalized weakness. There are currently no treatments for NM. The overarching goal of this proposal is to develop therapies for this devastating disease. Recessive mutations in NEB are the most common cause of NM. NEB encodes the giant protein Nebulin, which functions to regulate the length of the thin filament. Many NEB mutations cause single exon skipping or single exon deletion. Such mutations do not alter the RNA reading frame of NEB; however, despite only removing a very small part of an otherwise giant protein, they unexpectedly result in significant reduction (or even complete loss) of the whole Nebulin protein. The reason for this is not known. We hypothesize that the reason for this surprising observation is that these mutations remove an incomplete portion of repeat elements within NEB, the consequence of which is to make the Nebulin protein out of register, thereby preventing it from incorporating into the thin filament. We will test this hypothesis in Aim 1. Aim 1: we will use cutting edge imaging and biochemical strategies to study the Nebulin protein in a series of in-frame nebulin mutants. To accomplish this, we will employ the zebrafish model system, which is ideal for this purpose because we can visualize the Nebulin protein in intact skeletal muscle in a living organism. Nebulin protein levels in patients with NEB mutations are correlated with disease severity (less protein = more severe disease). This fact means that a therapeutic strategy targeted at increasing Nebulin protein expression should be very effective. Based on our hypothesis above, we predict that we can accomplish this by removing more of the Nebulin RNA to take out complete repeats, the result of which should be to enable a shortened Nebulin to re-integrate into the thin filament and be stably maintained. We will test this idea, which we call “domain skipping”, in Aims 2 and 3. Aim 2: Using either morpholino mediated multi exon skipping or CRISPR/Cas9 genomic deletion, we will establish the feasibility and efficacy of “domain skipping” in zebrafish. Zebrafish allow us to rapidly and comprehensively examine this strategy across the entire nebulin gene. Aim 3: We will translate our findings from zebrafish to the mouse model and to patient cells, focusing specifically on two common Neb mutations (exon 55 deletion and a nonsense mutation in exon 61). The use of the mouse model will enable us to test efficacy in a mammalian system, and testing in human cells will provide vital proof of concept and reagent development necessary for clinical translation.