Spinal Muscular Atrophy SMN-/- Zebrafish Model

Spinal Muscular Atrophy - Disease Pathology

Spinal Muscular Atrophy (SMA) is a genetic condition where the ubiquitously made protein namely survival motor neuron (SMN) has undergone a mutation leading to lack of functionality or poor functionality.

SMN plays several important roles within the cell such as energy regulation, autophagy, translational regulation, ubiquitin pathways regulation, trafficking, and regulation of cytoskeleton polymerization. The complete or partial loss of the SMN protein therefore leads to death of specific neuronal cells called lower motor neurons. Loss of lower motor neurons affects the effector muscles closer to the trunk of the body around the spinal cord, leading to a low muscle tone and eventual muscle atrophy in that region.

The pathology is observed as curvature of the spine, limited mobility due to loss of motor functions. The lower motor neuron also controls certain voluntary functions such as feeding, swallowing, movement of tongue, breathing and movement of eye muscles and vocalization which also maybe affected. However, the SMA disease pathology outcome is based on the severity of mutation. Hence there can be a wide presentation of the pathology from the most severe form, complete loss of SMN to base pair deletions that are less severe. The SMN protein can also be made from an identical gene called SMN2. However, SMN from SMN2 is expressed to a smaller level.

Figure 1: Bright field image of SMN-/- zebrafish larvae showing curvature of the spine due to muscle weakness around the spinal cord

Zebrafish as a validated model for Spinal Muscular Atrophy

• Given that Zebrafish is an active animal, phenotype presentations such as lack of ability to move the muscles and other SMA pathology are highly evident which can be quantified through behavioural motor function tests

• Other aspects such as curvature of the spinal cord, can be visually observed in bright field microscope

• Quantification of voluntary muscle activity can be observed as dysphagia which is lack of ability to swallow and breathing shortness can be measured as resting ventilation rate

SMA Zebrafish Model Design for Therapeutic Strategies

SMN mutants are created by gene editing methods and genotype, phenotype screened. This screening selection is to ensure that the model zebrafish larvae present all pathological outcomes observed in human conditions of spinal muscular atrophy. Animals are curated by inbreeding or evaluation of markers adjacent to the sight of mutation

SMA Discovery Strategies using Zebrafish

• Gene therapy Strategies:
  

  • Gene therapy strategies have been continuously challenged by the inability of the gene therapy drugs to pass through the blood brain barrier, lack of long-term translation of the functional gene, toxicity, and challenge of administration of the drug intrathecally

  • Zebrafish blood brain barrier and human blood brain barrier have proven to be mechanistically conserved at the cellular and molecular level. This enables replication of the drug ability to pass through the blood brain barrier

  • Zebrafish is also highly sensitive to toxic build up and can detect nanomolar levels of toxic build up aiding selection of the safest gene therapy construct

  • Zebrafish is highly flexible to intrathecal and intracranial injections due to the thin and transparent nature of the bones. Hence the fish is a highly validated platform to not only replicate human condition of SMA but also evaluate the routes of gene therapy administration

• SMN2 splicing modifier:
  

  • The use of external agents that modify splicing mechanisms in zebrafish are highly proven. Splicing modifiers have been employed in several muscle diseases in zebrafish to modify proteins involved in muscle homeostasis

• Other Strategies:
  

  • Other strategies such as muscle homeostasis regulators, Oxidative stress inhibitors, Neuroprotection, Muscle mass enhancers can be either administered through I.P or oral feed formulations. Endpoints can be tailored specifically for these conditions and quantitatively measured over a time period

• Large Scale Screening for Repositioning - Pentagrit Zephys Platform:
  

  • Several existing drugs are being proven to have a degree of rescue effect of the SMN pathology. For instance, anti-inflammatory drugs have been proven to slow down muscle atrophy in SMA. Hence for repositioning studies, large scale Hight Though Put screens can be done using the "Pentagrit Zephys Platform" where several thousands of drugs can be efficacy screened in a 96 well plate format with results obtained within 48 hours of model exposure.

Spinal Muscular Atrophy - Endpoint Readouts