# MK4 Repositioning for IAHSP: Overcoming In Vivo Data Gaps through In Silico Refinement and In Vitro Validation

## TL;DR

This study explores using MK4 to treat a rare genetic disorder by combining computer simulations and lab experiments to overcome limited in vivo data.

## Contribution

The novel approach integrates computational modeling with in vitro validation using patient-derived cells to advance drug discovery for rare diseases.

## Key findings

- MK4 treatment rescued mitochondrial morphology and ALSIN levels in patient-derived fibroblasts.
- Mean Branch Diameter of mitochondria was identified as a specific marker for IAHSP.
- Computational and cellular methods were successfully combined to evaluate drug efficacy in the absence of in vivo models.

## Abstract

Infantile-onset Ascending Hereditary Spastic Paralysis
(IAHSP)
is an ultrarare, autosomal recessive form of Hereditary Spastic Paraplegia
(HSP), caused by mutations in the ALS2 gene, which
encodes the protein ALSIN. In a previous study, we proposed a personalized
therapeutic strategy for an Italian IAHSP patient (AO), aiming to
correct the aberrant function of the R1611W mutant ALSIN using Menatetrenone
(MK4). While our results supported compassionate-use approval for
a patient-specific therapeutic regimen, further investigation was
needed to highlight the treatment’s benefits in the absence
of tractable biophysical assays and in vivo models.
In this respect, we first characterized MK4’s interaction with
the mutation site through Molecular Dynamics simulations. Next, we
established and characterized a skin fibroblast cell line derived
from patient AO. We analyzed the expression and stability of the mutant
ALSIN protein in AO’s fibroblasts and observed elevated oxidative
stress levels. Using advanced microscopy and automated image analysis,
we identified a characteristic mitochondrial phenotype associated
with AO’s IAHSP. One specific morphological parameter of mitochondria
(Mean Branch Diameter) accurately reflected the IAHSP phenotype and
was selected as a cell marker. Treatment of IAHSP fibroblasts with
MK4 highlighted the rescue of Mean Branch Diameter and ALSIN levels,
supporting cellular efficacy. Overall, this work presents an approach
that integrates computational and cell-based methodologies to overcome
the data scarcity challenges of drug discovery in rare diseases. Our
study provides a framework for preclinical, alternative drug discovery
programs in monogenic rare disorders such as IAHSP.