Autophagy Process

Gene Therapy Strategies

• Gene augmentation: adding a new normal protein-coding gene to cells with non-functional gene
  • Can be classified into two categories: gene replacement therapy and gene addition therapy
• Gene silencing: the use of DNA or gene editing technologies to enhance the function of a healthy person
  • Examples: increased muscle mass, improved oxygen delivery, and accelerated recovery

Gene Doping

• The use of DNA or gene editing technologies to enhance the function of a healthy person
  • Banned by the World Anti-Doping Agency (WADA) for non-therapeutic forms of genetic manipulation
• Examples: introducing genes that promote muscle growth, genes that encode for erythropoietin (EPO), and genes that promote tissue repair and regeneration

Gene Augmentation

• Gene replacement therapy: the delivery of a correct copy of the gene to restore the production of a defective or missing protein
  • Example: Spinal Muscular Atrophy (SMA) gene replacement therapy
• Gene addition therapy: the delivery of a protein-coding gene to improve cellular function and homeostasis
  • Example: autophagy gene addition therapy for neurodegenerative diseases

Gene Replacement Therapy

• Spinal Muscular Atrophy (SMA) is a group of genetic disorders characterized by the degeneration of anterior horn cells and subsequent muscle atrophy and weakness
  • Caused by mutations in the SMN1 gene, which produces a functional protein essential for motor neuron development
• SMA patients have a non-functional SMN1 protein, and the only functional protein is produced by the SMN2 gene
  • The number of copies of the SMN2 gene modifies the severity of the condition
• Example: Zolgensma, a gene replacement therapy for SMA, delivers a functional copy of the SMN1 gene to motor neuron cells
  • FDA approved therapy with a marketing price of $2.125 million

Gene Addition Therapy

• Autophagy is a highly regulated cellular process that facilitates nutrient recycling and degrades damaged organelles and proteins
  • Important in neurodegenerative diseases, where abnormal protein accumulation occurs
• Mechanisms of autophagy:
  • Microautophagy: direct engulfment of cytosolic material through lysosomal invagination
  • Chaperone-mediated autophagy: selective degradation pathway targeting proteins with recognition motifs
  • Macroautophagy: formation of phagophore, autophagosome, and autolysosome to degrade cellular material
• Example: Machado-Joseph disease (MJD) or Spinocerebellar Ataxia Type 3 (SCA3), a polyglutamine disease caused by an unstable expansion of a CAG repeat sequence in the ATXN3 gene
  • Upregulation of the autophagy pathway as a therapeutic strategy to clear mutant ataxin-3 and improve neuronal homeostasis

Gene Silencing

• Methods of gene silencing:
  • Antisense oligonucleotides (ASOs)
  • RNA interference (RNAi)
• Antisense oligonucleotides (ASOs):
  • Synthetic, unmodified or chemically modified, single-stranded nucleic acid molecules that hybridize to their target mRNA
  • Exert action through RNAse H-dependent or RNAse H-independent mechanisms
• Example: Duchenne muscular dystrophy (DMD), an X-linked recessive disease caused by mutations in the DMD gene, resulting in a prematurely truncated dystrophin protein
  • Antisense oligonucleotides-based therapy for DMD