Gene Therapy

Define gene therapy

• A biological medicinal product that uses recombinant nucleic acids to regulate, replace, add, or delete genetic sequences 

Benefits of gene therapy

• Patients produce their own therapeutic gene product

• Treats the root cause of genetic diseases 

• Addresses disease with no viable cure 

Potential life-long treatment from a single injection

Gene Augmentation (form of gene therapy)

• Replaces a mutant, non-functional gene with a functional version or the addition of a second functioning copy 

Allows it to produce own therapeutic protein

Enzyme Replacement Therapy

Requires repeated injections of purified enzymes

In-vivo in the context of gene therapy

Direct administration of a vector into the patient

Ex Vivo

 Extraction of patient cells, undergoes genetic modifications in a lab, and engineered cells are re-infused

Physical Methods

Electroporation

Sonoporation

Microinjection

Gene Gun

Optoporation

Define electroporation

• Electrical pulses permeabilize membrane; high efficiency but reduced cell viability 

Sonoporation

Ultrasound-induced membrane disruption; variable efficiency but potential cytotoxicity

Microinjection

Microneedle delivery into cytoplasm/nucleus; precise targeting but low throughput + technically intensive

Gene Gun

DNA-coated metal microparticles are physical driven into cells; effective for resistant tissues but causes mechanical damage

Optoporation

Focused laser pulses create pores; high spatial precision but required specialized equipment + risk of cell damage

Non-viral Methods (Nanoparticles)

• Lipid-based nanoparticles

• Cationic nanoparticles

• Inorganic nanoparticles

• Extracellular vesicle

• Lipid-polymer hybrid nanoparticles

Lipid-based Nanoparticles (LNPs)

Clinically validated but prone to liver accumulation

Cationic Nanoparticles

• Highly tunable but toxicity is a challenge

Inorganic nanoparticles:

High structural stability + multifunctionality enable gene protection + imaging; biodegradability is concern

Extracellular vesicle:

• Low immunogenicity; scalability + heterogeneity limit translation

Lipid-Polymer Hybrid Nanoparticles:

Improved stability, circulation + gene loading. Complex formulation process

Key Characteristics of AAV

• Small, Non-pathogenic 

• Non-enveloped icosahedral capsid

• Linear, ssDNA genome

• Cannot replicate without a helper virus (Adenovirus, Herpesvirus) 

Lysogenic Life Cycle

• AAV enters the nucleus

• Endosomal escape 

  • Degradation

  • OR transit to nucleus 

• Integrates into the host genome (AAVS1 site on Chr 19)  OR remains episomal DNA

Lytic 

• Occurs during co-infection with a helper virus

• Leads to DNA replication 

• Virion release

Gene Therapy Using AAV

• Viral genome is cloned 

• Viral gene removed 

• Target gene is added 

• Transform bacterial cells with DNA 

• rAAV produced

Limitation of AAV-Mediated Gene Therapy

• High cost of treatment 

• High cost of viral vector production + purification 

• Ineffective re-administration due to vector-neutralizing antibodies