YB - Non-Viral Gene Therapy

Question 1

Q1: What are the key advantages and disadvantages of non-viral gene therapy? (6)

Answer 1

Advantages:

• Lower cost of production and easy scalability – does not require complex bioreactors.
• No viral shedding, eliminating risks associated with viral vector spread.
• Avoids immune responses associated with viral capsids or replication.

Disadvantages:

• Lower transfection efficiency compared to viral vectors.
• Poor stability and targeting in vivo.
• Struggles to overcome extracellular and intracellular barriers to gene delivery.

Question 2

Q2: What types of nucleic acids and complexes can be delivered using non-viral systems? (6)

Answer 2

• Plasmid DNA – circular DNA for gene expression.
• microRNA – regulates gene expression post-transcriptionally.
• siRNA/shRNA – silencing specific mRNAs.
• Antisense oligonucleotides – e.g., used in cytomegalovirus retinitis.
• Aptamers – e.g., Macugen, RNA aptamer targeting VEGF-165.
• DNA-protein or ribonucleoprotein complexes – for genome editing.

Question 3

Q3: How is non-viral genetic material delivered into cells? (4)

Answer 3

• Naked DNA – simple injection, but rapidly degraded and poor cell uptake.
• Cationic lipids (e.g., DOTMA, DOGS) – form lipoplexes that fuse with membranes.
• Cationic polymers (e.g., PEI, PLL, chitosan) – form polyplexes with DNA.
• Cationic peptides (e.g., TAT, GALA) – short peptides facilitate membrane penetration.

Question 4

Q4: What physical methods can aid non-viral gene delivery? (2)

Answer 4

• Gene gun – DNA-coated gold microparticles shot into cells.
• Electroporation – electric pulses create temporary pores in the membrane.

Question 5

Q5: What extracellular barriers limit non-viral gene delivery? (6)

Answer 5

• Skin is the first barrier (syringe)
• Nuclease degradation – DNA is degraded in the bloodstream.
• Plasma protein binding – alters delivery system and enhances immune clearance.
• RES (Reticuloendothelial system) entrapment – uptake by phagocytes.
• Embolization – particle aggregation blocks capillaries.
• Poor extravasation – limited diffusion through normal vasculature.
• EPR effect – enhanced diffusion in tumors due to leaky vasculature. (loosely packed endothelial cells)

Question 6

Q6: What are the intracellular challenges for non-viral gene delivery? (6)

Answer 6

• Binding to Cytoplasmic Membrane (pH 7.4) - delivery system needs to bind to the cell membrane to initiate uptake.
• Endocytosis and endosome formation – can trap and degrade cargo.
• Acidification in early/late endosomes (pH 6.5 → 5.5).
• Lysosomal degradation if escape fails.
• Endosomal escape is critical to avoid degradation.
• Disassembly of complexes to release DNA.
• Nuclear import of plasmid DNA for expression.

Question 7

Q7: How does the immune system interfere with non-viral gene delivery? (4)

Answer 7

Immune responses to the delivered nucleic acids

• Cytokine release (e.g., TNF-α, IL-1β) triggered by PEI/DNA complexes.
• Complement activation and IFN-γ induction by lipoplexes.
• TLR9 activation by unmethylated CpG motifs in DNA → inflammatory response.

Carrier-mediated immunity

• Cationic lipids and peptides can trigger immune cells.

Question 8

Q8: What ligands are used to target non-viral particles to specific cells? (5)

Answer 8

• Asialofetuin – targets ASGPr receptors in hepatocytes/hepatoma.
• Transferrin – overexpressed in cancers (e.g., breast, lung); improves tumor targeting.
• T7 peptide (HAIYPRH) – targets transferrin receptor in gliomas.
• EGF – targets EGFR in breast, prostate, and ovarian tumors.
• Fibronectin-binding protein – targets epithelial fibronectin.

Question 9

Q9: What are Affibody molecules and their uses in gene therapy? (6)

Answer 9

• Small, engineered non-antibody proteins with high affinity and specificity.
• Chemically modifiable and disulfide-independent.
• Can block protein interactions or deliver payloads.
• Stable and robust → ideal for medical use.
• In clinical trials for tumor imaging and inflammation.
• Useful for personalized medicine with diagnostic and therapeutic fusion.

Question 10

Q10: How is transcriptional targeting used in non-viral gene therapy? (4)

Answer 10

• Exploits tumor-specific transcription factor activity.
• Uses cancer-specific promoters (e.g., hTERT, hAFP) to restrict gene expression to tumors.
• IGF2 promoter used in bladder cancer to express cytotoxic genes like diphtheria toxin.
• Enhances gene expression only in cancer cells, minimizing off-target effects.

Question 11

Q11: What improvements are needed for ideal non-viral gene delivery systems? (8)

Answer 11

• Stability in serum
• Extended circulation time and proper biodistribution
• Immune evasion
• Targeted cell binding and uptake
• Efficient endosomal escape
• Nuclear import and persistence
• Long-term gene expression
• Transgene inheritance during cell division

Question 12

Q12: How is non-viral gene therapy applied in tissue repair (e.g., skin)? (5)

Answer 12

• DNA/polymer complexes are loaded onto a scaffold.
• Scaffold is transplanted into a deep skin defect.
• Localized gene expression stimulates tissue regeneration.
• Scaffold promotes vascularization and wound healing.
• Similar strategies are being explored for bone regeneration.

Question 13

Q13: What are trigger-responsive gene transporters? (3)

Answer 13

• Non-viral delivery systems that release DNA only upon specific stimuli.
• Examples: Ultrasound, magnetic fields, laser light.
• Improves precision, safety, and spatial control of gene expression.

Question 14

Q14: How are nanoparticles used to deliver gene editing tools like CRISPR? (3)

Answer 14

• Gold nanoparticles (CRISPR-NPs) can deliver Cas9 and guide RNAs.
• Various nanocarriers (lipid, polymer, inorganic) are optimized for HSCs (hematopoietic stem cells).
• Enables precise editing in stem cells for disease correction.

Question 15

Q15: What are recent developments in B cell-based gene therapies? (2)

Answer 15

Immusoft (ISP-001):

• Uses engineered B cells to express α-L-iduronidase for MPS I.

Be Biopharma (BE-101):

• Uses CRISPR-modified B cells to express F9 Padua variant for hemophilia B.

Question 16

Q16: What are examples of patient-compliant, less invasive gene delivery methods? (3)

Answer 16

Category 1 – Reduced Invasiveness:

• Electroporation, iontophoresis, ultrasound, magnetic fields.

Category 2 – Minimally Invasive:

• Microneedle arrays, nanodevices.

Category 3 – Non-Invasive:

• Nanoparticle patches, liposomes, permeation enhancers.