Delivery Systems and Cellular Uptake
The Delivery Problem
Core challenge
Engineering success depends not only on the genetic payload but also on delivering it to the correct cell type, tissue, subcellular compartment, and developmental stage.
Key bottleneck
Poor delivery is often the dominant limitation in translating an otherwise strong editing system into a real biological outcome.
Delivery Modalities
Major delivery strategies
Electroporation
- Transient membrane permeabilization
- Good for ex vivo cells
- Can reduce viability
Lipid nanoparticles
- Scalable and non-viral
- Useful for RNA or RNP delivery
- Payload and targeting limits
Which delivery approach is often favored for ex vivo editing of primary cells because it is transient and efficient?
Electroporation can introduce nucleic acids or ribonucleoproteins efficiently into cells outside the body.
Correct answer: Electroporation
What is one major advantage of delivering CRISPR as an RNP complex?
Short-lived delivery may reduce off-target editing and improve control.
Correct answer: It is transient and can reduce prolonged nuclease exposure
Viral vector considerations
Adeno-associated virus, lentivirus, and adenovirus differ in cargo capacity, integration behavior, immunogenicity, and persistence.
Delivery trade-offs
| Platform | Strength | Limitation |
|---|---|---|
| Electroporation | High transfection efficiency | Cell stress |
| AAV | Broad in vivo utility | Small cargo capacity |
| Lentivirus | Stable integration | Insertional concerns |
| Lipid nanoparticles | Scalable non-viral delivery | Tissue targeting challenges |
A major limitation of adeno-associated virus vectors is:
AAV has a relatively small packaging limit, which constrains payload size.
Correct answer: Their cargo capacity is limited
Why is tissue tropism important for in vivo gene delivery?
Targeting improves efficacy and reduces off-target exposure in unwanted tissues.
Correct answer: It determines which cell types the vector can reach efficiently