Presentation 5: Lipid Nanoparticles Flashcards
What is gene therapy?
Ability to make genetic improvements based on cthe correction of mutated genes or site-specific modifications in order to treat recessive gene disorders, genetically acquired diseases, and even viral infections
What do we need to consider when performing gene therapy?
1) Knowledge of disease at hand
2) Accessibility of cells requiring treatment
3) Target cell types
4) Vectors: Type, specificity, efficiency, immune response, large- scale production
What is vector design, what does it involve, and why is design important?
Extremely important to successful gene therapy strategies
Mostly involves recombinant DNA technology to insert the gene into the vector
Should demonstrate an increase in normal function following administration
What are the viral vectors?
Retroviruses, Adenoviruses, Lentiviruses, etc…
Great for invading host cells and altering genetic material, but immunogenicity, insertional mutagenesis, and difficult vector production large issues
What are the non-viral vectors?
Low immune response Cytosolic protein expression Easy to produce Includes biochemical material: Lipid particles, Naked DNA plasmids, Concerns over toxicity
What are LNPs?
Promising non-viral vector for gene therapy
Clinically advanced
Reduced toxicity compared to previous polymeric systems
Other effective administration of therapeutic mRNA
Why are LNPs used?
Highly efficient nucleic acid encapsulation Small size Ionizable Lipid component Molecular Design Several routes of administration
Why are LNPs used for in utero administration?
Allows for maximum dosage for fetal weight
Easily accessible to progenitor cells
Compare with LNP mRNA delivery
What makes up the LNP?
1) Cationic/ionizable lipids
- -> Nucleic acids complexation, membrane fusion
2) Structural helper lipids
- -> Bilayer support
3) Cholesterol
- -> Integrity, and endosomal release
4) PEG lipids
- -> Hydrophilic surface, steric hindrance
5) Non-bilayer forming lipids
- -> Endosome destabilization
In this experiment what was incorporated into the LNP?
Ionizable Lipids B4 and C12-200
Non bilayer: DOPE
PEG lipid and cholesterol
How was the LNP formulated?
Ethanol phase: B4 combined with cholesterol, DOPE, and ammonium salt
Aqeous phase: Luciferase mRNA in citrate buffer
Ethanol and aqueous phases were combined to obtain the LNP
How was the LNP library constructed?
16 LNP formulations
Ionizable Lipid, DOPE, cholesterol, and PEG at different ratios
How was the LNP encapsulation efficiency determined?
1) Dilution
2) Lysis
3) Plating
4) Plate reader
5) RNA quantification
How were the LNP formulations evaluated?
1) Murine serum
2) Murine amniotic fluid
3) Sheep amniotic fluid
4) Pig amniotic fluid
5) Human amniotic fluid
What determines LNP stability?
Dynamic Light Scattering
