Intracellular Delivery and Gene Therapy

main advantages

specific localization, reduced side effects, lower dose needed

intracellular delivery

drug delivery, dna/rna delivery, protein/peptide delivery, imaging/sensing, tailored coatings, cell type specificty, organelle/compartment targeting

gene therapy has huge potential

cure disease, not just symptoms

candidate diseases for gene therapy

SCID, hemophilia, cystic fibrosis, sickle cell, cancer, neuro diseases

biggest problem in gene therapy

delivery

viral gene therapy

original essential viral gene deleted and replaced with a therapeutic gene

helper virus

contains essential genes necessary for replication

helper virus has

packaging domain deleted

unlimited maximum insert size

naked/lipid-DNA

no concentration limitaiton

naked/lipid-dna

ex vivo route of gene delivery only

retroviral

integration

retrovirus, lentiviral, AAV (sometimes)

long duration in vivo

lentiviral, AAV

stability

naked/lipid-dna

easy to scale up

naked/lipid-dna

immunological problems extensive

adenoviral

pre-existing host immunity

adenovirus, AAV

safety problems

insertation mutagenesis, inflammation, toxicity

new viral gene therapy success

luxturna first approved

limitations to cargo size

small cargos only

redosing limitations

immune response

manufacturing limitations

$475,000

some viruses have

safety concern

non-viral gene therapy safety

minimal immune response, minimal cellular toxicity, not carcinogenic

non-viral gene therapy cargo capacity

large

non-viral gene therapy cell targeting

flexible

non-viral gene therapy resistance to repeated administration

low

non-viral gene therapy

ease of production and quality control

non-viral gene therapy drawback

ineffective

polyethylenimine (PEI)

is gold standard

nanoparticle makeup

postivie polymer + negative DNA

nanoparticle taken up through

endocytosis

nanoparticles binding to overexpressed receptor

internalization into cells

nanoparticles can be

labeled

endosome destabilization to break out

charge change, structural change, and tight tradable amine group

chloride ions that balance charge → saltier inside than outside endosome

osmotic pressure → rupture membrane

particle is nondegrdable

wait for electrostatic interactions to have the right effect

disulfide linkages that break

reducing environment of cytosol breaks nanoparticles

most common gene therapy materials

adenovirus

adneovirus phased out

due to immunogenicity and cancer risks

inorganic particles

calcium phosphate → dna to precipitate and sediment down on cells

peptides (polylysine)

binds DNA easily

polysaccharides (chitosan)

positively charged

lipids/liposomes (lipofectamine)

positive combo with dna again

cationic lipid nanoparticle

mRNA delivery popular

polymeers (PEI)

challenges: nondegradable, toxic

microparticles (PLGA or POE) are

degradable

dendrimers (polyamidoamine)

branched structure

poly(beta-amino ester)s

more biomaterial

cationic lipid

ionizable lipid + neutral lipid + cholesterol + lipid

a lot of these structures have

amine groups to bind negatively charged nucleic acid and encap it

polylysine

good at forming particles but breaks out of endosome poorly

chloroquine

destablizie endosomes

tertiary means

buffer endosome at pka range around six

hydrolytic polyester amines and reducible polyamido amines

amine monomer conjugate addition to diacrylates

HPEAs and RPAAs synthesis

high throughput

HPEAs and RPAAs purification

none needed; synthesis neat or in DMSO

HPEAs and RPAAs byproducts

no side reactions or byproducts

HPEAs and RPAAs tunability

high

biomaterial libraries

1000+ polymers in library and growing

biomaterial libraries roles

structural diversity, tune cargo binding and release, tune degrability, tune cell-material interactions, non-cytotoxic

systemic delivery clearance

avoid clearance by liver and spleen

nanoparticle size needs to be

smaller than 100 nm

nanoparticle surface

hydrophilic

nanoparticle surface charge

uncharged/neutral

protection of DNA from degradation

tight binding to DNA through electrostatic interactions/self-assembly

multivalent avidity interaction

many negative charges on DNA, many positive charges on polymer

multivalent avidity mechanism

each interaction weak → together strong

positive charges important

primary amines that encapsulate DNA so no enzymes can cut it up

double emulsion

further keeps DNA safe

dna uptake is inefficient

large and hydrophilic, highly charged

small particle

bit of positive charge on outside and sugar coat that’s negatively charged → binds to surface

ligand

bind receptors at cell surface → internalization

cytoplasm very viscous

diffusion of large DNA and nanopartic

use virus to hijack active transport

move along microtubules for fast movement to the nucleus while DNA/particles still in endosome

particle could be potentially

designed to bind to motor proteins

staying in the endosome is a problem

endosome still an outside space — it gets degraded or recycled if the particle doesn’t come out

low pH, degradation or recycled outside of cell

different methods of escape

proton sponge mechanism (secondary and tertiary amines that can buffer the endosome) — soaks more proton until endosome ruptures

membrane disrupting peptide

alpha helix that could destroy the membrane

bound dna cannot be efficiently transcribed

release can be due to thermodynamic unbinding/competition with other molecules

degradation release

hydrolysis, enzymes, disulfide reduction

nuclear import

DNA has to make its way to the nucles

cell nucleus is a major bottleneck

some cells don’t divide much

certain DNA sequences and proteins

can recruit endogenous nuclear import machinery

nuclear localization sequences

added to biomaterial to increase import

downstream steps

transcription and translation, biomaterial degradation/elimination

transcription and translation

certain nucleic acids and proteins can serve as transcription factors to enhance expression + recruitment of endogenous cell machinery

biomaterial degradation/elimination

important to minimize cytotoxicity

self assembled polymeric nanoparticles

form nanoparticles 100 nm

polymer buffering curves

acid or base added → polymers have good buffering capability in range 5 to 7

PEI good at buffering

per mass, but not along all range of pH

hydrolytic degradation in water

happen over hours

glutathione

disulfide reduction

increased ydrophobicity

improves delivery

transfection to GBMs dependent on

end group and base polymer hydrophobicity

nanoparticles passively target

tumor vasculature

ligand-mediated uptake to

target cells

biomaterial mediated

cell specificty

cell specific promoter

transcriptional targeting

gene product

preferentially active in target cells