Download Gene Therapy

Gene Therapy
Each of us carry about half a dozen defective genes
One in ten people has or will develop an inherited genetic
disorder.
Approximately 2,800 specific conditions are known to be
caused by defects (mutations) in just one of the patient’s
genes.
Diseases that can be traced to single gene defects account
for about 5% of all admissions to children’s hospitals.
Gene Therapy
Many inherited diseases are due to protein deficiencies or
defects.
Gene therapy could correct some genetic disorders through
gene replacement.
Gene-based therapies could also be applied in vaccines,
viral infections, cardiovascular interventions, etc.
Recombinant proteins pose manufacturing and
administration problems. Gene therapy involves delivering the
gene encoding the protein.
Gene Therapy
The goal of gene delivery is the production of a therapeutic
protein in sufficient quantity at the appropriate site to ellicit the
desired biological responses.
Various modes of delivery:
Viral delivery systems (modified, nonreplicating, viral
genomes carrying a specific transgene). An efficient
approach but has limitations due to its potential
immunogenicity and insertion mutagenesis.
Non-viral delivery-plasmid-based gene delivery
systems (utilizing lipids, polymers, or peptides to deliver
the gene).
Naked DNA.
Gene Therapy
Well understood gene delivery systems are required
Primary considerations for successful gene transfer
technologies are:
Manufacture of the gene delivery vehicle,
Delivery to the target tissue and cell surface,
Cellular internalization,
Intracellular trafficking,
Nuclear uptake,
Functional gene expression (with appropriate and
controlled levels of duration).
Gene Therapy
Safety of the delivery system, the expressed gene product,
and the associated immune response are also potential issues
needed to take into account.
Viral and plasmid based gene delivery vehicles can be used.
Viral delivery system consists of modified, nonreplicating, viral
genomes carrying a specific transgene.
Plasmid based gene delivery systems utilize a variety of
agents (lipids, polymers, peptides) complexed with DNA
encoding a transgene or “utilize” the naked DNA alone.
Gene Therapy
Gene delivery systems must successfully traverse multiple
barriers from the site of administration to their destination
(nucleus or target cell).
Barriers to gene delivery:
Extracellular trafficking
Uptake into target cells
Intracellular trafficking
Extracellular Trafficking
Systemic intravenous administration is the most
challenging route of administration.
A delivery system for IV administration requires stability
within the complex milieu of blood serum, and ability to avoid
clearance by phagocytic cells.
Immune clearance: (PEG and lipids conjugated to
adenovirus can abrogate the neutralizing effects of
neutralizing antibodies in vitro and in vivo.
Extracellular Trafficking
In plasmid systems serum can affect the biophysical and
biochemical properties of lipid DNA complexes by altering
size and charge (complex disintegration, DNA release, and
degradation).
The composition of lipids in plasma-based delivery systems
affects the transfection efficiency. Lipid composition is an
important factor in the recruitment of serum proteins.
Extracellular Trafficking
Retroviruses are sensitive to opsonization* and inactivation
by serum components following systemic delivery.
Packaging cell line origin can impact immune response
activation and affect viral clearance and stability.
In vivo stability limits the primary use of retroviruses in
humans to ex vivo applications.
*Antibody opsonization is the process by which a pathogen is marked for
ingestion and destruction by a phagocyte.
Extracellular Trafficking
Targeting a specific disease requires:
Knowledge of the appropriate tissue and cell types
necessary to express the therapeutic protein.
Understanding the delivery system and route of delivery will
achieve the clinical goal.
Biodistribution studies can provide a foundation for IV
delivery.
Delivery of the cystic fibrosis transductance regulator (CFTR)
through IV or intratracheal (IT) routes provides different
outcomes.
Extracellular Trafficking
Differential uptake and expression of cystic fibrosis
transductance regulator (CFTR) based on the delivery
route.
IV administration of a cationic lipid-based delivery
system: DNA was delivered to distal lung in the alveolar
region, including alveolar region, including alveolar type
II epithelial cells.
IT administration: DNA was found in the epithelial lining
of the bronchioles.
Extracellular Trafficking
 Differences in gene expression profiles based on delivery
route highlight another major issue confounding the
development of gene-based delivery vehicles (there is a
lack of in vitro to in vivo correlation with available models).
 Different routes of administration result in different
extracellular barriers for gene administration (and
expression), e.g.; blood components in IV delivery vs.
mucus barrier in IT delivery.
Extracellular Trafficking
 Direct administration of the gene delivery vehicle to the
tissue of interest could circumvent extracellular barriers
(intratumoral injection, intramuscular injection, etc.)
 Minimizing extracellular barriers for the delivery vehicles
is necessary but may not be sufficient for gene
administration. Other barriers to gene transfer can still
exist.
Cellular Uptake
Receptor availability can affect the outcome of adenovirus gene delivery.
Retargeting strategies can overcome lack of receptor
limitations and generate specificity for target cells.
Manufacture of viral antibody hybrids is a challenge to
overcome before targeted delivery is accomplished.
Cellular Uptake
Cell division is required for retroviral transduction but rapid
proliferation is not sufficient for transduction efficiency and
transduction may be limited at the receptor level.
Plasmid-based systems rely on ionic charge-based
interactions for initial cell binding and subsequent
endocytosis.
Much of the plasmid-based formulation technology
development has relied on empirical assessments.
Intracellular Trafficking
Endosomal entrapment and nuclear uptake are important
issues that need to be engineered in gene delivery.
Endosomal release and nuclear uptake are the primary
foci to improve transfection efficiency.
A prevailing hypothesis is that nuclear membrane
breakdown during mitosis is required for uptake of plasmid
DNA into the nucleus.
Intracellular Trafficking
Adenoviral vectors are able to transduce nondividing cells
suggesting that the viral genome has evolved a means to
pass through the nuclear membrane.
Adenoviruses utilize specific endogeneous molecular
motors to facilitate transport through the cytoplasm.
Understanding the intracellular trafficking of DNA from
cellular uptake to nuclear delivery should allow increases in
the efficiency of gene transfer.
Intracellular Trafficking
The exploitation of cytoskeletal components for
enhancement of plasmid-based gene delivery will generate
novel strategies for gene delivery systems.
Increased delivery efficiency will affect dosing regimes,
therapeutic indices, and safety profiles.
Incorporation of peptide nuclear localization signals (NLS)
into plasmid delivery systems can assist the transport of
plasmid into the nucleus.
DNA delivery from hydrogels
DNA delivery from a tissue engineered scaffold
is a versatile approach to promote the
expression of tissue inductive factors locally to
be used as signals to promote tissue formation.
Naked DNA or complexed DNA has been incorporated into
hydrogel scaffolds:
Collagen,
Pluronic-hyaluronic acid,
PEG-poly(lactic acid)-PEG
Engineered silk elastin,
Fibrin,
PEG-hyaluronic acid hydrogels
DNA delivery from hydrogels
Major limitation remains:
Gene transfer efficiency,
Gene transfer to MSCs seeded in 3-D has not
been previously investigated.
 MSC-like progenitor cells are believed to reside in most
adult tissues, and responsible for adult tissue
regeneration.
 Therefore, the design of hydrogel materials that allow for
cellular infiltration and deliver genes to infiltrating cells
would be ideal for regeneration of tissues in vivo.
Cellular infiltration is migration of cells from their sources of origin, or direct
extension of cells as a result of unusual growth and multiplication.
DNA delivery from matrix metalloproteinase
degradable PEG hydrogels
DNA/PEI polyplexes were encapsulated inside MMP-degradable PEG
hydrogels through mixing the polyplexes with prepolymer solution.
Fluorescently labeled polyplexes were loaded inside the hydrogel and imaged
by confocal microscope.
At higher concentration of polyplexes (50 g/100 L), aggregation was
observed.
Hydrogels with or without polyplexes have similar storage and loss moduli
(G’ and G’’), which are indications of elastic and viscous properties.
The release kinetics of encapsulated polyplexes were tested in PBS, trypsin,
and D1 conditioned mediums.
Activity of encapsulated polyplexes were measured through degradation of
the gel in the presence of trypsin, and then measuring the luciferase activity
Luciferase commonly is used as a reporter to assess the transcriptional activity in cells
that are transfected with a genetic construct containing the luciferase gene under the
control of a promoter of interest.
Y. Lei, T. Segura / Biomaterials 30 (2009) 254–265
DNA delivery from matrix metalloproteinase
degradable PEG hydrogels
The toxicity of encapsulated DNA/PEI polyplexes to
infiltrating cells was determined by cell viability assay.
No significant differences in viability was observed between
hydrogel samples with or without DNA.
The ability of cells grown inside degradable hydrogels to
internalize and express encapsulated DNA/PEI polyplexes
was studied.
pSEAP expression (expresses alkaline phosphatase) was
measured. This allowed to quantify the reporter gene
expression over time of the same hydrogel, by analyzing cell
culture media, which is ideal for long time period gene transfer
to be characterized.
Y. Lei, T. Segura / Biomaterials 30 (2009) 254–265
DNA delivery from matrix metalloproteinase
degradable PEG hydrogels
PEG hydrogels were formed with cysteine-containing matrix metalloproteinase sensitive peptides
(MMPxl) with four-armed PEG-vinyl sulfone pre-modified with cell adhesion peptides (PEG-RGD).
Polyplexes were encapsulated into hydrogel matrix by mixing with the precursor solution prior to
gelation. Cells were seeded as single cells or a cluster of cells (shown) inside the hydrogel matrix.
As the cells infiltrate the scaffold, they encounter polyplexes and are transfected.
Y. Lei, T. Segura / Biomaterials 30 (2009) 254–265
Distribution of DNA/PEI
polyplexes inside the PEG
hydrogel. DNA labeled with
TM rhodamine was used to
form the polyplexes prior to
encapsulation inside the
gel.
Polyplexes made with 15
mg DNA/100 mL gel at N/P
¼ 7.5 (A), 30 mg DNA/100
mL gel at N/P ¼ 7.5 (B), 50
mg DNA/100 mL gel at N/P
¼ 7.5 (C) and 15 mg
DNA/100 mL gel
at N/P ¼ 15 (D) were
encapsulated inside the
hydrogel scaffold. Confocal
microscopy was used to
take the images using a 40
objective over a 20 mm
thick section.
Y. Lei, T. Segura / Biomaterials 30 (2009) 254–265
Storage (G’) and loss modulus (G’’) of
PEG hydrogel with and without
DNA/PEI polyplexes measured using
plate-to-plate rheometry. Polyplexes
made with 15 mg DNA/100 mL gel at
N/P ¼ 7.5, 30 mg DNA/100 mL gel at
N/P ¼ 7.5, 50 mg DNA/100 mL gel at
N/P ¼ 7.5 and 15 mg DNA/100 mL
gel at N/P ¼ 15 (* in A) were
encapsulated inside the hydrogel
scaffold. G’ and G’’ were measured
under constant strain of 0.05 and
frequency from 0.1 to 10 Hz. Overall
G’ and G’’ (A), and G’ (B) and G’’ (C)
over the entire frequency sweep are
shown.
Y. Lei, T. Segura / Biomaterials 30 (2009) 254–265
DNA delivery from matrix metalloproteinase
degradable PEG hydrogels
Cumulative release kinetics of
DNA/PEI polyplexes encapsulated
inside MMPdegradable
PEG hydrogels. Hydrogels (100 mL)
containing 15 mg of DNA complexed
with PEI at an N/P of 7.5 were placed
in PBS, trypsin or D1 conditioned
medium. At the
indicated time points the releasing
medium was analyzed for DNA
content. Data is shown as a percent
of the total DNA found after complete
gel degradation.
Y. Lei, T. Segura / Biomaterials 30 (2009) 254–265
DNA delivery from matrix metalloproteinase
degradable PEG hydrogels
Activity of pEGFP-LUC/PEI polyplexes
encapsulated inside MMP-degradable
hydrogels (A). The activity of released
polyplexes (R) was normalized to that of
fresh polyplexes (C), and compared to fresh
polyplexes with trypsin added (T), fresh
polyplexes with PEG added (P), and fresh
polyplexes with both trypsin and PEG added
(T&P). Transfection using freshly prepared
complexes supplemented with free PEG-VS,
0.25% trypsin/EDTA, or a combination of
both at the same concentration found in the
degraded hydrogels. Dose–response curve
of DNA/PEI polyplexes transfection efficiency
normalized to the RLU found with 1 mg DNA
(B). The dotted line represents polyplexes
that had 37% of the RLU activity and
corresponds to 65% of the DNA being
present.
Y. Lei, T. Segura / Biomaterials 30 (2009) 254–265
DNA delivery from matrix metalloproteinase
degradable PEG hydrogels
A
B
C
D
E
F
G
H
Migration of D1 cells in
MMP-degradable PEG
hydrogels. Cells were
placed in the gel either as
a cluster for 24 h (A), 69 h
(B), 122 h (C) and 155 h
(D) or homogeneously for
48 h (E), 96 h (F), and 240
h (G). A representative
picture of cells migrating
out of a fibrin cluster at 312
h is shown (H, green stain
is actin, blue stain is the
nuclei).
DNA delivery from matrix metalloproteinase
degradable PEG hydrogels
•Cumulative SEAP expression
(expresses alkaline phosphatase)
was leveled off between days 7 and
10 for cells seeded using
homogeneous approach.
•Cumulative SEAP expression
continued to rise throughout the 21
day incubation for infiltrating cells.
Y. Lei, T. Segura / Biomaterials 30 (2009) 254–265
DNA delivery from matrix metalloproteinase
degradable PEG hydrogels
Phase images were taken at 4 days, 9 days,13 days, and 17 days.
Y. Lei, T. Segura / Biomaterials 30 (2009) 254–265
DNA delivery from matrix metalloproteinase
degradable PEG hydrogels
Zymogram gel electrophoresis
of D1 cell conditioned medium
at passages 3 (lane 1), 6 (lane
2) and 10 (lane 3). BSA (lane
4), 10 ng MMP-2 (lane 5) and
DMEM with 10% serum (lane
6) were run for comparison. All
conditioned medium samples
as well as BSA and DMEM
were run at a total protein
concentration of 27 mg as
determined by Bradford assay.
Y. Lei, T. Segura / Biomaterials 30 (2009) 254–265