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GENE THERAPY
Methods for treatment of human diseases
Current methods:
1 Medicine tablets by oral
2 Medicine by injection.
3.Medicine by nasal spray
4 Medicine by applying to skin.
Methods in the future:
5 Cell therapy
6 Gene therapy
GENE THERAPY
Gene therapy:
Transfer of therapeutic gene into the
diseased tissue
Hemophiliac dogs with
coagulation factor IX deficiency
Normal dog: blood clots
in about 8 to 10 minutes
Diseased dog: blood clots
in about 50 to 60 minutes
Dr. Kenneth Brinkhous (North Carolina Univ)
1-hour procedure
of GT infusion,
15 month of expression,
20 min for clotting
Diseases for applying gene therapy
Disease
Defect
Severe combined
Target cell
Adenosine deaminase 4
Bone marrow cells or
immunodeficiency
Hemophilia
T-lymphocytes
Factor VIII, Factor IX deficiency
Liver, muscle, fibrob.
Cystic fibrosis
Loss of CFTR gene
Airspaces in the lung
Hemoglobulinpathies
 or  globulin gene
Bone-marrow cells
1-antitrypsin deficiency
1-antitrypsin
Lung or liver cells
Cancer
Many causes
Many cell types
Neurological diseases Parkinson’s, Alzheimers
Direct injection into
the brain
Cardiovascular
Restenosis, arteriosclerosis
Vascular endothelium
Infectious diseases
AIDS, hepatitis B
T cells, macrophages,
Liver cirrhosis
Fibrogenesis
Autoimmune disease
Lupus, diabetes
Hepatocyte growth factor
MHC, 2-microglobulin
In humans
Cancer 69%
General concerns
The Food and Drug Administration (FDA) has not yet approved
any human gene therapy product for sale.
Four major problems with gene therapy:
1) Short-lived nature of gene therapy. Very hard to achieve any longterm benefits without integration and even with it.
2) Immune response. It reduces gene therapy effectiveness and
makes repetitive rounds of gene therapy useless
3) Problems with viral vectors . Toxicity, immune and inflammatory
responses, also fears that viral vector may recover disease-causing ability
4) Multigene disorders. Most commonly occurring disorders,
such as heart disease, Alzheimer's disease, arthritis, and diabetes,
are caused by the combined effects of variations in many genes.
Gene therapy could be
very different for different diseases
• Gene transplantation
(to patient with gene deletion)
• Gene correction
(To revert specific mutation in the gene of interest)
• Gene augmentation
(to enhance expression of gene of interest)
• Targeted killing of specific cells by introducing killer
gene
• Gene ablation – targeted inhibition of gene expression
Gene therapy
In vivo
Ex vivo
in vivo and ex vivo schemes
EX VIVO
IN VIVO
http://laxmi.nuc.ucla.edu:8237/M288/SChow_4_10/sld005.htm
In vivo gene therapy
1. The genetic material is transferred directly into
the body of the patient
2. More or less random process;
small ability to control; less manipulations
3. Only available option for tissues
that can not be grown in vitro;
or if grown cells can not be transferred back
Ex vivo gene therapy
1. The genetic material is first transferred
into the cells grown in vitro
2. Controlled process;
transfected cells are selected and expanded;
more manipulations
3. Cells are usually autologous;
they are then returned back to the patient
Transgenes
Integrated
- stable expression;
may provide a cure
- Random insertions
in heterochomatin
Gene can be inactivated;
Not integrated
for episomes (plasmids),
Random mutagenesis
Is not an issue
- Expression is transient;
Repeated treatments
nesessary
In euchromatin -Can disrupt important host genes;
Long-term consequences are unknown
How episomes and integrated
trasgenes behave in dividing cells
Integral transgene
Episome
Loss of plasmid
Methods of gene delivery
(therapeutic constructs)
1 Injection of naked DNA into tumor by simple needle and syringe
-- DNA coated on the surface of gold pellets
which are air-propelled into the epidermis
(gene-gun), mainly non applicable to
cancer
2 DNA transfer by liposomes
(delivered by the intravascular, intratracheal,
intraperitoneal or intracolonic routes)
3 Biological vehicles (vectors) such as viruses and bacteria.
Viruses are genetically engineered
so as not to replicate once inside the host.
They are currently the most efficient means of gene transfer.
Desirable characteristics
of gene delivery vector
1. High titer or concentrations (>108 particles/ml)
2. Easy and reproducible method of production
3. Precise and stable introduction of transgene
4. Vector should not elicit immune response
in the host
5. Transgene should be responsible
for its regulatory elements (on/off system)
6. Vector should be able to target specific cell types
Injections of naked DNA
Naked DNA gene therapy
Intramuscular
delivery
Intravascular delivery
 liver and muscle
Covalently closed circular form is more stable that open plasmid
-- Results in a prolonged low level expression in vivo
-- Very cheap
DNA vaccines
Antiviral
and antibacterial
But, traditional vaccines are better
when available)
Passive
to increase
the pre-existing immune
response
to the cancer
Cancer immunotherapy
Active
initiates an immune response
against an unrecognised
or poorly antigenic tumor
Current attempts with naked DNA
vaccination in infectious diseases
HIV
hepatitis B and C
Influenza
Papilloma
Cytomegalovirus
Tuberculosis,
Lyme disease
Helicobacter pylori
Malaria
T cells recognise liver cell
with malarial parasite inside
Produce IFN-gamma
IFN-gamma stimulate
antigen presentation
www.malaria-vaccines.org.uk
DNA vaccines (and other vaccines too)
prime immune system with properly presented antigen
immunologically important components of the malaria pathogen
several peptide epitopes
that we know are recognised by T-cells
(a so-called multi-epitope string)
DNA vaccine encoding
an immuno
recognisable insert
whole protein called
thrombospondin related
adhesion protein (TRAP).
Ballistic DNA Injection,
particle bombardment,
microprojectile gene transfer (gene guns)
Invented for DNA transfer to plant cells
Fully applicable to mamalian cells
plasmid DNA is precipitated
onto 1-3 micron sized gold
or tungsten particles.
Discharge: helium pressure,
or high-voltage electronic
Light micrograph of DNA-coated gold
beads in the skin after gene-gun
vaccination
Duchenne muscular dystrophy
(DMD)
X-linked recessive disorder; 1/3500 boys worldwide
About 30% of cases represent new mutations.
Absence of dystrophin, a cell membrane protein
(approximately 0.01 % of skeletal muscle protein).
All muscles involved
1. Generalized weakness and muscle wasting
affecting limb and trunk muscles first.
Wheels at 12 y.o.
Life threatening dysrhythmia or heart failure
develops in about 10 %.
Death at 10th-20th after pulmonary problems (breathing)
Why muscles are enlarged in DM
patients?
Increased fibrous connective tissue revealed by this trichrome stain.
There are larger overly contracted muscle fibers
with scattered small degenerating or regenerating fibers
Degenerated muscles
contain lots of
fibrous and adipose tissue
Normal muscles and DM
muscle
muscles stained for dystrophin with monoclonal antibodies
myofibers are circumscribed
by the darkly-staining dystrophin
dystrophin is not evident
wider variation
in myofiber diameters
increased connective tissue
Dystrophin
Provide links between
the intracellular cytoskeleton
and the actin filaments
with the extracellular matrix
Whole complex
stabilizes the membrane.
Laminin2α2:
congenital MD chr 6
Sarcoglicans:
Limb Girdle MDs (4 types)
Duchenne and Becker MDs
DM is good model disease as
ballistic GT is available for
muscles
Problem:
Native gene is 2,4 Mb in size (quite unusual)
mRNA is 14 kb in size (also too big for any vector)
IDEA: Dystrophin can retain significant function
even when missing large portions of its sequence (Becker’s phenotype)
Becker’s phenotype is anyway better than complete Duchenne !
Patient: exon 17–48 removed (48% of the coding region),
Deletion variants of dystrophin for GT
ABD=
actin-binding domains
most, but not all, of the spectrin-like repeats
are dispensable for the function of dystrophin.
Scott Harper et al., 2002
GT with dystrophin minigene in
mice with DM phenotype
MDX mice with premature stop codon in exon 23; no dystrophin
GT treated
Non-treated
Jun. 05, 2003
French Muscular Dystrophy Association (AFM) and Transgene
announced that the results of their Phase I trial
on gene transfer for Duchenne/Becker's Muscular Dystrophy
Nine patients in three groups:
a single injection of low dose of plasmid with dystrophin
Muscle segment
were taken out
for examination
a single injection of high dose of plasmid with dystrophin
Two injections of high dose each of plasmid with dystrophin
Expression of dystrophin is found in 1 to 10 percent of muscle fibers
for group 1 and 2 ; for all 3 patients in group 3
No immune reactions; no side effects !!!!
Liposomes
Next level idea – why naked DNA?
Lets’ wrap it in something safe
to increase transfection rate
Lipids – is an obvious idea !
Therapeutic drugs
Liposomes are formed by the self-assembly of
phospholipid molecules in an aqueous environment.
Anionic liposome
www.emc.maricopa.edu/faculty/ farabee/BIOBK/
Cationic liposomes
Positively charged lipid heads
positively charged lipid droplets
can interact with negatively charged DNA
to wrap it up and deliver to cells
Inside liposomes DNA is resistant to degradation
Lipofectin, lipofectamine, lipofectase….
Lab procedure for liposome
preparation
Liposome disadvantages
Liposomes are rapidly cleared from the circulation
and largely taken up by the liver macrophages
How to overcome it?
liposome surface ligands decrease degradation
(monosialoganglioside or polyoxyethyle)
Modified liposomes
(stealth liposomes)
hydrophilic polyoxyethylene lipids
incorporated into liposome
Increased half-life is due to
a reduced coating (opsonisation)
of these liposomes by plasma proteins
cholesterol,
polyvinyl-pyrrolidone polyacrylamide lipids,
glucoronic acid lipids are working the same….
So liver cells not able to uptake them
Complex multilayer liposomes
(Piedmont)
Able to transport medication
through the epidermal and dermal layers
of the skin via the
lipid-rich intercellular channels.
The medication can be directed
specifically to the targeted area
Cochleates – multilayer lipid rolls
1. Storageable without any problems – could be lyophilized
(at least one year as a lyophilized powder at room temperature)!
2. Durable – survive multiple membrane fusion event
(fuse-release drug-disengage-fuse-release..)
3. Can survive in GI tract
Cochleates have been shown to be an effective oral delivery system.
Immunoliposomes for active
targeting
Antibodies to intracellular myosin
target liposomes
to infarcted areas of heart
Antibody against
tumor specific molecules
will target them to tumors
Liposomes could serve as tumor specific
vehicles (even without special targeting)
Liposomes better penetrate into tissues
with disrupted endothelial lining
delivery of genes by liposomes
Cheaper than viruses
No immune response
Especially good
for in-lung delivery (cystic fibrosis)
100-1000 times more plasmid DNA needed
for the same transfer efficiency as for viral vector
Cystic fibrosis
most common lethal genetic disorder in Caucasian populations
(1 in 2000 live births.) . Among African and Asian is really rare
a defect in the CFTR gene
(cystic fibrosis transmembrane conductance regulator )
irregular chloride and sodium ion conductance
in epithelial cells of many organs
(increased uptake of sodium ions).
Sweat glands
(too much
salty secretion)
Pancreas is damaged
(leads to diabetes)
Digestive tract
(constipation)
Lungs create
thick mucus secretion
(prone to infections,
constant cough,
leading cause of death)
Lungs in cystic fibrosis
Normal lung
Normal alveolar appearance
CF lungs filled with mucus
CF lungs
dilated crypts
filled with mucus and bacteria.
lung did not collapse
when it was removed postmortem
Cystic fibrosis lungs are prone to
infections
Pseudomonas aeruginosa
easily colonise mucus in the dilated lungs
Neutrophiles are activated,
then overactivated
The battle between neutrophils and bacteria
leads ultimately to lung fibrosis and damage
Mucus protects bugs and
promotes hypermutation
"Hyperinflammation" as recruited neutrophils
unable to eradicate bugs, instead damage lung tissue
Pancreas in cystic fibrosis
Normal pancreas
Distended CF cripts filled with mucus
Pancreatic enzymes not able to leave the gland;
they damage the gland
Less insulin produced
Impaired glucose tolerance and diabetes
Survivors to 25 years old:
1/3 with impaired glucose tolerance and with 1/3 diabetes
Treatment of CF
1. Many different antibiotics are required to clear infections.
Staphylococcus aureus,
Haemophilius influenzae,
Aspergillus fumigatus
- the same picture…
CFTR gene (chromosome 7)
27 exons, 1480 aa
ATP binding domain
CFTR product
Delta F508 mutation
50% of all patients are
homozygotes with this mutation
30% are heterozygotes,
delta F508/X
an incompletely folded,
protease-sensitive form;
Rapidly degrades before
entering Golgi complex
Cystic fibrosis Gene Therapy
January 1995.
Results of intranasal CFTR-liposome spaying in CF patients.
12 patients, Temporary relief in 20% of patients.
Maximum on day 3, faded away on day 7. No immune reactions.
Monthly applications also work,
2000
Multiple Dose: Gene Transfer Assays
 Gene
Transfer Detected After Each Dose
MD 1
56% (5/9)
DNA
MD 2
66% (6/9)
mRNA
MD 3
66% (6/9)
Protein
Total
63% (17/27)
Function
 Good
60% (15/25)
44% (11/25)
28% (7/25)
22% (6/27)
Correlation Between Gene Transfer Assays
 mRNA
Positives All +ve For DNA
Cationic-lipid-mediated CFTR gene transfer can
significantly influence the underlying chloride defect
 Functional Positives All +ve For DNA, mRNA and Protein
in the lungs of patients with cystic fibrosis.
MOST COMMON VIRAL VECTORS
Retroviruses
can create double-stranded DNA copies of their RNA genomes. Can
integrate into genome. HIV, MoMuLV, Rous sarcoma virus
Adenoviruses
dsDNA viruses that cause respiratory, intestinal, and eye infections
in humans. Virus for common cold
Adeno-associated viruses
ssDNA viruses that can insert their genetic material
at a specific site on chromosome 19
Herpes simplex viruses
dsDNA viruses that infect a neurons. Cold sores virus
Retroviral vectors are able
to infect dividing cells only
Preintegration complex of retroviruses
non able to penetrate nuclear membrane.
In dividing cells nuclear membranes are broken down,
so viral genome can enter and integrate into the chromosome
Infection of dividing cells only
Good for cancer gene therapy
Retroviruses are most often used vectors
for common disease gene therapy
Amphotropic retroviruses
Moloney murine leukaemia virus (Mo-MLV),
capable of infecting both mouse cells and human cells
Treatment could be tested in mouse
Safety features:
1. Propagation only in packaging cells
2. All regions of homology with the packaging virus should be removed
to prevent recombination resulting in
replication competent retroviruses
Some replication competent retroviruses
do occur at a low frequency….
After removing of all non-essential parts
carrying capacity for retroviral vectors is approximately 7.5 kb
(not enough for some approaches)
Tissue tropism still a major issue
even for amphotropic retroviruses
In humans, retroviruses use
sodium-dependent phosphate transporters
Pit-1 and Pit-2 for entry
Unfortunately, in humans
this receptor expressed too widely.
Many approaches invented
to improve and target the viral delivery
Modify env gene by
creation of a pseudotyped vector
(Rhabdoviridae)
Vesicular
stomatitis virus (VSV):
phospholipid component of
membrane as a receptor
VSV G protein
hybrid virion
with “mixed” envelope
www.urmc.rochester.edu/smd/
()
What we gain :
1) Host range now determined by both envelopes
Retro-VSV hybryds are able to infect even
Fish, Xenopus, Mosquito, Butterflyes….
2. VSV envelope is very durable
pseudotyped virus has the ability to withstand the
shearing forces encountered during ultracentrifugation
high-titer retroviral vector stocks could be generated
Drawbacks of using a
pseudotyped retroviral vectors
1. Host range now is too broad.
Cell-specific targeting not possible,
but we can use it for ex vivo approaches.
2. G protein of VSV is very immunogenic
(so, it’s one-time approach)
3. G protein of VSV is toxic for cell
pseudotypes could be produced only
by already dying packaging cells
(overcome by inducible promotors)
Other pseudotypes
are available:
HFV – human foamy virus, HIV-1,
LCMV (lymphocytic chiriomeningitis) – non toxic for cells
Equipping retroviral particles
with cell-specific ligands
Specific binding is easy to achieve;
but virus uptake become less efficient.
1. Addition of part of the ligand to create env interaction
with cell specific receptor
50 aa from EPO added to env
makes it interacting with EPO-receptor
on EPO receptor bearing cells
Other additions: heregulin. Binds to HER-2 and HER-4 receptors
overexpressed on breast cancer cells
Real treatments performed with
retroviral system
Severe Combined Immunodoficiency (SCID):
ADA-SCID and X-linked SCID
What is Severe Combined
Immunodoficiency (SCID)?
> 8 new ear infections per year
> 2 serious sinus infections per year
> 2 pneumonias per year
> 2 month on antibiotics with little effect
lymphopenia (absolute lymphocyte count less than 200)
-- failure to gain weight and grow
-- recurrent deep skin and organ abscesses
What are the cause of the
SCID?
1) Chromosome 20–linked SCID (mutated ADA);
25% of all cases
2) Mutated gamma-C receptor for IL-7 cytokine (X-linked SCID)
3) 70 other causes (not monogenic)
Adenosine deaminase is a glycoprotein
and acts as a hydrolase,
catalyzing the deamination of adenosine into inosine.
Adenosine + H2O = Inosine + NH3
Adenosine is toxic for B- and T-cells
ADA is essential for the proper growth and function of
infection-fighting T and B lymphocytes.
SCID treatments
Life in germ-free envinronment
Histocompatible bone-marrow transplantations
(with potential graft vs host disease)
Enzyme replacement therapy
with weekly injections of the PEG-ADA (ADAGEN)
VERY expensive; not a cure; temporary effect
An excess of adenosine deaminase leads to hemolytic anemia
GENE THERAPY
ADA gene therapy story
ADA protein has been characterized in the late 1970s
Three separate laboratories published the gene sequence in 1983
W. French Anderson (NIH); in the late summer of 1990, the FDA
was sufficiently convinced by the preliminary laboratory data
to approve the first human gene therapy trials
using the MoMLV-based delivery vector
Mature T-cells GT
Ashanti DeSilva; advanced stage of SCID; 4 yr old;
Cynthia Cutshall
What precisely has been done to
Ashanti DeSilva?
Her T cells were:
-- placed in tissue culture
-- stimulated to proliferate
(by treating them with the IL-2)
-- infected with the retroviral vector
MoMLV-ADA
-- returned to her in a series of treatments
the injections had to be repeated
because T cells live
for only 6-12 months in the blood
Both girls continued to receive ADA-PEG
so the actual benefit of the gene therapy was unclear
Radical approach:
make more room for transgenic T-cells
by suppressing host bone marrow
By non-myeloablative conditioning
“you don't really wipe out the bone marrow,
you just give one of the drugs used in for a transplant,
at a much lower dose,
to make 'space' for engineered T-cells to seize, expand and grow better,"
Two children in this study never got PEG-ADA
Results: improved immune functions
(including antigen-specific responses),
lower toxic metabolites.
Both patients are currently at home and clinically well,
Aiuti A et al., 2002 (Science)
with normal growth and development.
Umbilical cord blood
(gene therapy of stem cells)
Donald Kohn, a pediatrician,
diagnosed 3 children with ADA-SCID in utero.
Umbilical cord blood samples were collected
Shortly after infusion of the altered cord blood cells
about .01 to .10 percent of the T cells in these infants
were expressing the transgene.
As the PEG-ADA has been reduced,
the overall proportion of T cells  1-10% a 100-1,000-fold increase!
X-linked SCID (bubble
disease)
"bubble boy" disease, named after David Vetter, a Texan
who lived out his 12 years in a plastic, germ-free bubble.
More severe than ADA-SCID,
as X-SCIDs have no B-, T-, NK cells
Il-7 needed for T-cell proliferation;
T cell helps B-cell
David got a bone marrow transplant
from his sister; she was EBV positive.
David dies.
Gene therapy trial for X-linked SCID
successed in 2000;
8 of 10 patients significantly improved
and live normal life.
Photo: Courtesy of Duke Medical Center News Office
Results of X-SCID gene
therapy
3.5 years after stem cells GT
These X-SCID children (14 out of 15)
• are able to live normal lives at home instead of inside a
sterile "bubble";
• have normal numbers of T cells of both the CD4 and CD8
subsets;
• have responded to several childhood immunizations,
including diphtheria, tetanus and polio by producing both T
cells and antibodies specific for these agents.
• Antibody production is sufficiently good that they have no
need for periodic infusions of immunoglobulin (IG).
Leukemia in X-SCID treated
patients
In 2 of 15 cases therapeutic gene insert itself
near the LMO2 proto-oncogene
LMO2 = LIM domain transcription regulator
playing role in angiogenesis
Rearranged in T-ALL.
Transgenic mice with enforced expression of LMO2
in their thymocytes develop T cell leukemias…
One of them underwent gene therapy at the age of six months, and
contracted chicken pox at two-and-a-half.
Probable reason of stimulation….
The US Food and Drug Administration (FDA) halted 27 gene therapy
Lentiviral vectors
Lentiviruses are retroviruses
that can infect both dividing and nondividing cells
Preintegration complex of lentiviruses can get through
the intact membrane of the nucleus of the target cell.
Able to infect nondividing or terminally differentiated cells
such as neurons, macrophages, hematopoietic stem cells,
retinal photoreceptors, and muscle and liver cells
Example of lentiviruses:
HIV-1 (infects T-helper cells) – AIDS.
Good feature – no immune response!
Safety features for lentiviral vectors
replication competent lentiviruses could induce AIDS!
Even in the earliest studies HIV lentiviral vectors
produce no self-replicating particles
1) removing vpr gene from packaging plasmid.
This vector can not produce AIDS,
but also not able to infect macrophages
2) Self-inactivating lentiviral vectors
(deletions in LTRs made this virus not able to produce viral RNA,
but still able to integrate)
3) Use non-human lentiviral vectors
(feline immunodeficiency virus (FIV)
infects 2-20% domestic cats, produces AIDS-like disease
ADENOVIRUSES
non-enveloped viruses
containing a linear double stranded DNA genome
40 serotypes known;
most producing respiratory infections in humans
subgroup C serotypes 2 or 5 are predominantly used as vectors
can infect both
dividing and nondividing cells
12 antenna-like fiber projections
for virus attachment
www.nobel.se
Problems with adenoviral
vectors
1. Cannot integrate with the host cell genome
expression from adenoviral vectors is transient
(5-10 days) due to immunoclearance of the virus
Days posttreatment
in vivo hepatic gene delivery to hemophilia B dogs.
Transient nature of expression
in adenoviral vector
any therapy based on adenoviral gene transfer
would require long term application of the vector
increased risk of recombination,
especially if wild type infection occur simultaneously
severe inflammatory cellular and serological
immune responses possible
Adenovirus vector problems:
coxsackievirus-adenovirus receptor
(CAR)
CAR important
for cell-cell adhesions
Adenoviral receptors
Very common everywhere
Less common in the airway epithelium
and cancer cells
Topically administered Adenovirus
anyway will move to other tissues,
that produces distant toxic effects,
especially in the liver
(where virus is cleared)
Needs escalating doses
More toxicity
Safety features for adenovirural
GT
1. Should not able to propagate itself (E1A deletion)
2. Should be as non-immunogenic as possible
(get rid of most of the viral proteins)
3. Should be as non-recombinable as possible
(get rid of most of the viral proteins
that could be homologous to cells)
at high titres (>1011/ml) viruses are produced in special cell lines
with a helper virus (episomal or intergated in genome).
E1A integration in 293 cells
Adenovirural "gutless" vectors
"gutless" vectors which contain no viral coding sequences
The helper virus supplies the structural proteins
required for gutless vector replication and packaging (293 kidney)
Attachment via CAR,
internalization via integrins
Adenoviral particles
are disrupted in endosome
Cells that have less than normal
CAR expression
Mature skeletal muscle and smooth muscle (DM gene therapy) !!!
Endothelial cells (all cardio diseases)
Airway epithelium (cystic fibrosis) !!!
Lymphocytes
Dendritic cells
Fibroblasts
Hematopoietic cells !!!
Most cancer cells !!!
In the same time
other, non-target cells actively sequester the virus !!!!
Unwanted side effects again
How to manage tissue specificity
in adenoviral vector
1. to express the therapeutic gene under the control
of a tissue-specific promoter
(infect everything, express in the point).
2. stimulating the target cells to express an appropriate integrin
αVβ3 and αVβ5 are best integrins for this goal
Treatment with histone deacetylase inhibitor FR901228
increases expression of αVβ3 and leads to
at least a 10-fold increase in transgene expression
3. CAR important for cell-cell adhesions
When adhesion is broken, CAR is more available as receptor,
so AdV transfer to damaged tissue is more effective
Clinical gene therapy with adenoviruses
ornithine transcarbamylase (OTC) gene
for OTC deficiency (X-linked disorder)
OTC is a key urea cycle enzyme www.med.monash.edu.au/biochem/
(break down and removal of nitrogen
from the body)
OTC deficiency
hyperammonemia in the blood
Ammonia is neurotoxic
vomiting, refusal to eat meat,
and coma
OTC frequency:
incidence of 1:30,000 in the U.S
Severe form of disease in boys; mild in girls, often not detected.
in children and young adults
(encephalitis + liver failure after aspirin + viral infection)
Mortality 15 - 85% is caused by white matter edema
and demyelination
.
OTC-deficient sparse fur mouse as a model available.
Mice treatment with Ad-OTC vector was very successful.
Human trial for OTC deficiency
6 escalation doses; up to 1013 at the dose level 6
E-1, E-4-deleted third generation Ad-OTC vector
NIH's National Gene Vector Laboratories' facility in U. Penn
Jesse Gelsinger , 18 year old from Arizona
died after fast developing fever and organ failures
Probable source of problem:
1) Grade 3 toxicities in two patients at the 4th dose level
(level 6 should never be administered)
2) High level of ammonia in J.G.
3) Probable undetected parvoviral infection in J.G.
4) Recombination of adenovirus to wild-type
Conclusions form J.G. death:
1. Adenoviral vectors are better to use for killing cells
(as in case of cancer gene therapy) than to cure a disease
2. Dose escalation studies should be better controlled
3. Completely gutless vectors should be used
Good to remember: 90% of i.v. adenoviruses
go to the liver and produce liver toxicity
Liver have lots of CAR receptor. So, only way to solve this problem,
is to re-target adenoviruses away from CAR
Other adenoviral trials on their
way
Atherosclerosis: regional angiogenesis
Goal: improve perfusion of ischemic limbs
or heart by the induction
of collateral vessel formation
claudication
AdV with VEGF-121 in patients
with intermittent claudication of limb arterias
A single dose of Ad-VEGF will be administered
as 20 intramuscular injections
throughout the area of the lower limb
Walking impairment will be compared
in low-dose(109), high-dose (1010)and placebo groups
University of Michigan Health System
Adeno-associated virus (AAV)
Can be ideal as:
-- does not stimulate inflammation in the host
-- does not elicit antibodies against itself
-- can enter non-dividing cells
-- integrates successfully into one spot in the genome of its host
(on chromosome 19 in humans).
How to make expression tissue specific?
Binary system of AAV-based vectors
1 January 1999 issue of Science, James M. Wilson
Carry genes for the components of the transcription factors
needed to turn the target gene on.
Chimeric gene that encode p65 (transcativator, not able to bind DNA)
+ FRB that binds the drug rapamycin.
Chimeric gene that encode ZFHD1 (binds specifically EPO promoter)
but that by itself cannot activate transcription of the gene;
+ FKBP12 that also binds to rapamycin.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/
Vector 2
Target gene encodes EPO – erythropoietin –
that stimulate production of red blood cells
-- Treat severe anemia, e.g. after chemotherapy;
-- Instead of blood transfusions for Jehovah's Witnesses
-- Protect neurons during the stroke
-- Doping in sport (cyclists)
To enchance EPO expression
Experimental animals injected with both vectors into skeletal muscles
Than injected with rapamycin (clinical immunosuppressant)
If both vectors are in the same cell  EPO transcription is activated
RESULTS of experiment:
In mice:
Two vectors without rapamycin – harmless and no influence on Hb level
After injection:
Fast production of EPO (200 times induction)
Hematocrits (number of red blood cells) increase from 42% to 60%
Stable effect – still working 5 month after vector injection
So, with this system we can
deliver therapeutic construct once
– but have a prolonged effect
Curing Insulin-Dependent Diabetes
Mellitus (IDDM)
in mice and rats
Mice with inherited diabetes
Rats after chemical destruction of their insulin-secreting beta cells
AAV
Glucose -sensitive
promoter
enhancer
Intronless insulin gene
Signal sequence for secretion
Constructs injected into hepatic portal vein
Animals gained control over their blood sugar level
and kept this control for over 8 months.
Bleeding Disorders
A deficiency of a clotting factor can lead to
uncontrolled bleeding.
-- not enough of the factor OR
-- mutant version of the factor
•von Willebrand disease
(the most common)
•hemophilia A
for factor 8 deficiency
•hemophilia B
for factor 9 deficiency.
•hemophilia C
for factor 11 deficiency
Part of the
clotting
cascade
Hemophilia A and B
The genes encoding factors 8 and 9 are on the X chromosome.
Thus their inheritance is X-linked (males sick).
Treatments:
1) Extraction of a factors 8 and 9 from donated blood
(>1000 donors), than purification.
Injections of this material stops bleeding in hemophiliacs.
Drawback: AIDS, hepatitis C.
90% of hemofiliacs in 90s were HIV+
2) recombinant factor 8 and recombinant factor 9
made by genetic engineering are now available
Produced in mammalian cultures (very expensive; low yeild).
Production in E.coli is not good as glycosylation needed
Hemophilia treatments:
3) Transgenic animals.
female sheep transgenic for the human factor 9 gene.
The human gene is coupled to the promoter
for the ovine milk protein beta-lactoglobulin.
4) Liver transplants
5) Gene therapy
Avigen, Inc
Curing Hemophilia B in
mice
Mice were hemophiliacs due to knockout of gene for clotting factor IX
AAV
Liver specific
promoter
Intronless factor IX gene
The rats proceeded to make factor IX and
were no longer susceptible to uncontrolled bleeding.
HUMAN TRIAL: modest improvement after injection with
their own cells transformed by factor 8 ex vivo.
Number of needed injection lesser
a defective adeno-associated virus (AAV) (Avigen, Inc)
CANCER GENE THERAPY
Suicide GT
And other experimetal cancer
therapies
(113 trials currently open in US
in immunotherapy of cancer)
54% of immunotherapy trials
dedicated to melanoma
Delivery of the tumour-suppressor gene
TP53 accounts for the next largest group
Genetic prodrug activation therapy
(GPAT) tumor-specific promotor + drug activating gene
Major flaw of the current chemotherapy: lack of selectivity.
If drug-activating genes could be inserted and expressed only in cancer cells,
then treatment with an appropriate prodrug could be highly selective.
Normal breast cells
do not possess factors
that lead to
overexpression of ERBB2.
Cytosine Deaminase gene
under ERBB2 promotor.
Tumor-specific
Suicide
Active only in tumor cells.
It allows activation
of the harmless 5-FC prodrug
to the cytotoxic 5-FU
and consequent cell death.
http://www.sghms.ac.uk/depts/ogem
Ganciclovir conversion by HSV-TK
Examples of suicide schemes
Suicide gene
Prodrug
Active drug
Viral thymidine kinase
Ganciclovir
Ganciclovir triphosphate
Cytosine deaminase
5-fluorocytosine
5-fluorouracil
Linamarase
Amygdalin
cyanide
nitroreductase
CB 1954
nitrobenzamidine
Linamarase = beta-glucosidase, to convert the cyanogenic glucoside substrate,
into
glucose and cyanide.
From cassava
Examples linamarin,
of suicide
gene/prodrug
combinations
and the active
cytotoxic drug
selectively
produced
in the
target
cell. membranes.
Production of the
cyanide ion
that diffuses
freely
across
In culture 10% lis-positive glioma cells are sufficient
to eliminate the entire glioma cell culture in 96 h.
Two targeting strategies of
suicide gene
1.
Transcriptional targeting
regulatory sequences of genes overexpressed in cancer cells
(promotor) + suicide gene
e.g. ERBB2 promoter in breast cancer or
tyrosinase promoter in melanoma.
2.
Transduction targeting
relies on preferential delivery of vectors constitutively expressing
a suicide gene into actively dividing cells only.
e.g. glioma cells vs normal neighbouring central nervous system cells.
Like chemotherapy but may be topical (theoretically)