Gene therapy: Transfer of therapeutic gene into the
diseased tissueHemophiliac 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 Target cell
Severe combined Adenosine deaminase 4 Bone marrow cells or
immunodeficiency T-lymphocytes
Hemophilia 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 Hepatocyte growth factor
Autoimmune disease Lupus, diabetes MHC, 2-microglobulin
In humans
Cancer 69%
General concernsThe Food and Drug Administration (FDA) has not yet approved
any human gene therapy product for sale.
Four major problems with gene therapy:
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.
1) Short-lived nature of gene therapy. Very hard to achieve any long-term benefits without integration and even with it.
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
http://laxmi.nuc.ucla.edu:8237/M288/SChow_4_10/sld005.htm
IN VIVO
EX VIVO
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
In 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
Ex vivo gene therapy
Select normal cells that are GT resistant to chemotherapySelect hematopoietic cells resistant to paclitaxel (taxol)
after introducing an MDR1 pump
Resistant to alkylating agents after introducing an
O6-alkylguanine-DNA-alkyltransferase
Resistant to methothrexateafter introducing a mutant DHFR enzyme
Blood vessel
Transgenes
Integrated Not integrated
- stable expression;may provide a cure
- expression is transient;repeated treatments
nesessary
- random insertions in heterochomatin can be inactivated;
In euchromatin -- Can disrupt important host genes; Long-term consequences are unknown
for episomes (plasmids) random mutagenesis
not an issue
How episomes and integrated trasgenes behave in dividing cells
Integral transgene Episome
Loss of plasmid
Influences on choice of vector
high efficiency viral vectors for gene replacement
short term gene expression To prime an immune response
To sensitise cells to radiotherapy
…Liposomal Delivery…
therapy of monogenic diseases
(cystic fibrosis; SCID; hemophilia…)
Desirable characteristics of gene delivery vector
1. High titer or concentrations (>108 particles/ml)
3. Precise and stable introduction of transgene
2. Easy and reproducible method of production
4. Vector should not elicit immune response in the host
6. Vector should be able to target specific cell types
5. Transgene should be responsible for its regulatory elements (on/off system)
Methods of gene delivery (therapeutic constructs)
-- Injection of naked DNA into tumor by simple needle and syringe
-- DNA transfer by liposomes (delivered by the intravascular, intratracheal,
intraperitoneal or intracolonic routes)
-- DNA coated on the surface of gold pellets which are air-propelled into the epidermis
(gene-gun), mainly non applicable to cancer
-- 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.
Injections of naked DNA
Naked DNA gene therapy
covalently closed circular form is more stable that open plasmid
Intravascular delivery liver and muscle
Intramuscular delivery
-- Results in a prolonged low level expression in vivo
-- Very cheap
-- DNA vaccines based on naked DNA are unaffected by pre-existing immunity e.g.
due to maternal antibodies
DNA vaccines
Cancer immunotherapyAntiviral and antibacterial
(traditional vaccines are better when available)
Passive to increase
the pre-existing immune response
to the cancer
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 CInfluenzaPapillomaCytomegalovirus
Tuberculosis,
Lyme disease
Helicobacter pylori
Malaria
T cells recognise liver cell with malarial parasite inside
www.malaria-vaccines.org.uk
Produce IFN-gamma
IFN-gamma stimulate antigen presentation
Important: too much IFN-gamma is also too bad.
(it is pro-inflammatory)
DNA vaccines (and other vaccines too)
prime immune system with properly presented antigen
several peptide epitopes that we know are recognised by T-cells
(a so-called multi-epitope string)
immunologically important components of the malaria pathogen
whole protein called thrombospondin related
adhesion protein (TRAP).
DNA vaccine encoding an immuno
recognisable insert
Ballistic DNA Injection, particle bombardment,
microprojectile gene transfer (gene guns)
Invented for DNA transfer to plant cells
Fully applicable to mamalian cells
Light micrograph of DNA-coated gold beads in the skin after gene-gun vaccination
plasmid DNA is precipitated onto 1-3 micron sized gold or tungsten particles.
Discharge: helium pressure, or high-voltage electronic
Duchenne muscular dystrophy (DMD)
1. Generalized weakness and muscle wasting affecting limb and trunk muscles first. Calves often enlarged. Wheels at 12 y.o.
X-linked recessive disorder; 1/3500 boys worldwide
About 30% of cases represent new mutations.
Life threatening dysrhythmia or heart failure develops in about 10 %.
Absence of dystrophin, a cell membrane protein (approximately 0.01 % of skeletal muscle protein).
All muscles involved
Death ay 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
Duchenne and Becker MDs
Sarcoglicans: Limb Girdle MDs (4 types)
Laminin2α2: congenital MD chr 6
Whole complex stabilizes the membrane.
DM is good model disease as ballistic GT is available for
musclesProblem:
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), ambulatory before age 61
Deletion variants of dystrophin for GT
ABD= actin-binding domains
Scott Harper et al., 2002
most, but not all, of the spectrin-like repeats are dispensable for the function of dystrophin.
GT with dystrophin minigene in mice with DM phenotype
GT treated Non-treated
MDX mice with premature stop codon in exon 23; no dystrophin
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 200 mkg of plasmid with dystrophin
a single injection of 600 mkg of plasmid with dystrophin
Two injections of 600 mkg each of plasmid with dystrophin
Muscle segment were taken out for examination
Expression of dystrophin is found in 1 to 10 percent of muscle fibers
for group 1 and 2 ; for all 3 patient in group 3
No immune reactions; no side effects !!!!
Phase 1 trial for safety
LiposomesNext level idea – why naked DNA?
Lets’ wrap it in something safe to increase transfection rate
Therapeutic drugs
Lipids – is an obvious idea !
Liposomes are formed by the self-assembly of
phospholipid molecules in an aqueous environment.
www.emc.maricopa.edu/faculty/ farabee/BIOBK/
Anionic liposome
Cationic liposomes
positively charged lipid dropletscan interact with negatively charged DNA
to wrap it up and deliver to cells
Positively charged lipid heads
Lipofectin, lipofectamine, lipofectase….
Inside liposomes DNA is resistant to degradation
Lab procedure for liposome preparation
Liposome disadvantages
Liposomes are rapidly cleared from the circulation and largely taken up by the liver macrophages
liposome surface ligands decrease degradation(monosialoganglioside or polyoxyethyle)
How to overcome it?
Modified liposomes (stealth liposomes)
hydrophilic polyoxyethylene lipids incorporated into liposome
Increased half-life is be due to a reduced coating (opsonisation) of these liposomes by plasma proteins
So liver cells not able to uptake them
cholesterol, polyvinyl-pyrrolidone polyacrylamide lipids,glucoronic acid lipids are working the same….
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 tractCochleates 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)
www.pharmj.com/Editorial/19990828/ education/parenteral.html
Liposomes better penetrate into tissues with disrupted endothelial lining
DNA 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 fibrosismost 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)
Lungs create thick mucus secretion(prone to infections,
constant cough, leading cause of death)
Digestive tract (constipation)
Lungs in cystic fibrosisNormal lung CF lungs
dilated crypts filled with mucus and bacteria.
Normal alveolar appearance
CF lungs filled with mucus lung did not collapse when it was removed postmortem
Cystic fibrosis lungs are prone to infections
The battle between neutrophils and bacteria leads ultimately to lung fibrosis and damage
Pseudomonas aeruginosa easily colonise mucus in the dilated lungs
Neutrofiles are activated, then overactivated
"Hyperinflammation" as recruited neutrophils unable to eradicate bugs, instead damage lung tissue.
Mucus protects bugs and promotes hypermutation
Pancreas in cystic fibrosis
Normal pancreas Distended CF cripts filled with mucus
Impaired glucose tolerance and diabetes
Survivors to 25 years old: 1/3 with impaired glucose tolerance and with 1/3 diabetes
Pancreatic enzymes not able to leave the gland; they damage the gland
Less insuline prodiced
Treatment of CF
Staphylococcus aureus, Haemophilius influenzae, Aspergillus fumigatus - the same picture…
1. Many different antibiotics are required to clear infections.
CFTR gene (chromosome 7)27 exons, 1480 aa
ATP binding domain
CFTR productDelta 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 Goldgi complex
Cystic fibrosis Gene TherapyJanuary 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 reasctions.
Non CF CF
CF:Treated CF:Treated
Monthly applications
also work,2000
Multiple Dose: Gene Transfer Assays
Gene Transfer Detected After Each Dose
MD 1 56% (5/9) DNA 60% (15/25)
MD 2 66% (6/9) mRNA 44% (11/25)
MD 3 66% (6/9) Protein 28% (7/25)
Total 63% (17/27) Function 22% (6/27)
Good Correlation Between Gene Transfer Assays
mRNA Positives All +ve For DNA
Functional Positives All +ve For DNA, mRNA and Protein
Cationic-lipid-mediated CFTR gene transfer can significantly influence the underlying chloride defect
in the lungs of patients with cystic fibrosis.
MOST COMMON VIRAL VECTORS
Retroviruses
Adenoviruses
Adeno-associated viruses
Herpes simplex viruses
can create double-stranded DNA copies of their RNA genomes. Can integrate into genome. HIV, MoMuLV, v-src, Rous sarcoma virus
dsDNA viruses that cause respiratory, intestinal, and eye infections in humans. Virus for common cold
ssDNA viruses that can insert their genetic material at a specific site on chromosome 19
dsDNA viruses that infect a neurons. Cold sores virus
Retroviral vectors are able to infect dividing cells only
Good for cancer gene therapy
Nevertheless, retroviruses are most often used vectors for common disease gene therapy
Every therapeutic construct should include safety features
In dividing cells nuclear membranes are broken down, so viral genome can enter and integrate into the chromosome
Preintegration complex of retroviruses non able to penetrate nuclear membrane.
Infection of dividing cells only
Amphotropic retroviruses
capable of infecting both mouse cells and human cells
Moloney murine leukaemia virus (Mo-MLV),
2. All regions of homology with the packaging virus should be removed to prevent recombination resulting in
replication competent retroviruses
Treatment could be tested in mouse
Safety features:
1. Propagation only in packaging cells
….Anyway, 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.
(With ironical exception of hematopoietic stem cells)
Many approaches invented to improve and target the viral delivery
Modify env gene by creation a pseudotyped vector
Vesicular stomatitis virus (VSV):
phospholipid component of membrane as a receptor
(Rhabdoviridae)
VSV G protein
www.urmc.rochester.edu/smd/
hybrid virion with “mixed” envelope
1) Host range now determined by both envelopes
pseudotyped virus has the ability to withstand the shearing forces encountered during ultracentrifugation
Retro-VSV hybryds are able to infect even Fish, Xenopus, Mosquito, Butterflyes….
2. VSV envelope is very durable
high-titer retroviral vector stocks could be generated
()
What we gain :
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.
3. G protein of VSV is toxic for cell pseudotypes could be produced only by already dying packaging cells (overcome by inducible promotors)
2. G protein of VSV is very immunogenic (so, it’s one-time approach)
Other pseudotypes are available:
HFV – human foamy virus, HIV-1, LCMV (lymphocytic chiriomeningitis) – non toxic for cells
Modify env gene for ligand directed targeting
Drug resistance gene transfer exclusively to hematopoietic cells
Suicide gene transfer exclusively to cancer cells
1. Remove unwanted side effects,
of non-specific transfer
2. Specificity increase efficiency
In Ex vivo approaches
1. Colocalisation of cells and viruses on a specific matrix
Best matrix is retronectin (derivate of fibronectin)
Fibronectin contains specific adhesion domains for stem and progenitor cells and retroviral vectors
Local titer of viral particles increases
Takara Inc.
Equipping retroviral particles with cell-specific ligands
1. Addition of part of the ligand to create env interaction with cell specific receptor
50 aa from EPO added to envmakes it interacting with EPO-receptor
on EPO receptor bearing cells
Specific binding is easy to achieve; but virus uptake become less efficient.
Other additions: heregulin. Binds to HER-2 and HER-4 receptors overexpressed on breast cancer cells
Complete substitution of env surface subunit (SU)by cell specific ligand
Problem: conformational changes in Env are strong,
so resulting chimera not able to effeclively trigger internalisation
Single-chain antibodies as a ligands are especially perspective
Linker cleavable by protease
Binary systems (ligand for binding;
env for internalisation)
After cleavage, local titer of virus is high
Drawback: systemic applications of protease is no good.
1. Made this sequence cleavable for internal human protease
2. Made the linker flexible (Proline – rich), to move away without cleavage
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 month on antibiotics with little effect
> 2 pneumonias per year
-- failure to gain weight and grow
-- recurrent deep skin and organ abscesses
lymphopenia (absolute lymphocyte count less than 200)
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.
ADA is essential for the proper growth and function of infection-fighting T and B lymphocytes.
Adenosine + H2O = Inosine + NH3
Adenosine is toxic for B- and T-cells
SCID treatments
An excess of adenosine deaminase leads to hemolytic anemia
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
GENE THERAPY
ADA gene therapy story
Three separate laboratories published the gene sequence in 1983
ADA protein has been characterized in the late 1970s
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
September 14, 1990. Mature T-cells GTAshanti DeSilva; advanced stage of SCID; 4 yr old;
Cynthia Cutshall January 31, 1991
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
Aiuti A et al., 2002 (Science)
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 marrow 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, 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.
early 1993
As the PEG-ADA has been reduced, the overall proportion of T cells 1-10% -
a 100-1,000-fold increase!
Shortly after infusion of the altered cord blood cellsabout .01 to .10 percent of the T cells in these infants
were expressing the transgene.
Umbilical cord blood samples were collected
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.
Photo: Courtesy of Duke Medical Center News Office
Gene therapy trial for X-linked SCID successed in 2000;
8 of 10 patients significantly improved and live normal life.
More severe than ADA-SCID, as X-SCIDs have no B-, T-, NK cells
David got a bone marrow transplant from his sister; she was EBV positive.
David dies.
Il-7 needed for T-cell proliferation; T cell helps B-cell
Results of X-SCID gene therapy
• 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).
3,5 years after stem cells GT This X-SCID children (14 out of 15)
Alain Fischer at Necker Hospital, Paris
Leukemia in X-SCID treated patients
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….
In 2 of 15 cases therapeutic gene insert itself near the LMO2 proto-oncogene
The US Food and Drug Administration (FDA) halted 27 gene therapy
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…
Lentiviral vectorsLentiviruses 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 vectorsreplication competent lentiviruses could induce AIDS!
Even in the earliest studies HIV lentiviral vectors produce no self-replicating particles
ANYWAY
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
equine infectious anemia virus
ADENOVIRUSESnon-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
in vivo hepatic gene delivery to hemophilia B dogs.
Days posttreatment
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 is very promiscousMHC class I molecule
coxsackievirus-adenovirus receptor (CAR)
Adenoviral receptors
Very common everywhere
Less common in the airway epithelium and cancer cells
Topically administered Adenovirusanyway will move to other tissues, that produces distant toxic effects,
especially in the liver (where virus is cleared)
Needs escalating doses
More toxicity
CAR important for cell-cell adhesions
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 wt)
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
How to suppress immunoclearance of adenoviral vector
1. transient immunosuppressive therapies to patient
2. induce oral tolerance by feeding the host UV inactivated vector
Better to manipulate with vector, not with the host
"gutless" vectors which contain no viral coding sequences
The helper virus supplies the structural proteins required for gutless vector replication and packaging (293 kidney)
In preparateshelper virus represents less than 0,5% of particles,
but they are immunogenic anyway
Helps to overcome a liver toxicity
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 Fibroblasts Hematopoietic cells !!!
Dendritic 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
2. stimulating the target cells to express an appropriate integrin
1. to express the therapeutic gene under the control of a tissue-specific promoter
(infect everything, express in the point).
αVβ3 and αVβ5 are best integrins for this goal
3. CAR important for cell-cell adhesionsWhen adhesion is broken, CAR is more available as receptor,
so AdV transfer to damaged tissue is more effective
Treatment with histone deacetylase inhibitor FR901228 increases expression of αVβ3 and leads to
at least a 10-fold increase in transgene expression
Comparison of targeting startegies
Population of chemical conjugates is always heterogenous,
so clinical certification is difficult
Exogenous recombinant genes (anti-knob scFv + anti-receptor scFv)
are homogenous(CAR as anti-knob part could be used)
Changes in the adenoviral knob itself
Clinical gene therapy with adenoviruses
ornithine transcarbamylase (OTC) gene for OTC deficiency (X-linked disorder)
OTC is a key urea cycle enzyme (break down and removal of nitrogen
from the body)
hyperammonemia in the blood
OTC deficiency
vomiting, refusal to eat meat, progressive lethargy, and coma
Ammonia is neurotoxic
www.med.monash.edu.au/biochem/
OTC deficiency: treatment options
1. restriction of dietary protein
2. L-citrullin (to provide substrate for arginine synthesis)
3. During viral infections (when body produce more ammonia)protein intake should be stopped,
and glucose is given either by mouth or intravenously.
incidence of 1:30,000 in the U.S
OTC frequency:
Severe form of disease in boys; mild in girls, often indetected.Reye syndrom in children and young adults
(encephalitis + liver failure after aspirin + viral infection)
Mortality 15 - 85% is caused by white matter edema and demyelination
Even with this treatment, mortality rates in these children are about 50%.
Jesse Gelsinger , an 18 year old from Arizonadied after fast developing fever and organ failures
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 i.h. ; 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 UPenn
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.
Probable source of problem
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
AdV with VEGF-121 in patients with intermittent claudication of limb arterias
Claudication
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)
-- 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).
Can be ideal as:
How to make expression tissue specific?
Binary system of AAV-based vectors1 January 1999 issue of Science, James M. Wilson
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/
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.
Vector 2
Target gene encodes EPO – erythropoietin – that stimulate production of red blood cells
-- Treat severe anemia, e.g. after chemotherapy;
-- Doping in sport (cyclists)
-- Instead of blood transfusions for Jehovah's Witnesses
-- Protect neurons during the stroke
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:
Two vectors without rapamycin – harmless and no influence on Hb level
In mice:
Hematocrits (number of red blood cells) increase from 42% to 60%
After injection:
Fast production of EPO (200 times induction)
Stable effect – still working 5 month after vector injection
So, with this system we can deliver therapeutic construct once
– but have a prolonged effect
Mice with inherited diabetesRats after chemical destruction of their insulin-secreting beta cells
Intronless insulin geneGlucose -sensitive
promoterAAV
Signal sequence for secretionenhancer
Animals gained control over their blood sugar level and kept this control for over 8 months.
Constructs injected into hepatic portal vein
Curing Insulin-Dependent Diabetes Mellitus (IDDM) in mice and rats
Bleeding Disorders
•von Willebrand disease (the most common) •hemophilia A for factor 8 deficiency •hemophilia B for factor 9 deficiency. •hemophilia C for factor 11 deficiency
A deficiency of a clotting factor can lead to uncontrolled bleeding.
-- not enough of the factor OR -- mutant version of the factor
Part of the clotting cascade
Hemophilia A and BThe genes encoding factors 8 and 9 are on the X chromosome.
Thus their inheritance is X-linked (males sick).
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
Treatments:
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
Intronless factor IX geneLiver specific
promoterAAV
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
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
Suicide GT
Genetic prodrug activation therapy (GPAT) tumor-specific promotor + drug activating gene
http://www.sghms.ac.uk/depts/ogem
Normal breast cells do not possess factors that lead to overexpression of ERBB2.
Cytosine Deaminase gene under ERBB2 promotor.
Active only in tumor cells.It allows activation of the harmless 5-FC prodrug to the cytotoxic 5-FU and consequent cell death.
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.
Tumor-specific Suicide
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
Examples of suicide gene/prodrug combinations and the active cytotoxic drug
selectively produced in the target cell.
Linamarase = beta-glucosidase, to convert the cyanogenic glucoside substrate, linamarin, into glucose and cyanide. From cassava
Production of the cyanide ion that diffuses freely across membranes. 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
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.
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)
1.
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