GENE THERAPY

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)

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