at the University of Pécs and at the University of Debrecen Identification number

(1)

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(2)

IN VIVO AND EX VIVO GENE THERAPY

Zoltan Balajthy

Molecular Therapies- Lecture 4

in the Teaching Material of

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(3)

Learning objectives of chapter 4. We are going to learn several methods by which we can introduce genetic material into a cell to treat disease, and in this manner, the aim of gene therapy is to introduce therapeutic material into the target cells, for this to become active inside the patient and exert the intended therapeutic effect.

Topics in chapter 4 4.1. Gene therapy

Main criterias of the effective gene therapy In vivo gene therapy

Ex vivo gene therapy

4.2. Types of gene transfer, vectors for gene therapy Liposomes, naked DNA

Retrovirus vector Adenovirus vector

Adeno-associated viral vector (AAV)

4.3. General gene therapy strategies Targeted killing of specific cells

Targeted inhibition of gene expression Targeted mutation correction

4.4. 3.4. Human gene therapy

Severe combined immunodeficiency (SCID) Genetic defects in the LDL receptor

Cystic fibrosis (CF)

(4)

4.1. Gene Therapy

Gene therapy may be used for treating, or even curing, genetic and acquired diseases by using normal genes to supplement or replace defective genes or

to bolster a normal function.

• Somatic : gene is introduced into specific somatic cells;

not heritable

• Germline : gene is introduced into emryonic cells or fertilized egg; heritable (ethical, legal and religious

questions in human use)

(5)

Main criterias of the effective gene therapy

Well-designed and manufactured gene

The gene introduction into the right cells

Safe integration of the gene into the chromosome without disturbing the surrounding genes

The control of the gene, so the protein is produced only when it is needed

(6)

Somatic gene therapy targets

Cancer: main target of the current clinical trials

Muscle: easily accessible, has a good blood supply and abundant tissue

Endothelium: can directly secrete the therapeutic protein into the bloodstream

Skin: skin grafts also can secrete the necessary proteins Liver: has many functions and great regeneration

Lung: easily accessible with aerosol sprays

Nervous tissues: many illnesses and injuries can affect it,

not easy to modify the neurons

(7)

In vivo gene therapy

Recombinant virus

Plasmid DNA DNA liposome

(8)

Ex vivo gene therapy

Therapeutic gene

Therapeutic gene is inserted into a specially engineered

virus Target cells are removed from the patient and grown in a large number in tissue culture plates

Cultured cells are

mixed with the virus The cells are returned to the patient to replace the function lost due to the

inheritance of mutant gene

(9)

Properties of the ideal gene therapy vector

Safe (no side effects) Immunologically inert

Can be targeted to a specific cell type or tissue Can be used to deliver any gene whatever its size Easy large scale production

Cost effective

The ideal vector does not exist (yet)!

(10)

4.2. Types of gene transfer

Non-viral gene transfer Liposomes

Naked DNA Viral gene transfer Retroviruses Adenoviruses

Other viruses (Herpes simplex virus etc.)

(11)

Liposomes

hidrophyli c head

hidrophobic tail

phospholipid phospholipid

bilayer

Liposome

+ H₂O

Advantages: non-pathogenic, no immunity problems, no gene size limit Disadvantages: low transfection efficiency, low rate of stable integration

(12)

Naked DNA

Advantages: simplest

Disadvantages: very low transfection effectivity

Plasmids, PCR products

(13)

Advantages: same as liposome-mediated transfer, promising as a vaccination method

Disadvantages: limited to dermal tissue, low rate of stable integration, difficult to QC

Gene particle bombardment

(bioballistic delivery system)

(14)

Main criteria of the viral vectors

Well-designed and manufactured gene

The gene introduction into the right cells

Safe integration of the gene into the chromosome without disturbing the surrounding genes

The control of the gene, so the protein is produced only when it is needed

(15)

Retroviruses

surface glycoprotein transmembrane protein

integrase

reverse transcriptase protease

matrix capsid

nucleocapsid RNA genome

Env

Pol

Gag

(16)

-(A )n

-(A )n -(A )n

R T

IN

P R

(1 ) (3 )

(4 )

(5 ) (6 )

(8 ) (7 ) (9 )

(1 0 )

ea rly p h a se la te p h a se

(2 )

Life cycle of retroviruses

1. Attachment

2. Penetration and uncoating 3. Reverse Transcription

4. Transport of PIC to the nucleus 5. Integration

6. Transcription 7. Translation 8. Assembly 9. Budding 10. Maturation Gene therapy constructs

maintained at this stage

(17)

LTR gag pol env LTR reverse transcription

integration

integrated provirus transcription infection

infected cell

replication-competent retrovirus

virus assembly

ψ

translation

Moloney Murine Leukemia Virus Based Retroviral Vector I.

pol

ψ gag

LTR env LTR 3’

LTR THERAPEUTIC GENE LTR

A B C

ψ

retroviral genome

(18)

transfectio n

retroviral vector infectious, but replication incompetent

LTR ψ LTR

gag pol env

gag/pol protein s

envelop proteins

THERAPEUTIC GENE

LTR ψ LTR

packaging cell line

proviral therapeutic plasmid

Moloney Murine Leukemia Virus Based Retroviral Vector II.

(19)

MoMLV-based retroviral vector

(A) The retroviral genome contains the genes gag (structural proteins), pol (reverse polymerase), and env (envelope proteins). Ψ the packaging signal distinguishing cellular RNA from viral packaging proteins. The viral genome 5 flanked by long terminal repeats (LTR).

(B) Gag, pol, and env in the vector genome have been replaced by a therapeutic gene.

(C) Gag, pol, env are expressed by separate genes that are transfected into the packaging cell. lf the viral vector construct is cotransfected with the transgene into the packaging cell, the protein products of the vector genome recombine with gag/pol to form infectious viruses that cannot replicate.

(20)

Retroviral gene therapy

unhealthy cells

RNA

healthy gene

healthy cells

(21)

Advantages:

• stable and long term expression of transgene

• Very effective gene delivery for dividing cells (e.g. tumors)

• easy production

Disadvantages:

• maximum gene size 7-8kb

• difficult to purify so only used for ex-vivo methods

• can not be used for differentiated and non-dividing cells *

• complement cascade can inactivate it

• random integration may led to oncogenic activation

Properties of retroviral

gene therapy vectors

(22)

CMV/LTR ψ LTR 3’

5’ ^RRE ^cPPT ^CMV THERAP. GENE WPRE

pol gag

LTR ψ vif LTR

vpr

env tat

nef

rev

5’ 3’

pol

CMV gag _RRE polyA

RSV rev polyA

RSV VSV-G polyA

packaging cell lentiviral vector

packaging constructs

Lentiviral vector

A

B

C

wild type HIV

(23)

Lentiviral vector

(A) Schematic representation of the wild type HIV provirus. The HIV genome codes not only for gag, pol, and env, but also for proteins such as tat, rev, nef, vif, vpu and vpr. None of those, apart from rev, and tat, is needed for the in vitro propagation of the virus.

(B) The latest generation of SIN lentiviral contains a central polypurine tract (cPPT) to support the translocation of the vector into the nucleus. An additional WPRE sequence enhances the expression of the transgene. Nearly all viral elements have been deleted, apart from the LTRs (with a SIN deletion in the 3’-LTR, see arrow), RRE (essential for the nuclear export of viral RNA), and W which is needed for packaging.

(C) Gag, pol, tat (transactivates the HIV-LTR-promoter), while rev, enhances the export of unspliced genomic RNA from the nucleus after binding to RRE, and the envelope protein VSV-C are expressed by separate genes that are cotransfected into the packaging cell.

(24)

Adenoviruses

(25)

Evolution of adenoviral vectors

VA

IVa2

L1 L2 L3 L4

E3 L5

E4 ITR E2

E1A,B

ITR ψ

Ad5 genome

VA

IVa2

L1 L2 L3 L4 L5

E4 ITR E2

T. gén

ITR ψ

First generation vector; removed E1/E3

VA

IVa2

L1 L2 L3 L4

E4 ITR E2

T. gén

ITR ψ

Second generation vector; removed E1/E3/L5

CMV

E

4 ITR

Third generation vector; most of the genes are removed, helper-dependent

T. gén

ITR ψ ^CMV

(26)

Adenoviral vectors

(A) Schematic representation of a serotype 5 adenovirus (Ad5) on which most of the adenoviral vectors described here are based. The vector genome is flanked by inverted terminal repeats (ITRs). Ψ is the packaging signal. The adenoviral genes are highlighted in boxes.

(B) First-generation adenoviral vector in which the genes E1 and E3 have been deleted. The E1A plays a decisive part in viral replication as the initiator of the transcription of other viral transcription units. However, the gene is not needed for adenoviral replication within 293 cells, which makes those cells ideal for virus production. The E3 gene product is not essential for viral reproduction, although its role in immune modulation and suppression is important. The therapeutic gene is simultaneously transfected into the packaging cell, using a shuttle vector. It is than inserted into the adenoviral vector in exchange for the E1 gene.

(C) Helper-dependent adenoviral vectors in which parts of the adenoviral genome (flanked by loxP recognition sites, triangles) have been excised in order to avoid immune reactions in the host.

The expression of the therapeutic gene is driven by a promoter such as CMV.

(D) In order to avoid potentially violent immune reactions of the host to adenoviral proteins, mini or gutless adenoviral vectors have been produced in which most of the adenoviral genes have been deleted.

(27)

Adenoviral gene therapy

DNA genom

therapeutic protein

therapeutic gene

(28)

Advanteges:

• no risk of oncogenic activation (no DNA integration)

• can carry large genes (30 kb)

• infect dividing and non-dividing cells

• high level gene expression

• easy production

Dissadvantages:

• short term gene expression

• induce inflammatory and immune response

• Cell-specific targeting difficult to achieve

Properties of adenoviral

gene therapy vectors

(29)

Adeno-associated viruses

ITR

Adeno-associated viral genome

ITR rep cap _ITR

Recombinant vector genome Theraupetic gene

ITR

vector genomes

Rep/Cap construct

viral vector construct

Packaging cell

Packaging and replication proteins

Adeno-associated viruses

A B

C

(30)

Adeno-associated viral vector

(A) The AAV genome contains sequences which are essential for the transduction process, such as inverted terminal repetitions (ITRs) and the genes rep and cap.

(B) In the vector genome, rep and cap have been replaced by a therapeutic gene.

lf the therapeutic gene is larger than 4.5 kb, it is distributed over two concatemeric vector constructs.

(C) The REP and CAP proteins are expressed by the packaging cells and are needed for the production of single-stranded DNA genomes in a capsule consisting of proteins. A non-enveloped AAV virus collects in the nucleus. Helper proteins from adenoviruses, which are needed for replication, are also expressed in the packaging cell (not shown here). The AAV are released from the packaging cell through the lytic adenoviral replication process.

(31)

Advantages:

• not associated to human diseases

• effectively infects dividing and non-dividing cells

• able to integrate into the specific site (chromosome 19)

• small genom and easy to manipulate

• can obtain high titered virus stock (10

⁹

-10

¹⁰

/ml)

Dissadvantages:

• limited gene size ~ 4.5kb

Properties of adeno-associated

viral vectors

(32)

3.3 Gene therapy strategies I.

Gene augmentation

disease cells X gene

normal phenotype

disease cells toxin gene

disease cells prodrug gene

drug

cells killed by toxin

cells killed by drug

Direct cell killing

(33)

Gene therapy strategies II.

Indirect cell killing by immunostimulation

disease cells foreign

antigene gene

disease cells cytokine

gene

immun cells

killing of disease cells because

of enhanced immune response

(34)

Gene therapy strategies III.

Targeted inhibition of gene expression

disease cells with mutant or harmful gene antisense gene

antisense TFO, ODN

or

m

AAAA

N C block expression of pathogenic gene

Targeted gene mutation correction

disease cells with mutant gene x x gene

m

X X

corrected gene

normal phenotype

(35)

4.4. Gene therapy targets

cancer diseases cardiovascular

diseases

monogenic diseases

infectious diseases

other

(36)

Targeting of different organs by viral vectors

Adenoviruses

(tumors, hematopoietic cells)

Retroviruses

(tumors, stem cells, hematopoetic cells)

Herpes simplex virus

(CNS, hematopoietic cells, muscle, stem cells)

AAV

(liver, muscle, retina)

Alphaviruses

(tumors)

Lentiviruses

(CNS, liver, muscle)

Hematopoietic cells

stem cells

(37)

Severe combined immunodeficiency (SCID):

Lack of adenosine deaminase (ADA)

Accumulation of dATP blocks the

development of T and B-cells which leads severe immunodeficiency

„bubble boy” disease

deoxyadenosine deoxy-ATP

ADA deficiency

deoxyinosine

hipoxanthine

xanthine

uric acid

SYMPTOMS

STOP

(38)

Possible therapies for ADA-SCID

Life long germ-free tent (David Vetter) Regular injections of PEG-ADA

ADA is isolated from cow and conjugated with PEG Bone marrow transplantation

No rejection because of the defective immune system Transplanted T-cells can attack the graft recipient

Donor cells may be infected (David Vetter) T-cell gene therapy

Retroviral vectors, repeated injections because T-cells live 6-12 months

Stem cell gene therapy

Blood stem cells of the patients are transformed with ADA gene, while some of the bone marrow cells are destroyed. Then

transduced cells are injected back to build up new normal bone marrow cells

(39)

isolation of normal T-lymphocytes

lymphocyte growing

isolation of viral DNA and same restriction

cleavage

Isolation of DNA from normal cells

Restriction cleavage and isolation of

ADA gene Ligation of DNA fragments

Gene therapy for severe

immunodeficiency syndrom I.

(40)

T lymphocyte isolation from patient

viral vector production T lymphocyte

infection with virus

growing and testing lymphocytes (ADA)

injection of engineered lymphocytes into the patient

Gene therapy for severe

immunodeficiency syndrom II.

(41)

amino acids from food

liver

ammonia symptoms

mental retardation ATP

carbamoil-phosphate

citrulline

arginino- succinate

arginine ornithine

urea

urea cycle OTC

• most common disorder of urea cycle

• X-linked recessive disorder

• Low-protein diet and administration of medications scavenging nitrogen

STOP

Ornithine transcarbamoilase (OTC) deficiency

(42)

• had a mild OTC deficiency, which was controlled by diet and regular therapy

• volunteered for the OTC gene therapy where normal OTC gene was in vivo transferred into his liver using by adenovirus

• felt in coma after few hours of treatment then died 3 days afterwards

• he had extreme high virus level which caused strong immune response led to his death

Setbacks in gene therapy

Jesse Gelsinger (1999)

French X-SCID (2002)

• one of the eleven „bubble boys” did not respond to the treatment

• eight children cured

• leukemia was developed in two children

(43)

Limitating factors of gene therapy

• short-lived nature of therapy

- multiple periodic treatments required

• immune response

- hard for repeated treatments

• problems with viral vectors - could regain virulence

- could cause toxicity, immun and imflammation response - could control and activate genes

• multi-gene disorders

- challenge for gene therapy (high blood pressure, Alzheimer)

(44)

• Genetic disorder

- mostly resulted by the mutation of the LDL receptor gene - homozygous frequency 1: 500

- heterozygous frequency 1: 1 000 000

• High cholesterol and LDL levels in blood - homozygous have 6-7 X higher

- heterozygous have 2.5 X higher compared to normal values

• Early cardiovascular diseases (heart attack / stroke) - for homozygous in childhood at the age of 5 and 10 - for heterozygous at the age of 35 and 40

Familial hypercholesterolemia

(45)

Transport of lipids with plasma lipoproteins

chylomicrons ApoE

C-II B-48

free fatty acids

adipose tissue, muscle lipoprotein

lipase remnants intestine

exogenous pathway

dietary fats bile acids and cholesterol

remnant receptor

ApoE B-48

liver

dietary cholesterol endogenous

cholesterol

capillaries

VLDL

free fatty acids lipoprotein

lipase

adipose tissue, muscle capillaries

ApoE C-II B-100

IDL LDL

receptors

HDL

ApoA-I ApoE A-II

B100

Plasma LCAT (lecithine-cholesterol

acyl transferase)

endogenous pathway

LDL

ApoB-100

extrahepatic tissues

LDL receptors

(46)

Regulation of the mevalonate pathway

acetyl CoA + acetoacetyl CoA

HMG CoA mevalonate

cholesterol

isopentyl adenin (tRNS)

dolichol haem A ubiquinone farnesylations steroid hormons D vitamine

bile acids lipoproteins

LDL receptor

- -

reductase syntase

(47)

Main facts of the cholesterol question

• most of the cells permanently synthetize cholesterol

• daily cholesterol intake is significant even from a normal diet

• cholesterol does not degrade, removed with biles

• inhibition of the cholesterol synthesis could block the formation

of other important compounds which may lead severe side effects

(48)

Levels of the genetic deficiencies of LDL receptor

golgi

ER clathrin

coated pit

3

4 2

1

1. : no synthesis

2. : no transport to the membrane 3. :very low LDL binding

4. : no recirculation

Ch

Ch atherosclerotic plaque formation

(49)

Correlation between the LDL cholesterol level of blood and the number of LDL receptors in liver

0 1 2 3 4 5 6 7 8 9 14 100

80

60 40 20 0

LDL cholesterol level (mM)

Number of liver LDL receptors (%)

heterozygous

homozygous newborn

Increasing risk of cardiovascular diseases

normal adult

(50)

General treatments of the high plasma cholesterole level

• high HDL level

• change of the dietary mode

• block of the entherohepatic circulation of bile acids

• inhibition of HMG-CoA reductase by statins

no drugs bile acid depletion bile acid depletion +

reductase inhibitor

intestine liver plasma

bile acids cholesterol HMG

CoA

LDL

CoA

LDL LDL

CoA

LDL LDL LDL

(51)

• excision of left lobe

• cell separation with collegenase

• viral transduction of LDL receptor gene into the liver cells

• injection of modified liver cells into the liver vein

• modified liver cells adhere in the capillary veins of the liver and express LDL receptors

ex-vivo gene therapy protocol for

hypercholesterolemia

(52)

Cystic fibrosis

• autosomal recessive disease

- frequency 1: 3000 (in Caucasians)

• mutations in the CFTR gene

- cystic fibrosis transmembrane conductance regulator

protein (CFTR) responsible for the viscosity of mucus in

glands (lung, liver, pancreas, digestive and reproductive tract, skin);

- lack of CFTR results consistent mucus (infection risk)

• symptoms:

- digestive and absorption problems - poor height and weight gain

- cronic pneumonia

- infertility in the 95 % of the male

- diseases of vitamin deficiencies (ADEK)

(53)

• high energy diet with NaCl and vitamines(ADEK) supplements

• pancreatic enzyme supplements

• mechanical devices and medications to clear the mucus

• antibiotic treatment for bacterial infections

• lung transplantation

• gene therapy

- introducing normal CFTR gene into the affected cells

- liposomic and adenoviral vectors in aerosol spray (not enough efficient)

- 5 - 10 % infection would be needed to recover the normal function - Gentamicin treatment to support synthesis of full-length CFTR protein (promising results in experimental phase)

Treatments for cystic fibrosis

(54)

Some ethical concerns about gene therapy

Is it possible to distinguish „good” and „bad” gene therapy?

Who decides which traits are normal and which constitute a disability or disorder?

Is it available only for the rich?

Could the comprehensive use of gene therapy make people less accepting of different individuals?

Should people be allowed to use gene therapy to change their basic human traits (height, intelligence etc.) ?