Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
IN VIVO AND EX VIVO GENE THERAPY
Zoltan BalajthyMolecular Therapies- Lecture 4
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
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 therapyMain criterias of the effective gene therapyIn vivo gene therapyEx vivo gene therapy
4.2. Types of gene transfer, vectors for gene therapyLiposomes, naked DNARetrovirus vector Adenovirus vectorAdeno-associated viral vector (AAV)
4.3. General gene therapy strategies Targeted killing of specific cellsTargeted inhibition of gene expressionTargeted mutation correction
4.4. 3.4. Human gene therapy Severe combined immunodeficiency (SCID)Genetic defects in the LDL receptor Cystic fibrosis (CF)
TÁMOP-4.1.2-08/1/A-2009-0011
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)
TÁMOP-4.1.2-08/1/A-2009-0011
Main criterias of the effective gene therapy
TÁMOP-4.1.2-08/1/A-2009-0011
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
Somatic gene therapy targets
Cancer: main target of the current clinical trialsMuscle: easily accessible, has a good blood supply and abundant tissueEndothelium: can directly secrete the therapeutic protein into the bloodstreamSkin: skin grafts also can secrete the necessary proteinsLiver: has many functions and great regenerationLung: easily accessible with aerosol spraysNervous tissues: many illnesses and injuries can affect it, not easy to modify the neurons
TÁMOP-4.1.2-08/1/A-2009-0011
In vivo gene therapy
Recombinant virus
Plasmid DNA
DNA liposome
TÁMOP-4.1.2-08/1/A-2009-0011
Ex vivo gene therapy
Therapeuticgene
Therapeutic gene is inserted into a specially engineered
virus Target cells are removedfrom 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
TÁMOP-4.1.2-08/1/A-2009-0011
Properties of the ideal gene therapy vector
Safe (no side effects)Immunologically inertCan be targeted to a specific cell type or tissueCan be used to deliver any gene whatever its sizeEasy large scale productionCost effective
The ideal vector does not exist (yet)!
TÁMOP-4.1.2-08/1/A-2009-0011
4.2. Types of gene transfer
Non-viral gene transferLiposomesNaked DNA
Viral gene transferRetrovirusesAdenovirusesOther viruses (Herpes simplex virus
etc.)
TÁMOP-4.1.2-08/1/A-2009-0011
Liposomeshidrophyli
c head
hidrophobic tail
phospholipid phospholipid bilayer
Liposome
+ H2O
Advantages: non-pathogenic, no immunity problems, no gene size limitDisadvantages: low transfection efficiency, low rate of stable integration
TÁMOP-4.1.2-08/1/A-2009-0011
Naked DNA
Advantages: simplestDisadvantages: very low transfection effectivity
Plasmids, PCR products
TÁMOP-4.1.2-08/1/A-2009-0011
Advantages: same as liposome-mediated transfer, promising as a vaccination methodDisadvantages: limited to dermal tissue, low rate of stable integration, difficult to QC
Gene particle bombardment (bioballistic delivery system)
TÁMOP-4.1.2-08/1/A-2009-0011
Main criteria of the viral vectors
TÁMOP-4.1.2-08/1/A-2009-0011
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
Retroviruses
surface glycoproteintransmembrane protein
integrasereverse transcriptaseprotease
matrixcapsidnucleocapsid
RNA genome
Env
Pol
Gag
TÁMOP-4.1.2-08/1/A-2009-0011
-(A)n
-(A)n
-(A)nRT
IN
PR
(1) (3)
(4)
(5) (6)
(7)(8)(9)
(10)early phase late phase
(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. Translation8. Assembly 9. Budding10. MaturationGene therapy constructs
maintained at this stage
TÁMOP-4.1.2-08/1/A-2009-0011
LTR LTRgag pol env
reverse transcription
integration
integrated provirus
transcription
infection
infected cell
replication-competent retrovirus
virus assembly
ψ
translation
Moloney Murine Leukemia Virus Based Retroviral Vector I.polgagψ
LTR LTRenv 3’
LTR LTRTHERAPEUTIC GENE
A
B
C
ψretroviral genome
TÁMOP-4.1.2-08/1/A-2009-0011
transfection
retroviral vector infectious, but replication incompetent
ψLTR LTR
LTR LTRψ
gag pol
envgag/polproteins
envelop proteins
THERAPEUTIC GENE
THERAPEUTIC GENE
THERAPEUTIC GENEψLTR LTR
THERAPEUTIC GENEψLTR LTR
THERAPEUTIC GENEψLTR LTR
packaging cell line
proviral therapeuticplasmid
Moloney Murine Leukemia Virus Based Retroviral Vector II.
TÁMOP-4.1.2-08/1/A-2009-0011
MoMLV-based retroviral vector
(A) The retroviral genome contains the genes gag (structural proteins), pol (reverse polymerase), and env (envelope proteins). the packaging signal distinguishing Ψcellular 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.
TÁMOP-4.1.2-08/1/A-2009-0011
Retroviral gene therapy
TÁMOP-4.1.2-08/1/A-2009-0011
unhealthy cells
RNA
healthy gene
healthy cells
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
TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
ψCMV/LTR LTR 3’5’ RRE cPPT CMV THERAP. GENE WPRE
pol
gag
ψLTR LTR
vifvpr
env
tatnef
rev
3’5’
pol
gagCMV RRE polyA
RSV rev polyA
RSV VSV-G polyA
packaging cell
lentiviral vector
packaging constructs
Lentiviral vector
A
B
C
wild type HIV
TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
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.
Adenoviruses
TÁMOP-4.1.2-08/1/A-2009-0011
Evolution of adenoviral vectors
VA
IVa2
L1 L2 L3 L4E3
L5E4 ITRE2
E1A,BITR ψ
Ad5 genome
VA
IVa2
L1 L2 L3 L4 L5E4 ITRE2
T. génITR ψ
First generation vector; removed E1/E3
VA
IVa2
L1 L2 L3 L4E4 ITRE2
T. génITR ψ
Second generation vector; removed E1/E3/L5
CMV
E4 ITR
Third generation vector; most of the genes are removed, helper-dependent
T. génITR ψCMV
TÁMOP-4.1.2-08/1/A-2009-0011
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). is the packaging signal. The adenoviral genes are highlighted in boxes. Ψ
(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.
TÁMOP-4.1.2-08/1/A-2009-0011
Adenoviral gene therapy
TÁMOP-4.1.2-08/1/A-2009-0011
DNA genom
therapeutic protein
therapeutic gene
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
TÁMOP-4.1.2-08/1/A-2009-0011
Adeno-associated viruses
ITR
Adeno-associated viral genome
capITR rep ITR
Recombinant vector genome
Theraupetic geneITR
vector genomes
Rep/Cap construct
viral vector construct
Packaging cell
Packaging and replication proteins
Adeno-associated viruses
A B
C
TÁMOP-4.1.2-08/1/A-2009-0011
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.
TÁMOP-4.1.2-08/1/A-2009-0011
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 (109-1010/ml)
Dissadvantages:• limited gene size ~ 4.5kb
Properties of adeno-associatedviral vectors
TÁMOP-4.1.2-08/1/A-2009-0011
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 bydrug
Direct cell killing
TÁMOP-4.1.2-08/1/A-2009-0011
Gene therapy strategies II.
Indirect cell killing by immunostimulation
disease cells
foreign antigene gene
disease cells
cytokine gene
immun cellskilling of disease
cells because of enhanced
immune response
TÁMOP-4.1.2-08/1/A-2009-0011
Gene therapy strategies III.
Targeted inhibition of gene expression
disease cells with mutant or harmful gene
antisense gene
antisenseTFO, ODN
or
m
m
AAAA
N C
inhi
bitio
n
block expression of pathogenic gene
Targeted gene mutation correction
disease cellswith mutant gene x
x gene
m
mX X
corrected gene
normal phenotype
TÁMOP-4.1.2-08/1/A-2009-0011
4.4. Gene therapy targets
cancer diseases
cardiovasculardiseases
monogenic diseases
infectiousdiseases
other
TÁMOP-4.1.2-08/1/A-2009-0011
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)
Hematopoieticcells
stem cells
TÁMOP-4.1.2-08/1/A-2009-0011
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
TÁMOP-4.1.2-08/1/A-2009-0011
Possible therapies for ADA-SCIDTÁMOP-4.1.2-08/1/A-2009-0011
Life long germ-free tent (David Vetter)
Regular injections of PEG-ADAADA is isolated from cow and conjugated with PEG
Bone marrow transplantationNo rejection because of the defective immune systemTransplanted T-cells can attack the graft recipientDonor cells may be infected (David Vetter)
T-cell gene therapyRetroviral vectors, repeated injections because T-cells live 6-12 months
Stem cell gene therapyBlood 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
isolation of normal
T-lymphocyteslymphocyte growing
isolation ofviral 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.
TÁMOP-4.1.2-08/1/A-2009-0011
T lymphocyteisolation
from patient
viralvector
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.
TÁMOP-4.1.2-08/1/A-2009-0011
amino acids from food
liver ammonia symptoms
mental retardationATP
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
TÁMOP-4.1.2-08/1/A-2009-0011
Ornithine transcarbamoilase (OTC) deficiency
• 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 transferredinto 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
TÁMOP-4.1.2-08/1/A-2009-0011
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)
TÁMOP-4.1.2-08/1/A-2009-0011
• 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 hypercholesterolemiaTÁMOP-4.1.2-08/1/A-2009-0011
Transport of lipids with plasma lipoproteins
TÁMOP-4.1.2-08/1/A-2009-0011
chylomicrons
ApoEC-II
B-48
free fatty acids
adipose tissue, muscle
lipoproteinlipase
remnants
intestine
exogenous pathwaydietary fats bile acids and
cholesterol
remnantreceptor
ApoEB-48
liverdietary
cholesterol
endogenouscholesterol
capillaries
VLDL
free fatty acids
lipoproteinlipase
adipose tissue, muscle
capillaries
ApoEC-II
B-100
IDL
LDLreceptors
HDLApoA-I
A-IIApoEB100
Plasma LCAT(lecithine-cholesterol
acyl transferase)
endogenous pathwayLDL
ApoB-100
extrahepatic tissues
LDLreceptors
Regulation of the mevalonate pathway
TÁMOP-4.1.2-08/1/A-2009-0011
acetyl CoA + acetoacetyl CoA
HMG CoA
mevalonate
cholesterol
isopentyl adenin (tRNS)
dolicholhaem Aubiquinonefarnesylations
steroid hormonsD vitaminebile acidslipoproteins
LDL receptor
-
-
-
-
reductase
syntase
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
TÁMOP-4.1.2-08/1/A-2009-0011
Levels of the genetic deficiencies of LDL receptor
TÁMOP-4.1.2-08/1/A-2009-0011
golgiER clathrin
coatedpit
3
4
21
1. : no synthesis2. : no transport to the membrane3. :very low LDL binding4. : no recirculation
Ch
Ch atherosclerotic plaque formation
Correlation between the LDL cholesterol level of blood and the number of LDL receptors in liver
TÁMOP-4.1.2-08/1/A-2009-0011
0 1 2 3 4 5 6 7 8 9 14
100
80
60
40
20
0
LDL cholesterol level(mM)
Num
ber o
f liv
er L
DL
rece
ptor
s (%
)
heterozygous
homozygous
newbornIncreasing risk of cardiovascular diseases
normal adult
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 depletionbile acid depletion+reductase inhibitor
intestine
liver
plasma
bile acids
cholesterol
HMGCoA
LDL
bile acids
cholesterol
HMGCoA
LDLLDL
bile acids
cholesterol
HMGCoA
LDLLDLLDL
TÁMOP-4.1.2-08/1/A-2009-0011
• 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
TÁMOP-4.1.2-08/1/A-2009-0011
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)
TÁMOP-4.1.2-08/1/A-2009-0011
• 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 fibrosisTÁMOP-4.1.2-08/1/A-2009-0011
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.) ?
TÁMOP-4.1.2-08/1/A-2009-0011
