gene transfer and gene therapy dr greta sawyer 
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8/17/2019 GENE TRANSFER AND GENE THERAPY Dr Greta Sawyer
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GENE TRANSFER
AND
GENE THERAPY
Dr Greta Sawyer
8/17/2019 GENE TRANSFER AND GENE THERAPY Dr Greta Sawyer
http://slidepdf.com/reader/full/gene-transfer-and-gene-therapy-dr-greta-sawyer- 2/109
8/17/2019 GENE TRANSFER AND GENE THERAPY Dr Greta Sawyer
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Aims and Objectives
• To understand the complexity and risks of gene therapy
• To compare and discuss DNA vectors
• To explore the applications of gene transfer in research and medicine
• To understand the fundamental procedures in gene transfer
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Aims and Objectives
• To understand the complexity and risks of gene therapy
• To compare and discuss DNA vectors
• To explore the applications of gene transfer in research and medicine
• To understand the fundamental procedures in gene transfer
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Why?
• Investigate gene function and gene regulation
• Create animal models of human disease
• Produce commercial/therapeutic products
• Gene therapy
GENE TRANSFER
into
cells, organs & whole organisms
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Gene transfer into cultured CELLS
In the laboratory
Cells grown in flasks
• To investigate gene function, gene regulation.
• To develop DNA delivery systems
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Gene Transfer to cells
Neurons
Parkinson Disease
Liver cells
Metabolic disorders
HepatitisLiver transplants
Islets
Diabetes
Cell lines Primary Cells Primary tissue cultures
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How ?
Methods for transferring genes into cultured cells
Virus-mediated delivery Modified DNA and RNA viruses.
Non viral mediated deliveryPhysical
Polycations
Lipososmes
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Transgenic Animals
Genetically altered animals :
Alter the DNA of the germline to generate a modification or
mutation that is heritable.
Two methods used
Pronuclear injection
Gene targeted technology
Introduction of
exogenous gene
Modification of
a specific
endogenous
gene
Gene transfer into organisms creates
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Transgenic Mice
Useful disease models for research
• Small, easy/cheap/quick to breed
• Conserved gene structure and function
• Highly conserved biology vs humans
• Proves mutations are disease-causing
• Creates a disease model where a naturally occurringmodel does not exist
• Test therapies and treatments
• Study gene function in vivo
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Transgenic Mice
GOI
Knockout
KO
Overexpression
Tg
Cardiac myocytes
Tg KO
Global
Tg KO
Endothelial cells
Tg KO
Fibroblasts
Tg KO
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Pronuclear Injection
Microinjection of DNA into the pronucleus of a fertilised egg
Result
Addition of DNA by random, stable integration into chromosomes
Integrated into germline cells passed on to subsequent generations
Can control expression to some degree by tissue specific promoters.
Creation of transgenic animals
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Pronuclear Injection
• Inefficient• Multiple copies of gene – over expression
• Random integration – may get insertional mutagenesis
• Only gene “addition” is possible
Limitations
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Gene Targeting by Homologous Recombination
• Disruption of endogenous gene
expression by introduction of a non-
functioning version (Knockout)
• Addition or replacement of mutant gene
with the correct version to restore function
(Knock-in)
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DNA construct contains gene of interest, homologous
sequences, genes for antibiotic resistance e.g. neomycin (neo)
and drug sensitivity e.g. thymidine kinase (tk).
• Introduce DNA construct into embryonic stem (ES) cells
• DNA construct locates and recombines with theendogenous homologous sequences.
• Antibiotic resistance allows for selection of ES cells that
have the gene insertion.
• Drug sensitivity allows for selection of ES cells with
targeted gene insertion over those with random
gene insertion.
Gene Targeting by Homologous Recombination
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Most cells fail to take up thevector; these cells will be killed if
exposed to G418 (antibiotic). i.e.
cells killed if NO integration
In a few cells: the vector is inserted
randomly in the genome. In random
insertion, the entire vector,
including the tk gene, is insertedinto host DNA. These cells are
resistant to G418 but because they
have the tk gene they can be killed
by gancyclovir
Gene Targeting by Homologous Recombination
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In still fewer cells: homologousrecombination occurs. Stretches of
DNA sequence in the vector find the
homologous sequences in the host
genome, and the region between
these homologous sequences replacesthe equivalent region in the host
DNA. Thus tk is NOT integrated and
cells are resistant to gancyclovir
Gene Targeting by Homologous Recombination
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Alter gene in Embryonic Stem Cells (ES cells)in culture
Inject ES cells intonormal embryo
Chimaeric animal. If gonads are derived from ES
cells, these chimaeras produce sperm or eggs
carrying mutation. Inter-breeding produces non-chimaeric transgenic animal.
• ES from early mouse
embryos
• Transformation of ES cells
with selection of those withhomologous recombination
Gene Targeting by Homologous Recombination
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• Homologous Recombination is a rare event.
• Targeted insertion of non-functional gene to ‘knock -out’
target gene or replace defective gene ‘knock -in’
• Need to select for gene insertion by antibiotic resistance
selection e.g. neomycin and for targeted gene insertion, over
random gene insertion using drug sensitivity e.g.thymidine
kinase
• ES cells are essential, to enable selection of rare cells by cell
culture.
Gene Targeting by Homologous Recombination
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Animal models used for human disease
• Cystic fibrosis: insertional inactivation by targeting of CFTR
gene
• β-thalassaemia: insertional inactivation by targeting ofβ-globin gene.
• Alzheimer’s disease: insertion of mutant β-amyloid precursor
cDNA.
• Huntington disease: insertion of part of human huntingtin
gene with expanded trinucleotide repeat.
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Production of therapeutic proteins
using gene transfer
• Transgenic sheep and goats have been produced that express
foreign proteins in their milk. E.g. Clotting factors VIII and IX.
• Transgenic chickens are now able to synthesize human proteins
in the ‘white’ of the eggs. E.g. Gamma interferon
• Plants altered to produce therapeutic and commercial proteins.
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1. Objective - To understand the fundamental procedures
in gene transfer a) Name two methods by which transgenic animals can be made
b) State two reasons why transgenic mice are useful disease models for
research
c) Name two animals models used for human disease
Pronuclear Injection Gene Targeting by Homologous recombination
*Small, easy/cheap/quick to breed *Conserved gene structure, function
*Highly conserved biology vs humans *Proves mutations are disease-
causing *Creates a disease model where a naturally occurring model
does not exist *Test therapies and treatments*Study gene function in vivo
•Cystic fibrosis: insertional inactivation by targeting of CFTR gene•β-thalassaemia: insertional inactivation by targeting of β-globin gene
•Alzheimer’s disease: insertion of mutant β-amyloid precursor cDNA
•Huntington disease: insertion of part of human huntingtin gene with
expanded trinucleotide repeat.
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Aims and Objectives
• To understand the complexity and risks of gene therapy
• To compare and discuss DNA vectors
• To explore the applications of gene transfer in research and medicine
• To understand the fundamental procedures in gene transfer
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Gene Therapy
1980s ‘The ultimate cure for human disease’
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Gene Therapy
A brief History
1975
First human gene
isolated.
1990
First ex vivo
therapeutic
gene therapy study
2001
Human
Genome
sequenced.
1953
DNA structure
discovered
1973
DNA cloning
developed
1940
Genes = DNA
1993
First in vivo
therapeutic
gene therapy study
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Single Gene Disorders
• Addition of a normal copy of a defective gene
• Suppression of an abnormal gene product
Complex Pathology
Expression of a gene to influence a complex pathological process
• Cancer
• Chronic infections such as hepatitis B, AIDS
• Cardiovascular diseases, e.g. Atherosclerosis
• Brain diseases, e.g. Alzheimers
• Autoimmunity
What is gene therapy?
The delivery of genetic information to cells for therapeutic purposes
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What is the cause of a disease?
A) Genetic mutation leads to loss of function
loss of protein diseasee.g. Cystic Fibrosis
B) Mutation causes abnormal protein production disease
e.g Huntington’s disease
Transgene works alongside mutated
gene in same cell.
Widely researched and in clinical trials.
Homologous recombination required
to replace defective gene.
Transgenic production.
Not yet practical for gene therapy
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Single Gene Disorders
• Addition of a normal copy of a defective gene
• Suppression of an abnormal gene product
Complex Pathology
Expression of a gene to influence a complex pathological process
• Cancer
• Chronic infections such as hepatitis B, AIDS
• Cardiovascular diseases, e.g. Atherosclerosis
• Brain diseases, e.g. Alzheimers
• Autoimmunity
What is gene therapy?
The delivery of genetic information to cells for therapeutic purposes
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Germline transfer Somatic transfer
Two major areas of gene transfer
• Gene introduced in togamete or fertilised egg.
• New gene inherited.
• Considered inethical
• Gene directed at specific cellsor organs – non-germline.
• Not inherited.
Two approaches to targeting cells in Somatic gene therapy
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Ex vivoRemoval of target cell from body
Gene transfer
Re-infusion of modified cells
e.g. Genetic blood disorders
SCID, ADA deficiency
In vivoDirect application of gene
e.g. cystic fibrosis
• Systemic injection• Lung instillation
• Targeted organ perfusion
• Intramuscular etc….
Two approaches to targeting cells in Somatic gene therapy
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• Over expression of gene – production of too much of
protein- harmful effects
• Wrong cells transfected (healthy cells instead of cancer
cells)
• Cause cancer (insertional mutagenesis)
• Cause disease (e.g. viral vectors)
• Immune response (e.g. toxic shock)
Potential risks of gene therapy
2 Objective -To understand the complexity and risks of gene therapy
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2. Objective -To understand the complexity and risks of gene therapy
a) What is the main difference between germline and somatic gene
transfer?
b) What are the two approaches used to target cells in Somatic
gene therapy?
c) List two potential risks of gene therapy.
Germline – inherited, somatic not inherited
In vivo Ex vivo
• Over expression of gene – production of too much of protein-
harmful effects
• Wrong cells transfected (healthy cells instead of cancer cells)
• Cause cancer (insertional mutagenesis)
• Cause disease (viral vectors)
• Immune response (toxic shock)
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Aims and Objectives
• To understand the complexity and risks of gene therapy
• To compare and discuss DNA vectors
• To explore the applications of gene transfer in research and medicine
• To understand the fundamental procedures in gene transfer
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TWO KEY COMPONENTS
of gene delivery systems
• DNA expression constructs containing essential regulatory
elements
• Vectors carry the DNA to the cell and enable the DNA to traverse
all cellular and intracellular barriers to the nucleus
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What is the efficiency of gene delivery?
• What percentage of cells have taken up the transgene?
dependent on vector
• What is the level of gene expression?
dependent on DNA construct
dependent on site of integration
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Assessing efficiency of gene delivery
Use reporter genes to easily identify cells that have been transfected.
Examples
-galactosidase
Cloned from bacteria.
Adding substrate turns cells blue
Green Fluorescent Protein (gfp)
Cloned from jellyfish
Cells fluoresce green
Luciferase
Firefly
Adding substrate emits measurable light
Episomal vs Integrated Expression
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Nature Medicine (2001) 7, 33-40
Episomal vs Integrated Expression
Episomal DNA (independent
copy)
• DNA non-integrated
• Diluted out on cell division
• Short term (transient) expression
in dividing cells
Integrated DNA (integrated into chromosome)
• DNA replicated when host cell divides
• Usually long term expression
• Risk of insertional mutagenesis (carcinogenesis)
• Site-dependence of expression
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“Naked” DNA (no vectors)
• DNA injection: esp. into muscle
• Hydrodynamic (injection under pressure):
esp. to liver (hepatocytes)
• Electroporation (originally used to deliver
DNA to bacteria)
•
Ballistic DNA injection: the “gene gun”
• Ultrasound
• Calcium phosphate
!
(more cost effective, safe)
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Requirements 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 for dividing and non-dividing cells.
• Can be used to deliver any gene whatever size or function.
• Long term expression• Easy large scale production.
• Cost effective.
No single currently available vector can meet these
requirements - yet• Sustained and regulated expression of therapeutic product.
• Level and longevity expression controllable depending ontarget disease.
Something for the future?
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DNA Vectors
Viral vs Non-viral
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Viral DNA vector
Viruses are natural gene delivery systems.
They have evolved to be highly effective at getting into cells
and delivering their DNA to the nucleus.
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Virus :a) Attachment to the cell surface
b) Internalization by endocytosis
c) Disruption and exit from the endocytic vesicle
d) Traverse cytosol to nucleus
e) Active transport of DNA across nuclear membrane into nucleus
Entry pathway of a virus into the cell
- Endocytosis
(d) (e)
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RetrovirusAdenovirus
Viral vectors
• Retrovirus
• Adenovirus
• Adeno-associated virus
• Many others
Adenoassociated virus
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Retroviruses (retroviridae)
Oncoretroviruses (MLV) and lentiviruses (e.g. HIV)
• Enveloped virus containing an ssRNA genome.
• gag , pol and env genes responsible for infection and replication.
Group specific antigen (gag) – core and structural proteins
Polymerase (pol) – reverse transcriptaseEnvelope (env) – retroviral coat proteins
Retrovirus as a Gene Therapy Vector
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1. Remove genes for infection and replication (structural
genes) to make virus safe.
2. Insert gene of interest into space left by these genes.
Virus is now replication incompetent.
3.To make more viral particles for use in gene therapy need
to provide the structural proteins. This is done by using a
helper plasmid that can encode for the structural proteins but
not package them.
4.Put helper plasmid and vector genome into a packaging celltogether, the helper plasmid provides the structural proteins
to enable the formation of the viral particles, but these viral
particles are incapable of replicating themselves.
Retrovirus as a Gene Therapy Vector
Retrovirus as a Gene Therapy Vector
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Retrovirus as a Gene Therapy Vector
Wild-type provirus genome
Vector genome
Packaging cell genome
Retroviral vector prepared by removal of gag, pol and env genes
and insertion of your gene of interest.
N.B. LTRs and packaging signal retained
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Gag, pol and env are produced on a separate plasmids - the
packaging genome.
Helper
plasmid
No packaging signal
Vector
Packaging signal
Therapeutic viruses cannot replicate independently and are
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Replication-incompetent virus particles
p p p y
produced in a packaging cell line which provides all the viral proteins
required for capsid production and virion maturation
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Retroviruses cont.
• The ssRNA genome is converted to cDNA and integrates, via
Long Terminal Repeats (LTR), at a random point in host genome.
• MLVs can transfect only dividing cells, as they lack a mechanism
for nuclear translocation of the DNA.
• Lentiviruses can transfect both dividing and non-dividing cells
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Retroviruses
Advantages
• Usually stable, long-term gene expression because of
integration of therapeutic gene into host genome.
• High efficiency of gene delivery to many cells
Retroviruses
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• Maximum insert size 7 - 8 kb
• Oncoretroviruses (e.g. Mouse Leukemia Virus) can infect only
dividing cells. NOTE: for cancer, this is an advantage!
• Viral proteins can evoke immune response
• Recombination may produce replication-competent virus (disease
causing)
• Can be produced only in relatively low titres (~107 pfu/ml)
• Random integration may lead to oncogenic activation(insertional mutagenesis)
Disadvantages
G Th i l f S C bi d I d fi i (SCID)
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Gene Therapy trial for Severe Combined Immunodeficiency (SCID)
French Gene Therapy Clinical Trial
• SCID - Faulty immune system (gamma – c gene defective). Fatal if left untreated.
Children treated by bone marrow transplant but not ideal.
• Gene Therapy trial using retroviruses to deliver therapeutic gene (gamma-c) to
childs own bone marrow cells.
• 4/11 boys developed leukemia.
• Retroviral insertion of therapeutic gene near to the oncogene LMO2 whichactivated the gene and caused leukemia.
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Adenoviruses
• Non-enveloped icosahedral virus composed of nucleocapsid
containing a double-stranded linear DNA genome of ~36kb.
• Over 51 different serotypes in humans responsible for upper
respiratory infections.
• Viral genome not incorporated into host cell DNA. Left free in
nucleus and transcribed like any other gene (episomal).
• Not replicated upon cell division, (expression transient).
Vi l i E l ( l ) d L ( l)
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• Early genes can be removed and replaced with the therapeuticgene to produce replication-incompetent viruses. These are
“first generation” adenovirus vectors.
• Virtually entire genome (including all virus protein coding
sequences) can be removed and replaced with the therapeuticDNA. These are “gutless” adenovirus vectors. They require
helper viruses or sophisticated packing cell lines to produce
recombinant virus.
• Viral genome contains Early (regulatory) and Late (structural) genes.
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Adenoviruses
Advantages
• Ease of production
• High titres (>1012 pfu/ml) easily produced
• Efficient delivery of DNA to many cell types
• Infects dividing and non-dividing cells
• No integration of DNA, and therefore no risk of oncogenic activation• Can carry large genes (up to ~30 kb)
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Adenoviruses
Disadvantages
• Episomal delivery, and therefore gives transient
(short term) gene expression in dividing cells.
• Induces inflammatory and immune responses.
• Risk of generating replication-competent virus by
recombination
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Gene Therapy Trial for Ornithine Transcarbamylase Deficiency
• 1999, teenager Jesse Gelsinger volunteered for clinical trial for OTD,
liver gene deficiency.
• He died from toxic shock, an immune response evoked byAdenoviral vector carrying the transgene.
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Adeno-Associated viruses (AAV)
Small non-pathogenic human parvovirus
• Genome of single stranded DNA
• Wild type virus can insert genetic material at a specific siteon chromosome 19.
• Two genes, rep (replication) and cap (capsid
structure) together with terminal repeat containing a promoter.
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• Rep and cap genes replaced by
transgene.
• Production dependant on
a helper virus, usually adenovirus,
and helper cell line to proliferate.
• AAV combine advantages of both retroviruses and adenoviruses.
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Adeno-Associated viruses
Advantages
• Infects dividing and non-dividing cells
• Efficient delivery of DNA to many cell types
• Non-pathogenic, non-inflammatory, non-immunogenic
• Can integrate into host genome (wild type)
• Long-term expression observed
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Disadvantages
• Max insert size 5 kb
• Difficult to produce on large scale – it kills the packaging cells
• Potential problems with insertional mutagenesis
• Risk of generation of replication competent virus
Adeno-Associated viruses
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Retinal gene disorders
Duchenne Muscular Dystrophy
Haemophilia factor IX
Gene Therapy trials with AAV
No major incidents reported
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Pause
Q. Would you be happy to have gene therapy with a viral vector?
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Non-viral DNA vectors
h ll l
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Genes cannot enter the cell alone
- large
- anionic charge
Need to compact the DNA and encapsulate the negative charge
- how about a using a cation?
++ +
+
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++
+
How about artificial entry? – crude
Non viral DNA vectors
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Non-viral DNA vectors
• Cationic liposomes
• Polycations
• Polylysines
• Polyethylenimine
• Others
Cations for electrostatic interaction and condensation of DNA
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Liposomes
Most successful of non-viral systems
Phospholipid molecules with one end hydrophobic and other hydrophilic.
In aqueous solutions they spontaneously form lipid bilayers.
Hydrophobic surfaces facing inwards. Similar to a cell membrane.
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For many years they have been used to encapsulate drugs for clinical cellular
delivery e.g. Cancer treatments.
Liposomes What is their potential for gene therapy?
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Liposomes – What is their potential for gene therapy?
+
++
+
+ +
+
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+
_ _ _ _ _
_ _ _ _ _
Cationic liposomes Transgene
• Cationic synthetic phospholipid bilayer vesicles.
• Interact spontaneously with DNA and compact it to form condensed
vesicles.
• Interaction with cell membrane leading to endocytosis.• pH sensitive liposomes
• Clinical trials e.g. Cystic fibrosis: respiratory epithelium
Polycationic complexes
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Polycationic complexes
• Positively charged polymers or peptides (NOT liposomes!)
CH2
CH2
CH2
CH2
NH3
COC N
HH
x
Poly-L-Lysine
+
Polylysine
Branched PEI
Polyethylenimine (PEI)
Linear PEI
• Bind electrostatically to the negatively charged DNA
and condense it into compact particles that can be
taken up by the cell
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DNA
cation
Can bind electrostatically to the cell surface
Target to specific cells by addition of ligands
Non-viral vector mediated gene delivery with polycations
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Non-viral vector mediated gene delivery with polycations
• Enter cells by receptor mediated endocytosis.
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Polycationic peptide/DNA particles on surface of cells
Scanning Electron Microscopy
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Polycationic peptide/DNA particles
attaching and entering a cell
Transmission Electron Microscopy
Non-viral DNA vectors
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Non viral DNA vectors
Advantages
• Can be non-immunogenic
• Not infectious, and therefore fewer regulatory problems
• No limit to DNA size which can be delivered. HOWEVER,
large DNA is difficult to produce in quantity
• Easy to prepare, compared to recombinant viruses• Easy to scale-up
Disadvantages• Poor stability in vivo
• Transferred genes do not integrate – short term expression.
• Poor efficiency
• No insertional mutagenesis
Vectors currently used for gene therapy clinical trials
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3. Objective-To compare and discuss DNA vectors
a) What are the two key components of gene delivery systems?
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a) What are the two key components of gene delivery systems?
b) What is the main difference between episomal and integrated
gene expression?
c) Name the three main viral vectors used in gene therapy
d) List three advantages of non-viral vectors over viral vectors
DNA vector
Episomal gives short term (transient) expression
Integrated gives long term expression
Retrovirus Adenovirus Adeno-associated virus
• Not infectious, and therefore fewer regulatory problems
• No limit to DNA size which can be delivered.• Easy to prepare, compared to recombinant viruses
• Easy to scale-up
Viral vs non-viral vectors
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• Viral vectors
•Retrovirus
•Adenovirus
•Adeno Associated virus
• Non- viral vector
• Liposomes
• Polycationic complexes
High efficiency
High risk
Low efficiencyLow risk
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Aims and Objectives
• To understand the complexity and risks of gene therapy
• To compare and discuss DNA vectors
• To explore the applications of gene transfer in research and medicine
• To understand the fundamental procedures in gene transfer
In vivo gene delivery
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In vivo gene delivery
• Problems vary enormously depending on targettissue and route of administration
• Stability of non-viral vectors in blood is usually a problem
• Physical barriers – Endothelial cells
– Basement membranes
– Surface mucus
Delivering genes to tissues and organs in the body
Regulating gene expression for gene therapy
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Delivering DNA to the wrong cell could have
disastrous consequences
Ideally need to target your vector to specific cells or tissues
Regulating gene expression for gene therapy
Regulating gene expression for gene therapy
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Regulating gene expression for gene therapy
Targeted delivery
Targeted expression
Exogenous regulation of gene
expression
Localize delivery Target specific cell type
Regulating gene expression for gene therapy
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Regulating gene expression for gene therapy
Localise delivery to intended target tissue where
possible. Use specific routes of administration that leaddirectly to the target organ
• Inhalation to target lungs• Catheters to target liver and heart
• Direct injection into tumours and muscle
1. Targeted delivery
Regulating gene expression for gene therapy
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Regulating gene expression for gene therapy
Tissue-specific ligands (e.g. peptides, proteins or
antibodies) can be added to viral and non-viral vectorsto try to target gene delivery to specific cell types.
1. Targeted delivery
In most therapeutic applications the vector is
introduced into a mixed population of cells.
•Adeno-Associated viral vectors have been targeted to
bone marrow cells•Adenoviral vectors have been targeted to endothelial
cells
•Retroviral vectors have been targeted to cancer cells
Vi h l ffi i i ifi ll
BUT
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Viruses have natural affinities to specific cell types
via cell surface receptors
Natural tropism may not meet therapeutic need, natural viral tropism
may need to be modified so that it can no longer bind to its native
cellular receptors before it is engineered to add the targeting ligands
Respiratory cells
Liver and lung
Immune cells
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Liposomes can be protected from non-specific
delivery with PEG or with a ligand to reduce surface
charge
Delivery can be targeted to lung vasculature e.g. with
angiotensin converting enzyme
Non-Viral vectors
Regulating gene expression for gene therapy
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Regulating gene expression for gene therapy
2. Targeted expression• Tissue specific promoters in DNA constructs can
control in which cells the gene is expressed.
ExamplesLiver specific promoter such as alpha 1 antitrypsin
limits transgene expression to liver.
Endothelial specific promoter to target the vector to
endothelial cells e.g. vascular endothelial growth
factor receptor type 1
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Using a combination of targeted delivery AND
targeted expression can result
in
a synergistic improvementin the selectivity of gene expression.
Regulating gene expression for gene therapy
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• Glucose responsive promoters control insulin geneexpression
• Tet on/off system. Gene expression controlled by
administering tetracycline.
In this system expression of a target transgene is dependent on the
activity of an inducible transcriptional activator.
Gene expression is controlled by administering tetracycline (Tet) ordoxcycline (Dox, a more stable derivative)
Tet-Off activates expression in the absence of Dox/Tet
Tet-On activates expression in the presence of Dox/Tet
Regulating gene expression for gene therapy
3. Exogenous regulation of gene expression
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GENE THERAPY APPLICATIONS
Disease targets for gene therapy clinical trials
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Gene therapy treatment for Cancer
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py
To improve body’s natural ability to fight the disease or make
cancer cells more sensitive to other kinds of treatment such aschemotherapy.
1. Delivery of tumour suppressor genes2. Immunomodulation
3. Activated cytotoxicity
64% gene therapy trials are for cancer treatment.
Gene therapy treatment for Cancer
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1. Delivery of tumour suppressor genes
Tumour suppressor genes encode proteins that normally slow
down cell division, repair DNA mistakes, and trigger apoptosis.
Inactivation of tumour suppressor genes can lead to cells growing
out of control, which can lead to cancer.
Eg. p53 regulates cell division and apoptosis
Mutations of p53 are estimated to occur in up to half of all human
cancers and in approximately 20% – 30% of breast cancers
Delivery of p53 can trigger cell death
E.g. pRb regulates cell division. Mutations are found in 40% of
cancers. First identified from Retinoblastoma.
py
2 Immunomodulation
Gene therapy treatment for Cancer
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2. Immunomodulation
Stimulation of immune response to tumour cells
by transfer of immunoregulatory moleculesaids natural rejection of the tumour cells by
the body. Many tumour cells do not express
MHC which can limit their recognition by the
body’s T cells as foreign.
E.g. HLA gene injected into melanoma can induce a rejection
response towards the tumour cells
Cytokines are involved in the immune response. Tumour cells
can be modified to release cytokines that evoke an immune
response against the tumour.
E.g. IL2, IL4, IL6, IL7, IFNg TNFa
Gene therapy treatment for Cancer
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• What gene to deliver?
• Direct delivery to tumour cells
• Need to kill 100% cancer cells
• Leave normal cells unharmed
3. Activated Cytotoxicity
Delivery of “Suicide genes” to tumour cells. Cell death following
activation with prodrug.
py
Activated cytoxicity
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• Thymidine Kinase gene in retroviral vector injected into brain tumour.
• Retrovirus infects tumour cells but not non-dividing brain cells.
• Patient treated with ganciclovir. Selective killing of tumour cells.
• ‘Bystander effect’ – neighbouring tumour cells also killed.
HSV thymidine kinase gene activated by ganciclovir.
Gene therapy treatment for Cancer
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E.g. Multidrug resistance gene (MDR1) to bone marrow
cells to make them resistant to chemotherapeutic agents.
Gene therapy to make cancer treatments work better
E.g. make cancer cells more sensitive to particular treatments such as
chemotherapy or radiotherapy.
Gene therapy to block processes that protect cancer cells
Eg programmed cell death.Cancer cells can block the process of apoptosis.
Re introduce the ability for programmed cell death
py
Gene Therapy for Cystic Fibrosis
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py y
• Incidence 1 in 2500.
• Mutation in Cystic Fibrosis Transmembrane Conductance
Regulator gene (CFTR) lack of control of chloride ion flow.
Lung particularly affected.
• CFTR gene cloned.
• Extensive research to deliver gene to airway epithelium with
viral and non viral vectors (liposomes). Possible to ‘inhale’ vectorcontaining CFTR gene.
• Clinical trials in progress.
Application of gene therapy to treat Cystic Fibrosis
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Application of gene therapy to treat Cystic Fibrosis
Highly suitable for gene therapy treatment
• High incidence,
•
Well characterised,• Target cell accessible.
What level of gene expression is enough?
Need long term expression or repeat administration.
BUT
Phases of gene therapy clinical trials
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Healthy volunteers
for safety
Evaluate biological
effect
Safety surveillance after approvalValue in clinical practise
i
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Retroviral vectors
1990
First ever therapeutic study in humans
Ex vivo approach
Adenosine deaminase deficiency
Adenoviral vectors
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1993
First ever in vivo therapeutic study in humans
Cystic fibrosis
April 16 1993
Adeno Associated Viral vectors
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First Western approved gene therapy medicine
lipoprotein lipase deficiency (LPLD)
inability to digest fat properly, leading to pancreatitis
AAV vector with healthy LPL gene targeted to muscle cells
Glybera (alipogene tiparvovec)
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Treatment protocol:
Up to 60 intramuscular injections in the legs ina single session under anaesthesia with
immunosuppression given 3 days prior to and
for 12 weeks following the administration.
Uses AAV1 vector which has a strong tropism
for muscle cells
Glybera
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2005 Clinical trials started
2009 started submission for regulatory approval2012 Approval granted.
2013 Commercially available in
Brazil, China, Mexico and Russia.
2015 Commercially available in Europe and USCost?
2012 $1.6 million per patient
2015 $1 million per patient
The most expensive medicine in the world
4. Objective-To explore the applications of gene transfer
in research in medicine
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in research in medicine
a) Give two examples of how gene expression can be regulated
b) Name three ways in which gene therapy can be used to treat cancer
Targeted delivery Targeted expression
1. Delivery of tumour suppressor genes
2. Immunomodulation
3. Activated cytotoxicity
S
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Summary
• To understand the complexity and risks of gene therapy
• To compare and discuss DNA vectors
• To explore the applications of gene transfer in research and medicine
• To understand the fundamental procedures in gene transfer
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