GENE TRANSFER AND GENE THERAPY Dr Greta Sawyer

8/17/2019 GENE TRANSFER AND GENE THERAPY Dr Greta Sawyer

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GENE TRANSFER

AND

GENE THERAPY

[email protected]

Dr Greta Sawyer



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



Aims and Objectives







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



Gene transfer into cultured CELLS

In the laboratory

Cells grown in flasks

• To investigate gene function, gene regulation.

• To develop DNA delivery systems



Gene Transfer to cells

Neurons

Parkinson Disease

Liver cells

Metabolic disorders

HepatitisLiver transplants

Islets

Diabetes

Cell lines Primary Cells Primary tissue cultures



How ?

Methods for transferring genes into cultured cells

Virus-mediated delivery Modified DNA and RNA viruses.

Non viral mediated deliveryPhysical

Polycations

Lipososmes



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



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



Transgenic Mice

GOI

Knockout

KO

Overexpression

Tg

Cardiac myocytes

Tg KO

Global

Tg KO

Endothelial cells

Tg KO

Fibroblasts

Tg KO



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



Pronuclear Injection

• Inefficient• Multiple copies of gene – over expression

• Random integration – may get insertional mutagenesis

• Only gene “addition” is possible

Limitations



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)



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.




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




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




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




• 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.




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.



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.



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.



Aims and Objectives







Gene Therapy

1980s ‘The ultimate cure for human disease’



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



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



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



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



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



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



• 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



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)



Aims and Objectives







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



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



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



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



“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)



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?



DNA Vectors

Viral vs Non-viral



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.



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)



RetrovirusAdenovirus

Viral vectors

• Retrovirus

• Adenovirus

• Adeno-associated virus

• Many others

Adenoassociated virus



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



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.






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



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



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



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



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



• 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)



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.



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)



• 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.



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)



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



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.



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.



• 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.



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



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



Retinal gene disorders

Duchenne Muscular Dystrophy

Haemophilia factor IX

Gene Therapy trials with AAV

No major incidents reported



Pause

Q. Would you be happy to have gene therapy with a viral vector?



Non-viral DNA vectors

h ll l



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?

++ +

+

+++

++

+

How about artificial entry? – crude

Non viral DNA vectors




• Cationic liposomes

• Polycations

• Polylysines

• Polyethylenimine

• Others

Cations for electrostatic interaction and condensation of DNA



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.



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?



Liposomes – What is their potential for gene therapy?

+

++

+

+ +

+

++

+

_ _ _ _ _

_ _ _ _ _

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



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



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



Non-viral vector mediated gene delivery with polycations

• Enter cells by receptor mediated endocytosis.



Polycationic peptide/DNA particles on surface of cells

Scanning Electron Microscopy



Polycationic peptide/DNA particles

attaching and entering a cell

Transmission Electron Microscopy




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



3. Objective-To compare and discuss DNA vectors

a) What are the two key components of gene delivery systems?



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



• Viral vectors

•Retrovirus

•Adenovirus

•Adeno Associated virus

• Non- viral vector

• Liposomes

• Polycationic complexes

High efficiency

High risk

Low efficiencyLow risk



Aims and Objectives





In vivo gene delivery



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



Delivering DNA to the wrong cell could have

disastrous consequences

Ideally need to target your vector to specific cells or tissues






Targeted delivery

Targeted expression

Exogenous regulation of gene

expression

Localize delivery Target specific cell type





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





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



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



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





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



Using a combination of targeted delivery AND

targeted expression can result

in

a synergistic improvementin the selectivity of gene expression.




• 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


3. Exogenous regulation of gene expression



GENE THERAPY APPLICATIONS

Disease targets for gene therapy clinical trials



Gene therapy treatment for Cancer



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.




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




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




• 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



• 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.




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



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



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



Healthy volunteers

for safety

Evaluate biological

effect

Safety surveillance after approvalValue in clinical practise

i



Retroviral vectors

1990

First ever therapeutic study in humans

Ex vivo approach

Adenosine deaminase deficiency

Adenoviral vectors



1993

First ever in vivo therapeutic study in humans

Cystic fibrosis

April 16 1993

Adeno Associated Viral vectors



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)



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



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



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



Summary







[email protected]

GENE TRANSFER AND GENE THERAPY Dr Greta Sawyer

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