Principles and perspectives

of gene therapy

Prof. Józef Dulak, PhD, DSc

Department of Medical Biotechnology

Faculty of Biochemistry, Biophysics and Biotechnology

Room 3.025/3.07

Phone 664-63-75

Email: jozef.dulak@uj.edu.pl

Lectures can be downloaded from the web page –

Department of Medical Biotechnology

at http://biotka.mol.uj.edu.pl/zbm/

Conditions for positive outcome...

1. Learning and understanding the information delivered during the lectures

2. Asking the questions during and after the lecture!

3. Supplementary materials:

a) articles available in the library – in English

b) articles distributed during the course

b) „Gene transfer to animal cells” – several copies are in the library

For Polish students:

b) „Terapia genowa” red. Stanisław Szala (PWN, 2003) (

c) „Biotechnologia” – No 3/2007 (several copies are available in the library)

4. Passing the final exam …

atherosclerotic

artery healthy artery AAV vectors

adenoviral vectors

http://biotka.mol.uj.edu.pl/zbm

Treatment of atherosclerosis

Stimulation of new blood vessels

(helpful eg. in myocardial infarction)

without therapy with therapy

plasmid vectors

retroviral vectors

Department of Medical Biotechnology

Gene and cell therapy in vascular system

Dulak et al., Eur Surgery 2002, 34: 105-110; Józkowicz et al., Int J Artif Organs, 2003: 26: 161-169

Final exam

Test – single and/or multiple choice…

What is gene therapy?

Application of nucleic acids for treatment of diseases

Gene therapy

Therapeutic

gene

(transgene)

Vector Patient Expression of therapeutic gene

Protein

Which diseases could be cured with gene therapy?

Is gene therapy necessary?

HPRT-/- cells

HAT medium

Prof.Wacław Szybalski

McArdle Laboratory

for Cancer Research,

Wisconsin, Madison,

USA

Gene therapy was born in... 1962

HPRT+/+ cells

De novo and salvage pathways for nucleotide synthesis

De novo pathways

Salvage pathways

HAT medium

A selection medium for hybrid cell lines; contains hypoxanthine;

aminopterin; thymidine. Only cell lines expressing both

hypoxanthine phosphoribosyl transferase (HPRT+) and thymidine

kinase (TK+) can survive in this medium. Aminopterin inhibits de

novo synthesis of nucleosides, while HPRT and TK supply them from

hypoxanthine and thymidine.

HAT medium is used to

select hybridoma cells

producing monoclonal

antibodies

Lesch-Nyhan syndrome

HPRTHPRT

Inborn error of metabolism – deficiency of HPRT

allopurinol

What is Lesch-Nyhan Syndrome?

Lesch-Nyhan syndrome (LNS) is a rare, inherited disorder caused by a deficiency of the enzyme

hypoxanthine-guanine phosphoribosyltransferase (HPRT). LNS is an X-linked recessive disease-

- the gene is carried by the mother and passed on to her son. The lack of HPRT causes a build-up

of uric acid in all body fluids, and leads to symptoms such as severe gout, poor muscle control,

and moderate retardation, which appear in the first year of life. A striking feature of LNS is self-

mutilating behaviors – characterized by lip and finger biting – that begin in the second year of

life. Abnormally high uric acid levels can cause sodium urate crystals to form in the joints,

kidneys, central nervous system, and other tissues of the body, leading to gout-like swelling in the

joints and severe kidney problems. Neurological symptoms include facial grimacing,

involuntary writhing, and repetitive movements of the arms and legs similar to those seen in

Huntington’s disease. Because a lack of HPRT causes the body to poorly utilize vitamin B12,

some boys may develop a rare disorder called megaloblastic anemia.

Is there any treatment?

Treatment for LNS is symptomatic. Gout can be treated with allopurinol to control excessive

amounts of uric acid. Kidney stones may be treated by breaking up kidney stones using shock

waves or laser beams. There is no standard treatment for the neurological symptoms of

LNS. Some may be relieved with the drugs carbidopa/levodopa, diazepam, phenobarbital, or

haloperidol.

What is the prognosis?

The prognosis for individuals with LNS is poor. Death is usually due to renal failure in the first

or second decade of life.

Children suffering from deficiency

of HPRT-

Lesh-Nyhan syndrome

Children suffering from deficiency of HPRT- Lesh-Nyhan syndrome

Nat Genet. 1993 Mar;3(3):235-40. Links

Wu CL & Melton DW,

Production of a model for Lesch-Nyhan syndrome in

hypoxanthine phosphoribosyltransferase-deficient mice.Institute of Cell and Molecular Biology, Edinburgh University, Scotland.

The inherited disease Lesch-Nyhan syndrome, which is caused by a deficiency of the

enzyme hypoxanthine phosphoribosyltransferase (HPRT), is characterized by behavioural

alterations, including self-injurious behaviour and mental retardation. Although HPRT-

deficient mice have been generated using the embryonic stem cell system, no spontaneous

behavioural abnormalities had been reported. We examined whether mice were more

tolerant of HPRT deficiency because they were more reliant on adenine

phosphoribosyltransferase (APRT) than HPRT for their purine salvage. The administration

of an APRT inhibitor to HPRT-deficient mice induced persistent self-injurious behaviour.

This combined genetic and biochemical model will facilitate the study of Lesch-Nyhan

syndrome and the evaluation of novel therapies.

Why there is no effective treatment for Lesh-Nyhan syndrome?

-Reasons for neurological distrubances are not well known

(mouse models of HPRT-deficiency do not replicate human

phenotype)

- Gene therapy – not available

Devlopment of gene therapy

- Tools: vectors

- Mechanisms of diseases: genes are known

Main problems to solve in gene therapy

1. Efficient delivery of therapeutic gene

2. Safe delivery…

All is about vectors…

vectornucleic acid, which is used to deliver the therapeutic gene/therapeutic

nucleic acid

Vehicle

A chemical substance, which improves delivery of nucleic acid to the

cells

However, a term „vector” is also used interchangeably for

chemical vehicles.

Genetic syringes - vectors

Retroviral vectors Adenoviral vectors

AAV vectors

(adeno-associated)

Plasmid DNA

Vectors

Non-viral/plasmids Viral

RNA DNA

Retroviruses

(including

lentiviruses)

Adenoviral

AAV

Herpes

„naked” DNA

Lipoplexes

Viroplexes

(lipoplexes enriched

In specific viral proteins)

complexes with

other chemicals

DNA RNA

Genes

(DNA vectors)

Non-coding sequences

Antisense

oligonucleotides

DNA decoys

Genes

(RNA vectors)

Non-coding

sequences

ribozymes siRNA

Therapeutic nucleic acids

Gene therapy

Enhancing Substituting Suppresive

enhancement delivery of inhibition of

of gene expression the missing gene gene expression

Acquired diseases

Inherited diseasesAcquired diseases

Types of gene therapy

-Somatic

- germ line

Strategies of gene therapy – in vivo and ex vivo

Other useful terms

Transfection

Transduction

Molecular cloning

Introduction of foreign genes to eucaryotic cells by means of plasmid vectors

Transformation of bacteria

Introduction of foreign genes into bacteria

Introduction of genes by means of viral vectors

Production of numerous copies of a gene, by methods of genetic engineering,

eg. Transformation of bacteria with plasmid, PCR cloning

-

* physical methods (transfection)

* chemical methods (transfection)

* biological methods (transduction)

Nucleic acids can be introduced to the cells using

A. Crossing plasmalemma

* Cell membrane has a negative charge, as DNA or RNA, which blocks

attachment of nucleic acids to the surface of membrane.

* Both DNA and RNA are hydrophilic, which blocks the direct fusion

with lipid plazmalemma.

B. Release from phagosomes (if nucleic acid is taken by phagocytosis).

C. Dissociation of nucleic acid from the chemical carrier (if chemical methods

are used)

D. Transportation to the nucleus (can be improved by adding ORI sequence

from SV40 virus to the nucleic acid or adding to the chemical vehicle a

protein with nuclear localization signal, NLS, e.g. SV40 large T

antigen).

Most often, the rate limiting steps are B and D.

Barriers to be overcome by nucleic acid in the cells

Bariers decreasing transfection efficiency in vivo

I. Extracellular barriers II. Cellular/intracellular barriers

1. Opsonins 1. Cell membrane

2. Phagocytic cells 2. Endosomes/lizosomes

3. Extracellular matrix/mucus 3. Nuclear membrane

4. Digestion enzymes

Plasmids

the main tools of gene therapy

Plasmids are always in the begining…

Organisation of a typical plasmid vector

promoter

Plasmids are bacterial of origin, so they have to be modified to act in eucaryotic cells

- The most often used plasmids do not replicate in the eukaryotic cells.

- Some plasmids contain sequences, which enable replication in the permissive cells.

There can be:

* replicon from viruses of family Papovaviridae.

* sequence from simian virus SV40 (the most often used) - in monkey cells

(rhesus or green african monkey)

* sequence from mouse polyomavirus (MPV) - in murine cells

* sequence from bovine papilloma virus (BPV) - in bovine and murine cells

* sequence from human polymoma BK virus (BKV) - in human cells

- These sequence can be not sufficient to allow proliferation. For example for proper

work of SV40 replicon the presence of SV40 large T antigens is necessary. Thus:

* the plasmid should contain the sequence encoding large antigen T sequence

* experiments should be performed in COS cell lines (green african monkey

fibroblasts), containing integrated genome of SV40 virus. In this case, under optimal

condition even 100 000 copies of plasmids per cell can be obtained.

Self-replicative plasmids

- In most cases plasmid DNA exists as an episomal element and is gradually

degraded. Additionally it is lost during the replication of cells, thus the

percentage of transduced cells gradually decreases. Therefore the effects of

transfection are short-term.

Transient and stable transfection

Transient transfection

- Some vectors - especially retroviral vectors - introduce the transgene to the

host genome. Such DNA replicates together with the rest of chromosome and is

traded to the all doughter cells. Therefore, the effects of transfection are long-

term.

- Integration can occur also for other vectors e.g. plasmids or adenoviral

ones, but this process is very uneffective ranging from 1 per 100 to 1 to 1000

successfuly transduced cells.

- Transgene usually builds into more-less random site of genome.

- Site-specific integration using a homologous recombination mechanism is

possible, but more diffucul and rarerly used.

Stable transfection

How to construct a plasmid with a desired gene,

which we do not have...

Source of the gene:

Other scientists – simply ask... – the most common way...

Cloning on your own – use RT-PCR

Synthesize from scratch (on the basis of the sequence deposited

in GeneBank

RT-PCR is a preferable way for gene cloning

A piece of target DNA can be inserted into a plasmid if both the circular plasmid and the target DNA have been

cleaved by the same restriction nuclease in such a way as to create sticky ends. The newly created recombinant

molecule is stabilized with the DNA ligase enzyme which repairs nicks in the backbone of the DNA molecule.

DNA cloning

Isolation of DNA fragments from a mixture by cloning in a plasmid vector

Transformation of bacteria

DNA cloning

How to make a plasmid working

in mammalian cells?

Expression of foreign genes in eucaryotic cells

requires the eucaryotic promoter

in a plasmid vector

1. Viral promoters : CMV, SV40

2. Eucaryotic promoters

- constitutive:

- non-selective: -globin

- tissue specific:eg. Tie-2 (in endothelial cells;

MHC – myosin heavy chain – in cardiomyocytes)

- inducible

3. Complex

There will be more about promoters during one of the next lectures

What’s more?

Poly-A is required for expression of genes

in mammalian cells

AAUAAA

Poly(A) signal is located on the

pre-mRNA about 20 nt upstream

of actual site of cleavage.

Several cleavage and polyadenylation factors

bind to pre-mRNA.

Cleavage occurs (thick arrow) usually

at an A nucleotide.

A variant G is found at this position in the

prothrombin gene (1-2% of human population)

resulting in weaker cleavage efficiency (thin arrow).

The strength of a poly(A) signal can directly influence

the level of gene expression

increased risk of thrombosis

Gill DR et al., Gene Therapy 2009

Mammalian expression plasmid

Take home message

1. Gene therapy defintion

2. Diseases that gene therapy may treat

3. Beginings of gene therapy

4. Vectors for efficient gene delivery

5. Examples of effective gene therapy

6. Some limitations of gene therapy

Questions?

Information about gene therapy

European Society of Gene Therapy

www.esgt.org

American Society of Gene Therapy

www.asgt.org