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: [email protected]
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
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