Download Gene Therapy - Problems And Challenges

Gene Therapy - Problems and
Challenges
Ömer Faruk Bayrak
Gene Therapy
•
•
•
•
•
Background to Gene Therapy
Potential Benefits
Perceived Hazards and Risks
Regulations
Future?
2
What’s gene therapy?
• Imagine that you
accidentally broke one
of your neighbor's
windows.
I.
Stay silent: no one will ever find
out that you are guilty, but the
window doesn't get fixed.
II. Repair it with some tape: not the
best long-term solution.
III. Put in a new window: not only do
you solve the problem, but also
you do the honorable thing.
Many medical conditions result from flaws, or mutations, in
So, if a flawed
gene caused our "broken window,"
can you "fix" it? What are your
options?
one or more of a person's genes.
I.
II.
III.
Stay silent: ignore the genetic
disorder and nothing gets fixed.
Try to treat the disorder with drugs or
other approaches: depending on the
disorder, treatment may or may not
be a good long-term solution.
Put in a normal, functioning copy of
the gene: if you can do this, it may
solve the problem!
Background
• In the 1980s, advances in molecular biology had already enabled human
genes to be sequenced and cloned. Scientists looking for a method of
easily producing proteins, such as the protein deficient in diabetics —
insulin, investigated introducing human genes to bacterial DNA. The
modified bacteria then produce the corresponding protein, which can be
harvested and injected in people who cannot produce it naturally.
• Scientists took the logical step of trying to introduce genes straight into
human cells, focusing on diseases caused by single-gene defects, such as
cystic fibrosis, hemophilia, muscular dystrophy and sickle cell anemia,
optic nerve disease1, wound repair and regeneration2, and cardiovascular
disease3.
• However, this has been much harder than modifying simple bacteria,
primarily because of the problems involved in carrying large sections of
DNA and delivering it to the right site on the genome.
Gene Therapy
• Definition
The deliberate introduction of genetic material into
human somatic cells for therapeutic, prophylactic or
diagnostic purposes
– Addition of EXTRA genes
– Aim is to cure disease (or at least help the patient)
– First introduction of gene-modified cells into a patient was in
1989
– First gene therapy product approved for market in 2004
• Still very experimental and early in its development
5
What is gene therapy? Why is it used?
• Gene therapy = Introduction of normal genes
into cells that contain defective genes to
reconstitute a missing protein product
• GT is used to correct a deficient phenotype so
that sufficient amounts of a normal gene
product are synthesized  to improve a
genetic disorder
Diseases for applying gene therapy
Disease
Defect
Severe combined
Target cell
Bone marrow cells or
immunodeficiency
T-lymphocytes
Hemophilia
Liver, muscle
Cystic fibrosis
Lung Cells
Cancer
Many cell types
Neurological diseases
Parkinson’s/ Alzheimers
Infectious diseases
AIDS, hepatitis B
Nerve Cells
White Blood Cells
Defining Mutation Type
Oncogenes (active)
– Point mutations
– Amplification (Duplication)
– Formation of chimeric gene
Tumor Supressor Gene (inactivate)
– Loss of heterozygosity
Gene therapy could be
very different for different diseases
• Gene transplantation
(to patient with gene deletion)
• Gene correction
(To revert specific mutation in the gene of interest)
• Gene augmentation
(to enhance expression of gene of interest)
What is Vector?
• "Vector" is an agent that can carry a DNA
fragment into a host cell.
If it is used for reproducing the DNA fragment,
it is called a "cloning vector".
If it is used for expressing certain gene in the
DNA fragment, it is called an "expression
vector".
• Commonly used vectors include plasmid, Lambda
phage, cosmid and yeast artificial chromosome
(YAC).
•
Type of vectors:







•
Vectors designed for a certein purpose:




•
•
•
Plasmid
Lambda Phage
Cosmid
Fosmid
YAC
BAC
Viral Vectors
Expression Vectors
Plant Transformation Vectors
Epitope Tagging Vectors
Gene Silencing Vectors
Vector systems:
 Reporter Systems
 Selection Systems
 Inducible Systems
 Recombinase Based Systems
Special Vector Systems:
o Topo Vectors
o TA Vectors
o Gateway System
Transposons:
 Retrotransposons
 DNA transposons
Type of vectors:
Plasmid
Lambda Phage
Cosmid
Fosmid
YAC
BAC
Viral Vectors
Plasmid
• Plasmids are circular, indpendently replicating,
double-stranded DNA, most often found in
bacteria. They replicate quickly and are easily
manipulated in the laboratory. Plasmids are
typically 2-10 thousand base pairs in size
(Corbley, 1999). While this small size allows
plasmids the two aforementioned attributes, it
aslo means that plasmids are limited in the DNA
fragments they can clone. They are typically
limited to fragments around 5 thousand base
pairs (King, 2002).
Plasmid
Diagram of DNA sequence of a basic plasmid and incorporated construct.
Viral Vectors
• The efficient delivery of therapeutic genes and appropriate gene
expression are the crucial issues for clinically relevant gene therapy.
• Viruses are naturally evolved vehicles which efficiently transfer their
genes into host cells. This ability made them desirable for
engineering virus vector systems for the delivery of therapeutic
genes.
• The viral vectors recently in laboratory and clinical use are based on
RNA and DNA viruses processing very different genomic structures
and host ranges. Particular viruses have been selected as gene
delivery vehicles because of their capacities to carry foreign genes
and their ability to efficiently deliver these genes associated with
efficient gene expression.
• These are the major reasons why viral vectors derived from
retroviruses, adenovirus, adeno-associated virus, herpesvirus and
poxvirus are employed in more than 70% of clinical gene therapy
trials worldwide.
Retro Viral Vectors
•
Retrovirus vectors represent the most prominent delivery system,
since these vectors have high gene transfer efficiency and mediate
high expression of therapeutic genes.
• Efficient gene transduction and integration depend on the inclusion
in the retroviral vector of a number of cis-acting viral elements.
(1) a promoter and polyadenylation signal in the viral genome;
(2) a viral packaging signal (ψ or E) to direct incorporation of vector
RNA into virions;
(3) signals required for reverse transcription, including a transfer RNAbinding site (PBS) and polypurine tract (PPT) for initiation of firstand second-strand DNA synthesis, and a repeated (R) region at
both ends of the viral RNA required for transfer of DNA synthesis
between templates;
(4) short, partially inverted repeats located at the termini of the viral
LTRs required for integration. An important general consideration
in the design of retroviral vectors is the effect of viral replication
on vector structure. After one round of viral replication, the U3
regions in both LTRs are derived from the U3 region originally
present in the 3′LTR in the plasmid form of the vector, and both
U5 regions are derived from the U5 region originally present in
the 5′LTR in the plasmid. Ordinarily, R sequences should arise
primarily from the 5′plasmid LTR, but they may also include
3′plasmid LTR sequences.
Lenti Viral Vectors
• Lentiviral vector constructs have proven to be
very productive in terms of transduction due to
their ability to infect both replicating and nonreplicating cells, including stem cells. Lentiviral
vectors are becoming the vectors of choice for
short-interfering RNA (siRNA) delivery (Sachdeva
et al., 2007).
• The increased use of lentiviral vector constructs
in established and novel research applications
makes it essential for laboratory workers to
understand and protect themselves from related
exposure hazards.
Adeno Viral Vectors
• Adenoviruses are medium-sized (90–100 nm),
nonenveloped icosahedral viruses composed
of a nucleocapsid and a double-stranded
linear DNA genome. There are over 51
different serotypes in humans, which are
responsible for 5–10% of upper respiratory
infections in children, and many infections in
adults as well. When these viruses infect a
host cell, they introduce their DNA molecule
into the host. The genetic material of the
adenoviruses is not incorporated (transient)
into the host cell's genetic material. The DNA
molecule is left free in the nucleus of the host
cell, and the instructions in this extra DNA
molecule are transcribed just like any other
gene. The only difference is that these
extra genes are not replicated when the cell is
about to undergo cell division so the
descendants of that cell will
not have the extra gene.
Vector systems:
 Reporter Systems
 Selection Systems
 Inducible Systems
 Recombinase Based Systems
Reporter Systems
• A gene consists of two functional parts: One is a DNA-sequence that
gives the information about the protein that is produced (coding
region). The other part is a specific DNA-sequence linked to the
coding region; it regulates the transcription of the gene (promoter).
The promoter is either activating or suppressing the expression of
the gene.
• The purpose of the reporter gene assay is to measure the
regulatory potential of an unknown DNA-sequence. This can be
done by linking a promoter sequence to an easily detectable
reporter gene such as that encoding for the firefly luciferase.
• Common reporter genes are β-galactosidase,
β-glucuronidase and luciferase. Various
detection methods are used to measure
expressed reporter gene protein.
These include luminescence, absorbance
and fluorescence.
GFP
Luciferase
Selection Systems
• Selection markers are protein coding sequences that confer a
selective advantage or disadvantage to host chassis. For example, a
common type of prokaryotic selection marker is one that confers
resistance to a particular antibiotic. Thus, cells that carry the
selection marker can grow in media despite the presence of
antibiotic.
• Most plasmids contain antibiotic selection markers so that
researchers can ensure that the plasmid is maintained during cell
replication and division. (Cells that lose a copy of the plasmid will
soon either die or fail to grow in media supplemented with
antibiotic.)
• A second common type of selection marker, often termed a positive
selection marker, are those that are toxic to the cell. Positive
selection markers are frequently used during cloning to select
against cells transformed with the cloning vector and ensure that
only cells transformed with a plasmid containing the insert.
Positive-Negative Selection
• A strategy used in making gene knockouts designed to
enrich for homologous recombinants and select against
random integration of the targeting vector. Cells
transfected with the targeting vector incorporating two
selectable markers are first selected for their resistance
to an antibiotic such as G418 (positive selection) and
then for the loss of a second marker, such as HSV
thymidine kinase (HSVtk), which confers sensitivity to
ganciclovir (negative selection). HSVtk placed at one
end of the linear targeting vector is lost on
homologous recombination but is retained on random
integration.
Positive-negative selection using neomycin and
thymidine kinase (TK)
• What are G418 and ganciclovir used for?
• The neomycin gene confers resistance to G418 while TK
renders cells susceptible to ganciclovir. Neomycin is used as a
positive selection marker while TK is used as a negative
selection marker.
• Generally, they are used
together in a strategy
commonly referred to as
‘positive-negative selection’.
Gene therapy
In vivo
Ex vivo
Different Delivery Systems are Available
• In vivo versus ex vivo
– In vivo = delivery of genes takes place in the body
– Ex vivo = delivery takes place out of the body, and
then cells are placed back into the body
in vivo and ex vivo schemes
EX VIVO
IN VIVO
http://laxmi.nuc.ucla.edu:8237/M288/SChow_4_10/sld005.htm
In vivo gene therapy
1. The genetic material is transferred directly into the
body of the patient
2. More or less random process;
small ability to control; less manipulations
3. Only available option for tissues
that can not be grown in vitro;
or if grown cells can not be transferred back
Ex vivo gene therapy
1. The genetic material is first transferred
into the cells grown in vitro
2. Controlled process;
Genetically altered cells are selected and expanded;
more manipulations
3. Cells are then returned back to the patient
• In vivo techniques usually utilize viral vectors
– Virus = carrier of desired gene
– Virus is usually “crippled” to disable its ability to cause
disease
– Viral methods have proved to be the most efficient to date
– Many viral vectors can stable integrate the desired gene
into the target cell’s genome
Side Effects could be…
Replication defective viruses adversely affect the
virus’ normal ability to spread genes in the body
• Reliant on diffusion and spread
• Hampered by small intercellular spaces for transport
• Restricted by viral-binding ligands on cell surface 
therefore cannot advance far.
• Retroviruses convey a risk of insertional mutagenesis
Gene Therapy Vectors
• Vectors deliver genes to cells
Therapeutic gene
(Transgene)
Transcription
Vector for efficient gene delivery
Translation
Therapeutic
protein
34
Types of Gene Therapy Vectors
• Non-viral vectors
– Naked DNA
– Liposomes/DNA
– Polymer/DNA complex
(polyplex)
– Liposome/Polymer/DNA
(lipopolyplex)
• Viral vectors
– DNA viruses
• Adenovirus
• Herpes Simplex Virus
– RNA viruses
• Retrovirus
35
D. Limitations of Gene Therapy
• Gene delivery
– Limited tropism of viral vectors
– Dependence on cell cycle by some viral vectors
(i.e. mitosis required)
• Duration of gene activity
– Non-integrating delivery will be transient
(transient expression)
– Integrated delivery will be stable
Liposomes
Next level idea – why naked DNA?
Lets’ wrap it in something safe
to increase transfection rate
Lipids – are an obvious idea !
Therapeutic drugs
DNA delivery of genes by liposomes
Cheaper than viruses
No immune response
Cytotoxicity
100-1000 times more plasmid DNA needed
for the same transfer efficiency as for viral vector
Gene Therapy Strategies
1) Gene Replacement
•
•
•
•
Replace ‘faulty’ genes with normal genes
Corrects inherited genetic errors
Provides a missing function
Monogenic diseases e.g. cystic fibrosis,
haemophilia, X-SCID
2) Gene Addition
•
•
Delivers genes to provide a new function
Polygenic diseases e.g. cancer
39
PTQA April 2008
40
December 19, 2007
Boy gets leukaemia after gene treatment
to cure ‘bubble baby syndrome’
• 3 year-old with X-linked severe combined immunodeficiency
(XSCID) - immune system fails to develop
• Treated with genetically modified virus to correct the faulty DNA that
causes X-SCID
• Inserting the replacement DNA activated another gene that promotes
cancer
• Now an acknowledged risk of gene therapy
Also seen in 4 / 11 patients in a French trial
One has died while 3 are in remission
Retrovirus vector
APPENDIX 6 GENE THERAPY
•
•
•
•
•
Facilities
Documentation
Labelling
Training
Aseptic
processing
•
•
•
•
Cleaning
Storage
Transport
Waste
Disposal
• Spillage
42
Facilities
– Gene therapy should not be manipulated in clinical
areas
– Basic Principles - Containment
- Knowledge/ understanding/skill
- Validated procedures
• Persons handling the product should be masked and gloved
• All disposable equipment and materials used for prep & admin handled as biohazardous
• Dedicated facilities required
– -ve pressure isolators or Class II BSC
– +ve pressure room or lobby
– Containment level > 2
43
Clean room suite
designed to
provide protection
to the cleanroom
Aseptic Manipulation
– Doses
• Calculation/dilutions/multiple dilutions
• Needle stick injury risk
• Units
– Particle Units/ml (PU/ml)
– Plaque Forming Units/ml (PFU/ml)
– Infectious particle Units/ml (IU/ml)
– Gene Transfer Units/ml (GTU/ml)
– Stability
• Container compatibilities - Plastic/glass
adhesion
• Expiry date - Time to administration from thawing
45
Decontamination
• Cleaning
– Virucidal detergents (validated against GT vectors)
• Cleaning Validation
– Specific Detection methods needed for viruses that
are virus specific and highly sensitive
• Waste Disposal
• On site validated autoclave for re-usable
equipment
• Inactivation on-site for Class 3 vectors
– Validated autoclave
– Incineration
– Disinfectant treatment
46
Accidental Exposure
• Spillage
– Specific to GT vector
– Spillage kit
• Contents ( gloves, masks, aprons, goggles,
disposable shoe covers, virucidal detergents,
absorbent material, disposable forceps &
biohazard incineration bag)
• Positioned in all GT handling areas
• Notification to HSE
47
SOPs needed
– Safe handling & protection
– Storage
– Operators
(Not pregnant, breastfeeding or immunosuppressed)
– Training
– Facilities
– Spillage, contamination & needle stick
– Waste disposal, cleaning and transport
48
Risk Assessment
• Assess each product individually
– Cytolytic viruses
– Non-cytolytic viruses
– Replication competent
– Replication deficient
– Class I, II or III
49