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Gene Therapy: An Overview of Delivery, Control and Testing
1. Gene therapy
Definition: delivery and expression of genetic
material leading to an alteration in the instruction
set of a cell for a therapeutic purpose
1960’s ‐ idea of gene therapy proposed
1990 ‐ first human gene therapy clinical trial
2010 ‐ still no gene therapy product commercially
available
Somatic vs germ‐line gene therapy
Gene therapy ‐ making it work
What genetic material to deliver?
Where to deliver it?
How to deliver it?
How to control it?
How to test it?
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2. Key goals for a gene therapy strategy
Easy to administer.
Long‐term production of a therapeutic protein or
therapeutic effect following a single application.
Highly specific, targeted to disease cells/tissue only,
thereby little or no side‐effects.
What genetic material to deliver?
Genes and RNA interference sequences
Correcting faults by:
Replacing a non‐functional gene with a functional copy
Silencing an abnormally functioning gene
Altering the phenotype of a cell
Need to know something about the disease process
Inherited or acquired, acute or chronic
Treatment outcome
Cure, alter natural history, alleviate symptoms
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3. Where to delivery it?
During & Tipene-Hook,
New Ethicals 1997 May, 57-65
Factors to consider
Many diseases affect multiple sites
Effects on circuitry e.g brain
Gene may need to be delivered to widespread areas
or restricted to a specific organ/group of cells
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4. Getting genes into cells: gene delivery
Ex vivo ‐ gene transfer performed
in cell culture before
transplantation
In vivo ‐ gene transfer performed
in situ
Therapeutic gene ‐ transgene
Success of gene therapy
dependent on efficient gene
transfer
Gene transfer mediated by gene
delivery vehicles (=vectors)
www.biochem.arizona.edu/. ../Lecture25.html
Gene delivery‐ non‐viral vectors
Plasmid DNA
Liposomes
DNA mixed with cationic lipids and polymers
(polylysine, protamine) becomes encased
within a lipid bubble.
Cellular uptake via an endocytic process.
No restriction on size of gene, easy and
cheap to manufacture.
Main disadvantages are relative inefficiency
in gene delivery and transient expression
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5. Gene delivery ‐ viral vectors
Exploits natural ability of viruses to infect
mammalian cells
Genetically engineered to carry a transgene
cassette and deposit it within a cell.
Engineered for a single round of host cell infection
by removal of viral genes involved in replication.
VIRUS
VIRAL PROTEINS
ASSEMBLY SPREAD OF
INFECTION
VIRAL GENES
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6. Life cycle of a viral vector
VECTOR
GENE OF INTEREST
DISPLACED VIRAL GENES
PROTEIN OF
INTEREST
NO VIRAL GENES NO NEW VIRUS
OR VIRAL
PROTEINS
Engineering vectors from viruses
Molecular cloning techniques used to genetically
engineer a virus to carry a transgene cassette
e.g. AAV rep and cap genes are removed and
replaced with a transgene gene cassette
Therapeutic genes are controlled by promoters,
regulatory elements
Cell lines are used to package vector particles
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7. AAV vector transgene cassette
e.g AAV expression cassette
ITR rep cap ITR
Wild-type AAV
ITR transgene expression cassette ITR
Recombinant AAV
145 1100 <1600 600 300 145bp
ITR promoter transgene RE pA ITR
ITR – AAV inverted terminal repeat
RE – regulatory element, e.g. woodchuck postregulatory transcriptional element (WPRE)
pA – polyA signal
Viral vectors ‐ types
Retrovirus
Herpes simplex virus
Adenovirus
Adeno‐associated virus (AAV)
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8. Retrovirus
7‐11kb ss RNA; Following target cell entry, RNA genome is reverse
transcribed into ds DNA which integrates into the cell chromatin
Efficient integration of transgene into chromosome leading to stable
gene expression which persists in parent and daughter cells.
Good for ex vivo approaches.
Main disadvantage ‐ potential for random integration near an
oncogene. Also unable to infect non‐dividing cells as it requires one
mitotic division for integration and expression of the transgene.
Lentivirus (HIV) ‐ subclass of retroviruses, infects both dividing and
non‐dividing cells, stable and long‐term gene expression.
Adenovirus
36kb dsDNA
Transduces non‐dividing and dividing cells
High titer vector produced
Main disadvantage ‐ transient transgene expression
due to immune responses directed against
adenoviral proteins; may be overcome by producing
“gutless” adenoviral vectors
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9. Herpes simplex virus (HSV1)
152kb linear ds DNA
Broad host cell range
Main disadvantage ‐ intrinsic toxicity and contamination of vector
stocks with wild‐type (pathogenic) virus. Difficulties in maintaining
long‐term gene expression
Adeno‐associated virus (AAV)
4.7kb ss DNA
>40 serotypes isolated from human, primates
Non‐pathogenic
Transduces non‐dividing and dividing cells
Long‐term gene expression (>2.5 yrs)
Main disadvantage ‐ small size
How do you choose the right vector for
the job?
Dependent on cell targets
Dividing vs non‐dividing cells, e.g stem cells and
post‐mitotic cells difficult to transduce, rapidly
dividing epithelial and cancer cells easy to transduce
Transduction = vector‐mediated gene transfer
Vector tropism = selectivity for a cell type
Dependent on the ligand present on a vector and
whether the target cell expresses the appropriate
cell‐surface receptor
Gene insert size
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10. Achieving specificity ‐ vector targeting
Needed for:
Safety – minimises potential toxicity of gene product in healthy cells
Increases gene transfer efficiency – want to minimise amounts of
vector needed to produce gene product at therapeutic levels
Achieved by:
Mode of delivery e.g. direct injection into site of interest
Vector selection
Exploit or modify specificity of viral vectors for certain cell types
e.g AAV serotypes (human, primate)
Pseudotype vectors
Modify capsid shell that allows binding to different receptors
AAV2
AAV1/2
eGFP expression in the hippocampus brain region following AAV2
vector or AAV1/2 vector-mediated gene transfer
How to control gene expression?
Why is it necessary?
Figure 2. Transduction of the target cell.The vector
particle containing the therapeutic gene sequences
binds to a cell, generally through a receptor-mediated
process and then enters the cell, allowing the genome
to enter the nucleus. The vector genome may go
through complex processes but ends up as dsDNA that,
depending on the vector, can persist as an episome or
become integrated into the host genome. Expression of
the therapeutic gene follows.
Kay et al. 2001. Nat. Med., 7, 33-40.
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11. Gene expression
Recombinant protein
Level of transgene expression
determined by promoter choice
and inclusion of other cis‐acting
DNA elements.
Aim is to achieve stable, long‐
term transgene expression.
Gene therapy
Regulating transgene expression
Gene therapy
Essential in maintaining
transgene protein expression at
steady therapeutic levels
Enables flexibility in adjusting
dosage as disease evolves or for
therapies tailor‐made for
individual patients
Built in safety mechanism
Gene therapy - regulated
Clackson, Gene Therapy, 7, 120-125, 2000.
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12. Regulatory systems
Endogenous promoter‐regulated transcription systems that are responsive to
physiological stimuli
e.g. glucose responsive element ‐ control insulin release
Exogenous drug‐regulated transcription system
e.g. Rapamycin, ecdysone, tet system
Ideal regulatory system
Low basal expression
Be inducible to high levels over a wide dose range to provide a useful dose
responsiveness
Induction should be a positive effect ‐ i.e administer drug to switch it on
Drug should be active following oral administration and have no pleiotropic
effects in mammalian cells
Regulatory protein should have no effects on endogenous gene expression
and be of human origin to minimise immunogenicity
Tet On/Off regulatory system
Transactivator ‐ regulatory
element – expresses the
tetracycline‐controlled
transactivator (rtTA) which is
a chimeric protein composed
of the Tet repressor fused to
the VP16 activation domain
of HSV.
Tet‐responsive element
(TRE) is upstream of a silent
promoter driving the
transgene of interest.
Adapted from: http://www.bdbiosciences.com/clontech/tet/index.html
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13. How to test a gene therapy strategy?
Test functionality of therapeutic gene cassettes
In vitro and in vivo animal models of disease.
How adequate are those models and do they reflect
disease processes in humans?
Need measurable endpoints to assess whether
gene therapy has therapeutic effect and benefit
Assays for quantifying and/or visualising
therapeutic protein levels
Current status of gene therapy
Toxicity of some viral vector systems
Immune responses directed against cells containing foreign
proteins (viral) leading to elimination of the therapy. Thus the
therapy may be short‐lived.
Enhanced immune responses against vectors encountered
previously may mean problems in re‐dosing.
Potential for some vectors to recover ability to cause disease.
Vector targeting not optimised – transgenes expressed in
healthy and diseased cells
Safety mechanisms/control of gene expression not sufficiently
optimised and long‐term effects unknown
Multigene disorders
Best candidates for gene therapy are those that arise from
single gene mutations. Many common diseases (e.g Alzheimer’s,
heart disease) involve effects on a variety of genes.
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14. Viral vectors: tools for gene function
studies
Gene overexpression
Express poorly characterised genes to gain a better
understanding of their physiological effects
Generating animal models of disease e.g.Parkinson’s
Huntington’s, Alzheimer’s diseases
Gene knockout
RNA interference
Advantages over transgenic animals
Can be engineered to express single or two foreign genes, different
regulatory elements and promoters
Can be administered at any developmental stage ‐ from in utero to adult
to senescent animals
Either short or long‐term CNS gene expression
Expression can be obtained in crucial brain regions while possible side‐
effects associated with widespread overexpression of the gene can be
avoided
Not host‐specific ‐ rats, primates, mice
Inexpensive and rapid to generate compared with classical transgenics
Higher levels of transgene expression obtained
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15. Combined use of germline and viral
transgenic methods
Can be combined with transgenic mouse models for
studies of interactions of disease‐causing proteins
with other cellular proteins
Confirm gene specificity with transgenic knockouts.
Use viral vector‐mediated gene transfer of the
missing gene to rescue the phenotype
Observe further potentiation or downregulation of
gene effects
Affect regulation of genes in different tissues or
brain regions
Rat and primate models of Parkinson’s
disease
AAV vectors used to overexpress wild‐type and
mutant forms of α‐synuclein in the substantia nigra
of rats and primates
Show many of the pathological features of PD
• protein inclusions
• dystrophic neurites
• progressive loss of TH cells in the SNpc
• drug‐induced rotation and motor deficits
2
16. Kirik D & Bjorklund A, Trends Neurosci. 26(7)
2003, 386-392
RNA interference (RNAi)
A form of post‐transcriptional gene silencing in which specific sequences
of double‐stranded RNA (dsRNA) can be used to knock down the
expression of a gene target
Adapted as a tool to investigate gene function
History of RNAi
http://www.invitrogen.com
/content.cfm?pageid=10088
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17. Biochemical mechanism of RNAi
1. dsRNA is introduced into the cell
2. DICER digests dsRNA into ~21bp
dsDNA (short‐interfering RNAs;
siRNAs)
3. The siRNAs are integrated into
the RNA Induced Silencing
Complex (RISC)
4. siRNAs undergo strand
separation. The antisense strand
binds to its complementary/
target mRNA
5. Argonaute ‐ endonuclease
within the RISC degrades the
http://www.ambion.com/techlib/tn/101/7.html
targeted mRNA
Using RNAi as a tool for manipulating
gene expression in mammalian cells
In mammalian cells, introduction of long dsRNA initiates
a cellular interferon response that ultimately causes cell
shutdown and leads to apoptosis
RNAi can be achieved in mammalian cells by direct
delivery of:
synthetic short‐interfering RNA (siRNA)
synthetic short‐hairpin RNA (shRNA)
microRNA (miRNA)
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18. siRNA
21‐23 bp of double‐stranded RNA with a 3’ dinucleotide overhang
Algorithms are used to design RNAi sequences (guidelines provided e.g
30‐50% G/C content).
3‐5 siRNA sequences covering the gene of interest are selected and tested
for the degree of suppression of gene expression.
siRNA ‐ synthesis, delivery, effect
Synthesis in vitro
Chemical synthesis
In vitro transcription systems
Delivery
Cell culture ‐ transfection, electroporation
In vivo models ‐ injection or direct
application
RNAi effect ‐ transient (3‐7 days), partial to
full knockdown of gene expression
Davidson BL, Paulson HL.
Lancet Neurol. 2004 Mar;3(3):145-9.
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19. shRNA
siRNA template
Termination sequence
Promoter/enhancer
• Hairpin siRNA
• Pol III: string of 3-5 T’s
• Pol III, (U6, H1)
• Sense and antisense
• Minimal SV40 poly A
• Pol II, (CMV)
signal
strand of siRNA
shRNA
Delivery
Plasmids
Viral vectors
Effect
Cell culture ‐ transient knockdown
In vivo ‐ long‐term (>1 week) effects
using viral vectors
Davidson BL, Paulson HL.
Lancet Neurol. 2004 Mar;3(3):145-9.
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20. MicroRNA (miRNA)
Endogenous RNAi pathway in animal cells
Endogenous ~21‐mer small RNA molecules from non‐coding RNA (introns,
independent miRNA genes)
Regulate gene transcription by binding to the 3’‐untranslated regions of
specific mRNAs
Key regulators of early development, cell proliferation, cell death,
apoptosis, cell differentiation, brain development
miRBASE ‐ http://microrna.sanger.ac.uk/
3000 miRNA sequences from various species
Sequences of many miRNAs are homologous between species
800 unique miRNAs in humans with 400‐500 conserved in mice
Regulate expression of at least 30% of protein‐coding genes
miRNA processing
Transcription of pri‐miRNA
Processing into pre‐miRNA in the
nucleus by Drosha
Export of pre‐miRNA to the
cytoplasm
Dicer complex processing
miRNA strand selection by RISC
http://www.ambion.com/techlib/resources/miRNA/mirna_pro.html
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21. miRNA reduce steady state protein levels
Translational repression
Imperfect duplex
Reduces protein expression
without impacting on
corresponding mRNA
levels. (mechanism still
unclear)
mRNA degradation
Perfect duplex
Transcriptional regulation
guiding chromatin
methylation
http://www.ambion.com/techlib/resources/miRNA/mirna_fun.html
Using artificial miRNA as a tool for gene
function studies
Similar to applications for siRNA/shRNA
Invitrogen ‐ BLOCK‐iT™ system
https://catalog.invitrogen.com
/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=12492
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22. Potential Applications of RNA Interference
Testing hypotheses of gene function
Functional screening and target identification
Target validation for drug development
Potentially new therapeutic approaches to treating
diseases ‐ a new approach to antisense and new
possibilities for gene therapy
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23. Gene therapy for Parkinson’s disease
Parkinson’s disease
Progressive neurodegenerative disorder affecting
~1% of the population over the age of 65
Two types: sporadic and familial
Clinical symptoms: disorders in movement (resting
tremor, rigidity, akinesia, bradykinesia). Non‐motor
symptoms include depression and dementia.
Pathology
Selective degeneration of dopaminergic neurons within the substantia nigra
pars compacta (SNpc) that project axons to the striatum
Lewy bodies ‐abnormal protein aggregates in the cytoplasm of neurons
Dopamine depletion in the striatum causes disorders in movement
Focal pathology but has impact on overall basal ganglia circuitry
Adapted from Lindvall & Bjorklund. Nat. Med. 2000. 6, 1207-1208
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24. What causes these dopamine cells to die?
Still largely unclear. Possible contributors:
Oxidative stress
Mitochondrial abnormalities
Excitotoxicity
Disturbances in calcium homeostasis
Toxins
Environmental e.g pesticides
Cellular e.g. dopamine, α‐synuclein
Animal models of PD
Models have predictive value with regard to dopamine deficiency
Rodent models
6‐hydroxydopamine (6‐OHDA) injected unilaterally into the
striatum or SNpc
MPTP (systemic injection) ‐ MPTP converted to toxic MPP+ which
is selectively taken up by dopamine neurons, inhibits
mitochondrial respiration
Rotenone (chronic infusion of low doses) ‐ mitochondrial complex
I inhibitor
transgenic mice (e.g α‐synuclein)
Non‐human primate
MPTP
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25. Treatment strategies
Goals
Alleviate motor symptoms
Prevent ongoing cell death process in the SNpc
Current pharmacological treatments
Focus on augmenting striatal dopamine levels
L‐Dopa/carbidopa ‐ crosses the blood brain barrier and is
converted to dopamine by aromatic amino acid
decarboxylase (AADC)
Dopamine agonists and/or monoamine oxidase B inhibitors
which prevent dopamine breakdown, antioxidants,
glutamate antagonists may provide some benefits
Problems
Loss of efficacy over time, on‐off effects, dyskinesias,
hallucinations
Do not affect ongoing cell death process
3
26. Gene therapy strategies for PD
Three main strategies:
Biochemical augmentation ‐ alleviating symptoms
correct dopamine deficiency
Neuroprotection ‐ altering natural history of the
disease
growth factors (e.g. GDNF)
Resetting basal ganglia circuitry ‐ alleviating
symptoms/preventing further cell death?
silence overactive neuronal circuits by expressing glutamic
acid decarboxylase (GAD)
Biochemical augmentation
Express genes involved in dopamine biosynthesis
Tyrosine
Tyrosine hydroxylase (TH), tetrahydrobiopterin
L‐Dopa
AADC
dopamine
Vector systems used: HSV, Ad, AAV
Main disadvantage is inability to maintain dopamine concentrations
at appropriate therapeutic level
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27. Neuroprotection
Introduce growth factor genes that promote cell survival and/
or regeneration of remaining neurons e.g. glial‐derived
neurotrophic factor (GDNF), brain‐derived neurotrophic factor
(BDNF), sonic hedgehog
Useful in early stages of the disease when there is still a
significant dopamine neuron population in the SNpc
GDNF gene therapy in particular, looks promising ‐
improvements in neurochemical assessments and motor
symptoms in rodent and primate models of PD using AAV and
lentiviral vector systems
GDNF promotes survival and regeneration
GDNF promotes axon regeneration (sprouting) and correction of dopamine
deficiency in striatum
This effect only occurred when the vector was injected directly into the
striatum
Bjorklund et al., Brain Res., 886 (2000), 82-98
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28. Methods used to assess whether gene therapy has
therapeutic effect and benefit
Neurochemistry ‐ measure dopamine levels (tissue
punches, microdialysis)
Assays for quantifying and/or visualising therapeutic
protein levels ‐ e.g immunohistochemistry, RT‐PCR,
Westerns, ELISA
Cell survival ‐ e.g TH as a marker of dopamine neurons
Non‐invasive imaging e.g. PET, MRI
Behavioural ‐ changes in motor function as a functional
endpoint
Functional assessment in rodents
Spontaneous motor activity
Drug‐induced motor activity ‐
rotational behaviour
Unilateral lesions produce an
imbalance in striatal dopamine
levels between the right and left
brain hemispheres and
upregulation of postsynaptic
dopamine receptors and/or signal
transduction sensitivity on the
lesioned side.
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29. Rate of rotation (e.g turns/min) is directly proportional to
severity of the lesion and dopamine loss.
Assess rotation behaviour before and after treatment.
Expectation is that if a therapy works, will see a reduction in
rotation rate.
Blue squares = AAVGDNF in striatum
Red squares = AAVGDNF in SN
Green = AAVGDNF in SN and striatum
Open squares = control
Bjorklund et al., Brain Res., 886 (2000), 82-98
Resetting brain circuitry
Dopamine deficiency in striatum has consequences on overall basal ganglia
circuitry
Overactivity in the subthalamic nucleus (STN)
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31. Use a genetic approach to silence STN neurons by introducing
GAD (glutamic acid decarboxylase) ‐ enzyme involved in GABA
synthesis in these cells
Principle is similar to deep brain stimulation, an established
treatment for PD in humans
May also have a neuroprotective effect
GAD gene therapy in humans
World’s first gene therapy trial for PD approved by the U.S FDA
in 2003
Open label, safety and tolerability trial of AAV‐GAD injected
unilaterally into STN of 12 patients
First patient treated August 2003 with surgery on 12 patients
completed by July 2005
1 year follow‐up completed July 2006
Built‐in safety mechanism ‐ ablation of the STN is an
established treatment for PD.
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33. Functional assessment in humans ‐ GAD gene
therapy
Fluoro‐deoxyglucose PET imaging
MRI
Motor assessments ‐ Unified Parkinson’s disease rating scale
(UPDRS), Glosser QOL
Neuropsychological evaluation
Blood for haematology and chemistry, urinalysis, ECG
Blood for antibodies to AAV and GAD
GAD gene therapy led to improvement in
overall movement (UPDRS score)
“off “state “on” state
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36. Current status of PD gene therapy
Unravelling pathways involved in mediating death of
dopaminergic neurons will lead to identification of new
targets.
Areas to target: Ubiquitin‐proteosomal pathway (e.g parkin),
heat‐shock proteins
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