Medical Genetics - Q603 13

2014

Treatment of Genetic Disease

Overview and Gene Therapy
Stem Cells
 Overview
many levels where interventions
are possible

Text Fig 13-2

 Gene Therapy
doesn’t just apply to fixing a gene.

Transfer of genetic material with therapeutic intent

Goals: Alleviate symptoms

Correct gene defects

Potential for broad application:

Classic single-gene defects

Complex diseases (e.g., diabetes)
Acquired conditions (e.g., AIDS)
Cancer treatments
 Objectives

• Understand strengths and limitations of

current gene therapy technology.

• Understand the unlimited potential of gene

transfer technology to treat disease.
** Somatic Gene Therapy
there are ethical considerations

vs

Germline Gene Therapy

 Ethics

• Approved clinical trials of gene therapy target somatic cells.

• Germline gene therapy has not been approved for ethical reasons.

just the
Somatic Cells Repairs Patient treated

Patient, their progeny

Germline repairs and the gene pool
altered.
∴ not done in humans in US currently
 Disease candidates for gene therapy
• Substantial disease burden (untreated patients suffer from
severe morbidity or mortality) and favorable risk/benefit
ratio compared to alternative therapy
• Relevant gene/locus identified/cloned and have
sufficient knowledge of the disease process
– Preferably a single gene defect
– May include data from cell / animal studies ( Proof of concept )
important to consider the
• Appropriate target cell(s) and gene delivery method
– Ideally, cells can be manipulated ex vivo
– Long half life or good replication potential gene
– How to get the gene into the appropriate cell? (Delivery)
– Selective advantage to genetically altered cells methods
target cell/engineered cell has some advantage once its created
• Wide range of expression associated with a normal
phenotype you don’t have to provide exact amt of new material/enzyme. can give 10%,
wide range of acceptable levels of product that benefit patient w/o harm
 Two main types of gene therapy:
Ex Vivo In Vivo
(treat cells outside the body) (treat cells inside the body)

getting gene to cell target or tissue type

can use
viruses or
culture in vitro then Naked DNA
return to patient
 Methods for DNA delivery
Have advantages and disadvantages

Vector - system used to transfer exogenous

genetic material into the target cell
Classes:

T
1 • Non-viral
– Plasmids, naked DNA

2 • Viruses (various types)

• Retroviruses
• Adenoviruses
• Adeno-associated viruses
• Lentivirus (e.g., modified HIV)
• Other
Transfection of Plasmids
Most plasmids are degraded with time ∴ no potential for long term expression
Only a small minority make it to the nucleus

Plasmid!
90
80
70
get plasmid into 60
cell (this is a challenge) 50
40
30
20
10
0
0 1 2 3 4 5 6 7

Days!
 Choosing a viral vector
Consider
• Host range and tissue specificity where you want virus to deliver DNA?
• Transfer into dividing vs nondividing cells
• Integration into host genome vs maintained as episome
• Effects on target cell viability and potential in vivo
toxicity
a key concern is
• Potential to generate replication-competent virus
• Immunogenicity another concern w/ some viral infections
• Ease of manipulation
• Amount of DNA that can be accommodated in the virus
__________________________________
 Retroviral Gene Transfer

significant use in gene “Gene therapy vector production”

therapy protocols

Wild-type
virus

contains viral genes important for

replication, infection, and capsule production.

Helper
virus
want to remove viral oncogenes and replace w/ therapeutic sequence

Gene-therapy
construct
you can have a gene therapy fragment of DNA/RNA but you also have to have a
virus you can put it into (aka helper virus) which is deficient/can’t be packaged
into viral particle
 The therapeutic gene has a functional packaging
sequence and can be put into the viral particle.

only have a virus w/

therapeutic sequence
∴ don’t want virus to
+ replicate on its own

plasmid sequence
you can clone therapeutic sequence put into empty viral
using plasmids particles

infect cells w/ helper virus and then cells will build E.g., can t make viral
empty viral particles
Helper virus has genes to make viral envelope.
envelope but the viral DNA can t be put in.
 Retroviral Gene Transfer
RNA viral particle w/
therapeutic sequence

Packaging RNA
Cell Line
DNA

cell has therapeutic

seq and can build viral
env particle
Therapeutic
gag/pol
Protein

Vector TARGET CELL

 Retroviruses
• Advantages
– High transfer (transduction) efficiency
– Long-term expression (integration, provirus)
b/c the retrovirus integrates
and progeny of cell also get the gene into a provirus and you stably
produce transcripts. Will replicate
when cell divides producing
progeny cells
• Disadvantages
– Requires dividing cells
• Inefficient transfer to hematopoietic stem cells and other
non-dividing cells
– Risk of insertional mutagenesis if it goes into the wrong place it can
cause harm
– Limited / moderate insert size
any virus is limited to the amount of DNA that it can hold
 Adenoviruses
• Advantages
– Broad range of target cells
both
– Infect dividing and non-dividing cellsb/c they don’t actually
– Low risk of insertional mutagenesis integrate into genome. Can also
be a problem
– Easy to purify and concentrate, high titer
• May get thousands per cell
b/c they’re not integrated into genome,
• Disadvantages expressed as an episome ∴ no long-term
effects
– Transient expression of transgene (episome)
• Not passed on to progeny cells
– Immunogenic (the cells that you’re inserting the
genome into)
• May stimulate destruction of transgene containing cells
– Risk of replication competent virus
– Limited / moderate insert size as w/ retroviruses
 Pro s and Con s of Viral Vector Systems **

Vector Advantages Disadvantages Target tissues

Retrovirus Integrates Insertional mutagenesis Bone marrow and
Lentivirus Non-toxic other stem cell
T cells
Adenovirus High titer Non-integrating Cancer
Cycling independent Immune reaction Immunization
AAV High titer Small genome Muscle, liver, brain
Non-toxic Non-integrating
Herpes High titer Non-integrating Cancer
Large genome ? toxicity CNS

labs are currently modifying envelope genes

of viruses to control their infectivity
(pseudo-typing)

T
So the perfect gene therapy vector is....

It depends!
What target cell type(s) required?
Permanent or transient?
Which administration route used?
Some Applications
not the key use of gene therapy!
 Adenosine deaminase deficiency
• 20% of severe combined immunodeficiency (SCID)
• Both humoral and cellular immunity impaired, failure to
thrive, recurrent infections
• Purine metabolism defect (autosomal recessive)
– Accumulated deoxyadenosine impairs DNA replication
and cell division, toxic to lymphocytes
• Early death if untreated Bubble-boy
• Treatment
– Bone marrow transplant to improve immune competency of individuals
adenosine deaminase active enzyme is stabilized
– PEG-ADA (lifetime compliance) and injected ∴ can detoxify cells extracellularly
• Extracellular replacement of intracellular enzyme
– First inherited disease treated with gene therapy
• Ex vivo, replacement gene therapy
 1)take out bone marrow

4)
2)
hoping they would engraft
and provide sufficient
enzyme activity.

ADA gene/
cDNA
3) add adenosine deamines to cells using a retrovirus

PEG-ADA at 1/3 to 1/4 of pre-therapy dose

maintained low levels of injected enzyme at first for safety reasons
• 10 patients reported
• Median follow-up 4 yrs (1.8 –
8 yrs)
• 8 pts off enzyme were able to stop
enzyme addition
replacement
• 9 pts immune function
adequate
Severe combined immunodeficiency-X1

• X-linked
• Block in T and NK lymphocyte differentiation
• [gamma]c cytokine receptor deficiency
• Therapy used retroviral-derived vector with cDNA
had cDNA for receptor
• Ex vivo gene therapy (CD34+ cells)
∴ took cells out of patient, added genes into them w/
• 10 month follow up retroviral vector, then reintroduced cells to patients
– T and NK cells detected
– Correction of phenotype
• 9 of 11 patients cured
but
• 2 patients subsequently developed leukemia
– ? Insertional activation of oncogene ?
was there a problem w/ where the retrovirus was inserted into the genome?
as opposed to ex vivo
In vivo gene therapy

delivering directly to target

 In vivo example: Cystic fibrosis

- Only 10% of cells

needed to correct
goal was targeting
defect in order to help CF patients lung tissue

- Most trials used

adenovirus b/c it’s easier to get
adenovirus to deliver
to epithelia of lung than a retrovirus
- Failures due to
delivery issues and
poor transduction not much success
(thick mucus)
In vivo example: Factor VIII deficiency

Vector delivers Factor VIII into mm and

from there factor VIII is released into blood

- Muscle a good
target because cells
rarely divide
adeno-associated vectors
- AAV vectors have
primarily been used
 (delivering gene directly to body)
Early failure for in vivo gene therapy:
Ornithine transcarbamylase (OTC) deficiency
• X-linked, urea cycle mitochondrial enzyme
• Neonatal hyperammonemia
– Massive levels (>1000 uM)

• As many as 70% do not survival neonatal period

• 1999, Philadelphia, Gene therapy trial
– 1st generation Adenovirus in vivo with OTC gene
– Intrahepatic injection
– 18-year-old (Jesse Gelsinger) died 4 days after treatment
which is a key concern of
• Multisystem failure from strong immune response adenoviruses
• Primate preclinical toxicity data may have been improperly reported
• 3rd generation adenovirus (gutless) may not have this problem
viral vectors have been improved since then
 Gene transfer but not the gene you might think.
Viral delivery of glial cell line-derived neurotrophic factor improves
behavior and protects striatal neurons in a mouse model of
Huntington s disease
AD, loss of caudate due to repeat expansion of Huntingtin gene

McBride et al. PNAS 2006, 103:9345-9350

mouse model useful

Gene therapy is not simply replacement of

defective gene !
 Gene Therapy and Cancer
(Multiple Strategies)

• Enhance immune response

• Interfere with function of oncogene
• Replace tumor suppressor gene
• Introduce suicide gene into cells
– E.g., HSV thymidine kinase
• Enhance resistance of hematopoietic cells
to chemotherapeutics
• Other
 Example of cancer gene therapy
(using herpes simplex virus thymidine kinase)
uses a “suicide gene” Produce a virus w/ HSV thymidine kinase & that’s the therapeutic
sequence. Produce a vector that will express the gene in a tissue specific manner/certain location

- Vectors have tumor

specific promoter

- Brain a good target

because lower
immune reactions
and soft, localized
∴ can try to inject gene containing vector
tumors directly into tumor.
If cells of tumor takes up vector, virus infects
cell and thymidine kinase leads to conversion
of ganciclovir into a toxin (normal human thymidine
kinase doesn’t do this) and are killed—“Suicide
gene”, you could put diptheria toxin gene directly
 Engineering Oncolytic Viruses
(example for adenovirus)

! Normally when cell become infected with

adenovirus p53 is activated

! Unopposed p53 will cause cell death and prevent a

lytic infection p53 is helping prevent adenovirus from replicating and causing
infection

∴ adenovirus infecting a cell can be successful by downregulating p53

! Adenoviral gene E1b down regulates p53 so virus
can commandeer cell machinery, massively
replicate virus, eventually leading to lytic infection
(killing the cell)
 Engineering Oncolytic Viruses
Normal Cell Tumor Cell
E1b deleted virus!
from adenovirus

p53
p53+ deficient

cell that is deficient

cell then infected, in p53 (tumor) when
but won’t be lytic infected w/ adenovirus
will undergo lysis. Can
infect another cell (following
lysis) and kill other tumor
cells
Not permissive Permissive and lytic
for killing the cell
 Engineering Oncolytic Viruses
• Engineer virus to replicate only in cancer cells

• Adenovirus, Vaccinia, New Castle Virus and other

viruses under development

• Only approved gene therapy product (China)

• New vectors are modifying viruses to increase

tumor kill by expressing additional anti-tumor
genes putting a virus in that can kill a tumor cell
 Allow more aggressive chemotherapy w/kindscertain
of cancers
(e.g., increase the resistance of hematopoietic cells to chemotherapeutic agents)

BCNU (bis-chloroethylnitrosourea) isa chemotherapeutic alkylating agent that

causes interstrand crosslinks and impairs DNA replication and transcription
it alkylates guanine residues, leading to
interstrand linkages
BCNU damage can be repaired by MGMT
(O6–methylguanine methyltransferase)

methyl BCNU
sufficient damage to DNA of cancer cell
BCNU will kill the cell. Problem is hematopoietic stem cells
are also sensitive to the action of BCNU. Methyl guanine
methyl methyltransferase can repair the action.

C G C T A C G G C T A G G C T G
G C G A T G C C G A T C C G A C
MGMT

methyl MGMT
C G C T A C G G C T A G G C T G
G C G A T G C C G A T C C G A C
∴ introduce MGMT gene into bone marrow stem cell, reimplant them into patient, and ∴
protect patient from effects of BCNU which will now only kill the cancer cells

MGMT and Chemotherapy

Obtain Stem Introduce the

Cells by MGMT gene and
Pheresis transplant

Administer
BCNU
chemotherapy

Can we increase chemotherapy and cures?

 eg enzymes or
Rather than add something receptors or
factors
(e.g., replace a deficient factor or add a gene for a toxin)

it may be necessary to remove or

knock out/down something

• Why? in dominant-negative diseases where accumulation of something is

causing a negative effect, adding more protein won’t help
– Treat disease due to dominant-negative mutations
– Counteract infection (e.g., HIV)
– Shut down an oncogene (turn something off)
 How does one knock out/down ?

Various schemes are used that depend on the

specificity of nucleic acid interaction to target the
gene/sequence of interest

Antisense Therapy
Inhibitory Nucleic Acids
MicroRNA
Ribozymes
RNAi (RNA interference)
 Inactivate a harmful sequence
There are number of ways to potentially and specifically
interfere with and/or silence gene expression.
the idea is to inactivate or inhibit the expression of the harmful sequence.
Produce sections of DNA that can titrate away
Decoys a transcription factor (act as decoys) that is
necessary to express gene
Triplex

DNA RNA interference

RNAi

RNA
specific RNA molecules
Antisense w/ catalytic activity that
Sequence can cleave a target RNA
Ribozyme
Protein
general scheme is that rather than treating at gene level,
attack at RNA level. Prevent RNA (which is having a negative impact)
from doing its job
Target Sequences in the HIV Genome!

Stem Cells!
Patient w/ HIV. Take some stem cells then treat
them w vector that produces small amount of
inhibitory RNA or ribozyme that inhibits HIV gene

siRNA
Ribozyme
 *Relisten
RNA Interference
(RNAi) mRNA
5 ….. AAUGCACCUGCUCCGAU…..AAAA 3
Earlier version of concept
Anti-sense among other things/
names 3 ..UGGACGAGG..
5
Antisense transcript
Many genes have anti-sense partners
or oligonucleotide

Prevents translation of mRNA produce a complimentar

antisense nucleotide that
can hybridize…

5 ….. AAUGCACCUGCUCCGAU…..AAAA 3
3 ..UGGACGAGG..
5
 microRNAs
w/in human genome, naturally produce
• Small noncoding RNAs (~ 21 nucleotides long) from
endogenous genes make RNA molecules, some interact w/ smaller genomes and
impact ability of RNA to be expressed

• Regulate gene expression by binding to 3 -UTR of

messenger RNA
– Translational gene silencing or messenger RNA cleavage

• 1,000 microRNAs in human (Berezikov et al., 2005)

– One of the most abundant classes of regulatory genes
naturally
• Control 20-30% of human genes (Lewis et al., 2005)
– Suggestion that microRNAs control over 90% of genes

• Each microRNA can regulate multiple genes

– 100s of microRNA genes offer an enormous potential for
regulatory circuitry
START: Micro RNA Processing
a number of genes within the human genome
can lead to micro RNA
w/in transcript is the
sequence that will
eventually be a micro RNA
pre-micro RNA molecule is processed/cleaved to
produce a smaller molecule depicted
as hairpin
it is then exported from nuc
into cytoplasm

Translation repression or if there’s a complete match between

miRNA and target then it’s degraded
messenger RNA degradation
if there’s a partial
match between miRNA dicer further
and target then RNA molecule processes it,
has less efficient translation and cleaving loop
doesn’t properly produce protein structure and
mature micro RNA releasing small
interacts w/ proteins red fragment.
to produce RiSC. This Finally releasing
complex of protein a single stranded,
has partial homology w/ RNA specific RNA
and RNA can specifically interact
sequence. Some miRNAs can sequence =
w/ target RNA sequences.
attach to different genes. micro RNA
RiSC : RNA-induced silencing complex
 microRNA Regulates Gene Expression
messenger RNA
microRNA
completely matching miRNA ∴ degradation will occur

cleavage of target mRNA

Multiple Partial match -

Perfect match -
Effects
Degradation of target Inhibition of translation
∴ if you want to use this to shut off gene, you produce in some diseases, whether
a miRNA by delivering a pre-miRNA to the cell. Cell processes or not miRNA can bind
pre miRNA to form miRNA that can shut down target. is sufficient to cause disease
 Clinical Trail Phases
Phase I • Toxicity

Phase II • Estimate efficacy,

additional toxicity data

• Randomized, establish
efficacy, pivotal trial
Phase III pre-approval
Status of Gene Therapy

much of gene therapy is still in developmental stage

ETHICAL CHALLENGES
Gene Enhancement
• Advantaging the advantaged
• Resetting what is normal
• Eugenics
– Intellectual
– Emotional
– Moral
 Major Questions

• RISK BENEFIT RATIO

• What will be ethical if gene therapy
is the safest option?
• What is disease and what is
enhancement? what is treatment vs excess?
• Balancing the theoretical with what
technology can offer today –
addressing the ethical challenges of
today.
 Summary

• Gene therapy trials moving from Phase

I towards licensed product.
• Viruses are commonly engineered to
deliver the genetic payload. Target cell
will drive the type of vector to be used.
• Most trials in cancer but almost any
disease can be approached using gene
transfer technology.
• As safety is shown, the ethical issues
will become more, not less complex.
?
 Stem Cells
• Stem cells display the ability of self-renewal
(to divide and give rise to other stem cells)
and to differentiate along specific molecular
pathways to form cells of specialized
functions.
 STEM%CELLS%

ADULT%% EMBRYONIC%% INDUCED%

(SOMATIC)% (ES%CELLS)% PLURIPOTENT%(iPS)%

Self-renewing, pluripotent Self-renewing, pluripotent Self-renewing, pluripotent

cells derived from cells derived from pre- cells derived from re-
adult tissues implantation embryos programmed adult cells
 Collection of Adult
Stem Cells
Bone Marrow Harvest

to get SC:
Apheresis – Peripheral Blood
Stem Cells
 STEM%CELLS%

ADULT%% EMBRYONIC%% INDUCED%

(SOMATIC)% (ES%CELLS)% PLURIPOTENT%(iPS)%

Self-renewing, pluripotent
cells derived from pre-
implantation embryos

• ES cell derivation from pre-implantation embryos

• Ethical considerations
• Legal considerations
• NIH funding of ES cell research
Collection of Embryonic
Stem Cells

inner cell mass !

(trophectoderm)!

Hogan et al, Manipulating the Mouse Embryo, p 55, 1994

 Sources of Human
Pre-Implantation Embryos

Left-Over Embryos Following

Fertility Treatments
idea is to have partially developed embryos

Generation of Blastocysts Using

Somatic Cell Nuclear Transfer
 Somatic Cell Nuclear Transfer,
Nuclear Transplantation, Therapeutic Cloning
have a somatic cell that you can get Inject adult extracted nucleus
DNA from. Take out the nucleus. into Oocyte (becomes 2n)

Pluripotent ES Cells
Activates oocyte to start dividing
somatic cell forming a pre-implantation stage
genomic DNA

Engineering of tissue
immunologically compatible
can create SCs that with the donor
are identical to original
 (Nuclear transplantation)

stimulate it
to divide

develops a
clone (in mice)

NEJM 349, 275 – 286, 2003

August 9, 2001 – George W. Bush permitted the use of 60 existing stem
cell lines -- cell lines that had already been derived from human embryos.
President Bush stopped short of allowing federal funding for research using stem
cells derived from frozen embryos.

http://stemcells.nih.gov/policy/2001policy.htm

March 9, 2009 – Barack H. Obama issued Executive Order 13505:

Removing Barriers to Responsible Scientific Research Involving Human Stem Cells.
The Executive Order states that the Secretary of Health and Human Services,
through the Director of NIH, may support and conduct responsible, scientifically
worthy human stem cell research, including human embryonic stem cell (hESC)
research.

http://stemcells.nih.gov/policy/2009guidelines.htm
 Research utilizing human ES cells derived
from left-over embryos following
fertility treatments permitted using
NIH funds

Individuals donating embryos for research purposes

should do so freely, with voluntary and informed consent

Private funds must be used, however, to derive the

new ES cell lines
 STEM%CELLS%

ADULT%% EMBRYONIC%% INDUCED%

(SOMATIC)% (ES%CELLS)% PLURIPOTENT%(iPS)%
not as great a source
as originally thought

Self-renewing, pluripotent Self-renewing, pluripotent Self-renewing, pluripotent

cells derived from cells derived from pre- cells derived from re-
adult tissues implantation embryos programmed adult cells
2012 Nobel Prize in Physiology or Medicine

Shinya Yamanaka
Born: 1962, Osaka, Japan

Affiliation at the time of the

award: Kyoto University, Kyoto,
Japan, Gladstone Institutes, San
Francisco, CA, USA

Prize motivation: "for the

discovery that mature cells can be
reprogrammed to become
pluripotent"
Induced Pluripotent Stem Cells (iPS cells)

Zygotic ES Cell-Derived Somatic Cell-Derived

Nucleus Nucleus Nucleus
these cells are close to high
less pluripotency in adult/somatic cells
potency state

Nuclear Potency
Induced Pluripotent Stem Cells (iPS cells)

Zygotic ES Cell-Derived Somatic Cell-Derived

Nucleus Nucleus Nucleus
adult cells reprogrammed
to enhance pluripotency

Nuclear Potency
How were the necessary factors defined?
4 factors were introduced to
reprogram adult cells into pluripotency

Fbx15-directed
neomycin expression

Hypothesis: A combination of factors that permits Fbx15 expression (neomycin

selection) should reprogram the somatic cells to a pluripotent cell.

Takahashi and Yamanaka, Cell, 2006

 Generation of Reprogrammed
Induced Pluripotent Stem Cells (iPS)
KLF4
Oct4
Myc Sox2

directly treat adult cells w/ these factors to create an iPSC Induced Pluripotent
Stem Cells
somatic cell
genomic DNA

Engineering of tissue
immunologically compatible
with the donor
 Induced Pluripotent Stem Cells
(iPS Cells)
take a somatic
induce growth of embryo
nucleus from adult

Induced Pluripotent
Stem Cells
these steps are skipped
S c ells
era t e iP
START
m t o gen ∴ ethical concerns are removed
ro gra
R ep
Engineering of tissue
immunologically compatible
with the donor
 iPS (pluripotent SC) as a therepeutic model Mouse α-globin>human α-globin gene
Mouse β-globin>human βs (sickle) gene
engineered a sickle cell mouse w/
Transplant genetically corrected,
human type genes. Used as a model
iPS cell-derived hematopoietic of a person w/ sickle cell.
Stem and progenitor cells

∴ not creating a whole

mouse, just a specific SC
to fix the sickling problem

grown in culture

(oncogene)
reprogramming
getting cells to divide w/
these factors

introduced via vector/

gene therapy
Cells were transfected with targeting plasmid were produced from fibroblast of mouth
Containing the human βA (wildtype) globin gene Hanna et al., Science, 2007
 Benefits of iPS cells

• Immunologically matched source of

transplantable cells

• Potential to repair genetic abnormalities by

homologous recombination you’re able to “do it again”

• Potential to repeatedly differentiate iPS cells as

needed for continued therapy

• Side-step the ethical issues involved in generating

human ES cells
 Challenges with iPS Cells

• Need to bypass the use of oncogenes (Myc) as part

of the reprogramming factors due to worry about it getting out of
control later on (neoplasms due to
oncogenes).

• Avoid the use of retroviral vectors in gene delivery,

as they carry the risk of insertional mutagenesis
(due to random genomic integrations)

• Leaky expression of the reprogramming

transcription factors in differentiated progeny of
iPSCs eg overexpression of cMYC can cause leukemias
 BIG GOAL

• Transgene-free delivery methods

• Polycistronic expression of factors
• Inducible promoters to temporally drive factor expression
• Cre recombinase-based excision of factors
these have a disadvantage
b/c they’re degraded but in
• Transfection of plasmids and episomal vectors this case it’s an advantage
b/c plasmids go away once cell is reprogrammed
T
• Non-integrating viruses (adenovirus, Sendai RNA virus)
can use non-integration as an advantage
End