If you

1
If youre not willing to put 100 effort into
your academic studies, choose a career that
doesnt require a postgraduate education
2
Lecture 12 Gene Therapy

• Gene therapy the delivery of corrective genes
  to make up for a genetic defect

3
MMEP Class of 2000
4
Gene therapy as nucleic-acid-based treatment,
or transfer of DNA/RNA to somatic target cells in
the intention to treat serious illness
In vivo gene therapy is the transfer of
therapeutic genes directly to the patient. E.g.,
injection into the blood stream. In situ gene
therapy is a specialized form of in vivo therapy,
where the therapeutic gene is directly injected
into a selected tissue of the patient (e.g.,
muscle). Ex vivo gene therapy involves transfer
of therapeutic genes into cells grown in
culture. These cells are first removed from the
patient. The cells that are successfully
genetically engineered are selected and expanded
in culture before being returned to the patient.


5
Gene Therapy

• Gene therapy is more than the complementation of
  defective gene.
• Involves treatments to alter the course of
  infectious diseases such AIDS (Integrase
  inhibitors, Immunostimulates)
• Interfere with autoimmune processes such as
  arthritis
• Immunex Corp (IMNX)
• Induce effective immunity to cancer and
  microorganisms
• p53
• 4. Provide growth factors and hormones to the
  body
• hGH
• 5. Scavenge harmful substances or block harmful
  aspects of tissue injury and improve healing
  processes or tissue regeneration.

6
How do We make it Work?

• Delivery of the genetic payload (efficient, safe)
• Viral vs. nonviral transfer technologies
• Expression in the affected tissues only
  (specific)
• Regulated expression in target tissue
  (constitutive)
• 4. Prevent immune response

7
Viral Transfer Technologies

• 1) Retroviruses (e.g HIV-1, type of
  Lentivirus)
• Class of enveloped viruses
• Single stranded RNA molecule as the genome.
• Following infection, the viral genome is reverse
  transcribed
• into double stranded DNA, which integrates into
  the host genome is expressed as proteins.
• Problems
• 1) May cause insertional mutagenesis
• 2) Carrying capacity is 7.5 kb (too small for
  many genes even if cDNA is used)
• 3) Preference for dividing cells

8
Bacteriophage infecting a bacterium
Assembly of phage
9
Viral Transfer Technologies
2) Adenoviruses (attenuated or modified versions)
ds DNA

• Life cycle does not normally involve
  integration into the host
• genome
• Problems
• liver toxicity
• immune response

10
Viral Transfer Technologies

• 3) Adeno-associated viruses (AAV) ss DNA
• Non-pathogenic
• Generally no detectable immune response
• Dependant on a helper virus, usually adenovirus,
  to proliferate.
• Infect both dividing non dividing cells
• integrate into a specific point of
• the host genome (19q 13-qter)
• at a high frequency.

11
THM of Viral transfer safety risks

• Immune response
• Insertional mutagenesis
• toxicity

Need for alternative (non-viral) vectors
12
Nonviral Transfer Technologies

• Plasmid DNA Naked DNA. IM injection
• Sustained Expression!
• 2. DNA-Protein complexes IM imjection
• 3. Liposomes DNA Associates with Cationic
  lipids bind to plasma membrane. Add some
  anionic lipid- DNA is incorporated into liposome.
  Why is this an advantage?
• 4. Nanoparticles gold or polymers with
• associated DNA bombard into cells

13
Summary of the Ideal Gene Transfer Vector
14
Tricks to enhance GT

• Target tissue Muscle
• Add histamine to intravenous injections- natural
  vessel destabilizer .
• Add collagenase
• Add targeting proteins (antibodies) to liposome,
  virus
• Add a DNA binding protein- enters nucleus,
  enhances transcription
• Use muscle specific promoter

Ligands
Antibodies
15
Suicide therapy defined
Suicide genes code for enzymes that convert
nontoxic compounds (prodrugs) into toxic
products.
16
GDEPT (Gene-directed enzymatic prodrug treatment)

• A type of suicide gene therapy
• Delivery a gene for an exogenous enzyme express
  in tumor cells
• Deliver a prodrug (inactive) to tissue.
• Prodrug is converted to the active drug by the
  foreign enzyme expressed inside or on the surface
  of tumor cells.

17
Gene-directed enzymatic prodrug treatment, aims
to maximize the effect of a toxic drug and
minimize its systemic effects by generating the
drug in situ within the tumor.

18
Suicide genes code for enzymes capable of
transforming a non-toxic compound into a toxic
product. Suicide/prodrug systems are effective at
eliminating tumors in vivo, and are therefore one
of the most widely used approaches for cancer
gene therapy.
Suicide Gene Therapy
19
Suicide Gene Therapy
20
How Does HSV TK work?

• 1. GCV is a guanosine analog that can be
  phosphorylated by HSV1tk to GCV-MP.
• GCV (ganciclovir) is the prodrug or inactive
  form.
• GCV-MP is then converted to the GCV-diphosphate
  and GCV-triphosphate forms by endogenous
  kinases.
• GCV-triphosphate lacks the 3 OH on the
  deoxyribose as well as the bond between the 2
  and 3 carbons which are necessary for DNA chain
  elongation. As a result, GCV-triphosphate
  integration inhibits DNA polymerase and causes
  premature DNA chain termination and leads to
  apoptosis.

21
How does FCY1 cytosine deaminase therapy work?
fcy1 is the yeast (S. cerevisiae) gene encoding
the enzyme cytosine deaminase (CD). 1. CD
converts cytosine to uracil. This enzyme activity
is found in prokaryotes and lower eukaryotes, but
is absent in higher eukaryotes. The yeast CD
(fcy1) has superior activity than the bacterial
CD (codA). 2. Following transfection with fcy1.
Patients are treated with the prodrug
5-fluorocytosine (5-FC). Fcy1 is able to convert
the non toxic 5-FC into 5-FU, which kills cells
by disrupting DNA synthesis, thereby triggering
apoptosis.
22
One HSVtk-expressing tumor cell will destroy up
to 10 untransfected cellsWhy is the cellular
basis for this bystander effect?Hint think
of how adjacent cells communicate.
23
Bystander effect due to diffusion of drug through
gap junctions
24
(No Transcript)
25
Can Gene therapy be used to treat Muscular
Dystrophy?

• The IVth workshop on Duchenne muscular dystrophy
  gene therapy.Schwartz K, Leterrier F.Inserm
  U523, Institut de Myologie, Groupe Hospitalier
  Pitie-Salpetriere, Paris, France.
  s.tary_at_myologie.chups.jussieu.fr

26
Duchenne Muscular dystrophy
Guillaume-Benjamin Duchenne de Boulogne

• Clinical syndromes
• X-linked
• Death 15 - 25 years due to respiratory or cardiac
  failure
• Other clinical features
• Muscle pseudohypertrophy Especially calf
• Cardiomyopathy
• Mild mental retardation Mean IQ 88

27
Efforts to treat DMD using gene therapy have been
hampered by three major obstacles
1) the size of the dystrophin gene prevents its
administration by the common viral gene vectors
(dystrophin mRNA is 14 kb) 2) large numbers of
cells must contain the gene in order to improve
muscle strength 3) apparent immune rejection of
cells infected by the vector probably limits the
duration of the gene correction.
28
Dystrophin Gene is largest known genecDNA is
also large!
14kb dystrophin mRNA Encodes 3685 amino acid
427kDa protein Problem Carrying capacity of
retrovirus is 7.5 kb Carrying capacity of
adenovirus can hold complete copy of dystrophin

29
Where is the Dystrophin Protein located?Muscle,
(skeletal and cardiac), neuronal, others!
30
Immune Response
If you dont make dystrophin, and then it is
made following gene therapy treatment, the
protein may result in an immune response
31
  Schematic representation of the
dystrophin-associated glycoprotein complex.
The dystrophin-associated glycoprotein complex
What parts can you do without? H. Lee Sweeney
and Elisabeth R. Barton Department of
Physiology, University of Pennsylvania School of
Medicine, Philadelphia, PA 19104-6085
32
Can DMD be cured?
33
What about gene therapy with dystrophin?
34
AAVs are considered among the safest and most
effective vehicles for delivering genes to muscle
tissue, but adenoviruses can hold bigger payloads
35
Gene Therapy Strategies for DMD

• Develop novel adenoviral vectors (gutted)
• Modify conventional adenoviral vectors by
  selectively removing critical genes needed for
  viral replication and protein synthesis, and
  placing those genes into packaging cell lines to
  support growth  in vitro.  E.g No viral
  replication- less chance of immune response,
  fewer side effects.
•  GUTTED viruses can only replicate in a
  helper-dependent manner

Long Island biotech company OSI Pharmaceuticals
Promoter for Utrophin Cell Genesys (CEGE)
Patents on AAV gene delivery
36
Do the gutted adenoviral vectors work?

• Yes, and they appear to evade immune detection!
• Using a mouse model of DMD, Jeffrey Chamberlain
  and his group at the University of Washington in
  Seattle group has now shown that gutted
  adenovirus carrying a mouse dystrophin gene seems
  to provoke very little immune response. Also, the
  dystrophin is effectively delivered to muscles,
  and in force measurement tests, treated muscles
  showed a lasting improvement in endurance
  compared to untreated muscles. Highlights from
  the American Society of Gene Therapy
  meetingSeattle, May 30-June 3, 2001

37
Gene therapy- mini genes
To get the dystrophin gene small enough to fit
into an adeno-associated virus, Xiao Xiao and
colleagues miniaturized it but preserved its
important functions
Strategy concentrate on "rod domain," of the
protein. This section is thought to provide
some shock absorption and act as a link between
the anchored ends of the ropelike dystrophin
molecule.
Xiao Xiao and Juan Li in the Department of
Molecular Genetics and Biochemistry of the
University of Pittsburgh
38
Does the mini gene encoding only part of
dystrophin work?
39
Long term minidystrophin expression in mdx-mice!
untreated
treated

• Fig. 3.   Long-term minidystrophin expression in
  mdx mice treated at a young age (a) or as adults
  (c-h) with AAV vectors containing different
  minigenes under the control of different
  promoters. IF staining of minidystrophin (green)
  and myonuclei counterstaining with DAPI (blue)
  were performed on gastrocnemius muscles isolated
  from (a) MCK- 3849-treated 10-day-old mdx for
  6 months, (b) untreated 6-month-old mdx, (c) MCK-
  3849-treated adult mdx for 2 months, (d) MCK-
  3990-treated adult mdx for 2 months, (e) MCK-
  3849-treated adult mdx for 4 months, (f) MCK-
  3990-treated adult mdx for 4 months, (g) CMV-
  3849-treated adult mdx for 6 months, and (h) CMV-
  3990-treated adult mdx for 6 months.

40
End

• Thursday Angiogenesis and tumor growth
• Review for exam
• Friday Stem cells in treatment of disease
• Monday Immunotherapy
• Tuesday HIV
• Wednesday Exam 830 sharp

41
Construction of highly truncated minidystrophin
genes
Fig. 1.  . Dystrophin has four major domains the
N-terminal domain (N), the CR domain, the CT
domain, and the central rod domain, which
contains 24 rod repeats (R) and four hinges (H).
The minidystrophin genes were constructed by
deleting a large portion of the central rods and
hinges and nearly the entire CT domain (except
the last 3 aa). The minidystrophin genes
subsequently were cloned between an MCK promoter
(or a CMV promoter) and a poly(A) sequence in AAV
vectors.
42
Other methods to treat DMD

• Using chemicals known as antisense
  oligoribonucleotides (AOs), the re-searchers

The effects of the AOs are similar to those of
the drug gentamicin, which also changes the way
cells read genes. Gentamicin is being tested in
clinical trials in people with DMD and in people
with limb-girdle
43
Genetic correction of dystrophin deficiency and
skeletal muscle remodeling in adult MDX mouse via
transplantation of retroviral producer
cells.Fassati et al., JCI, 97
Use retroviral producer cell clones to relase a
vector which contains the 6.3 kb dystrophin
minigene FK 506 immunosupression
44
Protection of muscle plasma membrane integrity by
minidystrophin genes in mdx mice treated either
at 10 days of age (a) or as adults (b). (a) Three
months after AAV vector injection, either the
treated mdx mice or the age-matched controls
(normal C57/B10 and untreated mdx mice) were i.v.
injected with Evans Blue dye. The gastrocnemius
muscles then were collected and cryosectioned
either from normal C57/B10 mice from mdx mice
treated at 10 days of age with AAV-MCK vectors
 3849,  3990, or  4173 or from the untreated
mdx mice. Normal dystrophin and minidystrophin
expression was visualized by IF staining (Left,
green). The leaky myofibers were visualized by
the uptake of Evans Blue dye (Center, red
fluorescence). Note the mutual exclusivity
between dystrophin expression and Evans Blue dye
uptake as shown by the merged images (Right).
Photographs were taken with a 10 lens. (b) Adult
mdx gastrocnemius muscles were treated with AAV
vectors containing  3990 minigene. Evans Blue
dye tests were performed at 2 months after
AAV-MCK-  3990 treatment (Left) or at 6 months
after AAV-CMV-  3990 treatment (Right). Note the
widespread minidystrophin expression (green) and
the leaky myofibers (red), which were negative
for minidystrophin staining.
45
(No Transcript)
46
Items to consider when attempting gene therapy
Overexpression of gamma -Sarcoglycan Induces
Severe Muscular Dystrophy. IMPLICATIONS FOR THE
REGULATION OF SARCOGLYCAN ASSEMBLY.Zhu X,
Hadhazy M, Groh ME, Wheeler MT, Wollmann R,
McNally E J Biol Chem 2001 Jun 15276(24)21785-90
.
Department of Medicine, Section of Cardiology,
and Department of Human Genetics, Department of
Molecular Genetics and Cell Biology, and
Department of Pathology, The University of
Chicago, Chicago, Illinois 60637.
gene dosage and promoter strength should be given
serious consideration in replacement gene therapy
to ensure safety in human clinical trials