BASIC SCIENCE RESEARCH BLOCK #

Lecture 1: Post-Ischemic Neovascularization and  H 2O2 used to mimic reperfusion injury


Blood Flow  Lactate dehydrogenase (LDH) levels used to measure cell
I. OVERVIEW death (LDH is released by necrotic cells)
A. Current Study
 Test the importance of HO-1 (heme-oxygenase I) in post-
ischemic neovascularization and BF (Blood Flow) recovery
in HO-1-deficient mice
 Generated and successfully used a plasmid with human
HO-1 driven by 3 HRE (hypoxia response element)
sequences
 Demonstrated anti-apoptotic, anti-inflammatory, and pro-
angiogenic action of HO-1

B. Results
 BF recovery and neovascularization after hind limb
ischemia is impaired in HO-1-deficient mice
 Beneficial effects of HO-1 overexpressed in normoxic and
hypoxic endothelial cells
 Administration of pHRE-HO-1 (pre-emptive hypoxia
regulated HO-I) improves BF and reduces incidence of
necrosis in ischemic hind limbs
 Administration of pHRE-HO-1 diminishes inflammatory
response and apoptosis in ischemic skeletal muscles
3. Beneficial effects of HO-1 overexpressed in HYPOXIC
endothelial cells
 LDH released by pHRE-HO-1 - transfected HMEC-1 cells
treated with 500 mM H2O2 in hypoxic conditions  mildly
but significantly lower LDH release when compared with the
pHRE-empty vector transfected cells subjected to the same
treatment.

II. BACKGROUND
A. The Problem: Ischemic injury due to Peripheral arterial
disease
 Critical limb ischemia
o Usually due to atherosclerotic vessel block from
Peripheral Arterial Disease
o Treatment: Limited use of bypass surgery or balloon
angioplasty → need amputation
 Heme oxygenase-1 (HO-1) gene – a good gene therapy
candidate. Previous studies show that:
o Degrades heme to CO, biliverdin / bilirubin, and
ferrous iron
o Products: anti-oxidative, anti-apoptotic, and anti-  So far the y‟ve proven the beneficial effects of HO-1 gene in
inflammatory effects vitro. That is, its pro-angiogenic and it has cyto-protective
o Pro-angiogenic: Mediated by CO via stimulation of properties
guanylate cyclase in endothelial cells (EC‟s)  So, the next logical step would be, to test it in vivo
o Regulate myogenic regulatory factors (MRF’s) and
miRNA’s involved in myoblast differentiation 4. Administration of pHRE-HO-1 improves BF and reduces
o When treated with HO inhibitor, blood flow recovery in the incidence of necrosis in ischemic hind limbs
femoral artery ligated mice was impaired
B. The BAD: Uncontrolled overexpression of HO-1 (high • BF and Necrosis: In wild-type mice, hind limb ischemia
supply of heme) → high levels of reactive iron  oxidative induced endogenous HO-1 expression 1 day after the insult.
stress  solution: Manipulate hypoxia-regulated gene • Overexpression of HO-1  much better recovery of BF in
carriers → create beneficial threshold of HO-1 protein ischemic hind limbs at day 14 after hind limb ligation and
HO-1 gene transfer than in control animals treated with
C. Past Experiments pHRE-empty.
 Hypoxia-regulated HO-1 gene transfer
o Reduced cellular damage during myocardial ischemia-
reperfusion (I/R) injury
 Mice treated with HO inhibitor Ferritin protoporphyrin X
blunted blood flow (BF) recovery after femoral artery
ligation (FAL)

III. EXPERIMENTAL DESIGN AND METHODS

Nucleofection of HMEC-1  Western Blotting  analysis of cell
death  analysis of cell migration  murine hind limb ischemia
and gene delivery  histological analysis  total RNA isolation
 RT-PCR eval of gene expression and miRNA levels  ELISA

IV. RESULTS AND DISCUSSION

1. BF reco very and neovascularization after hind limb ischemia
is impaired in HO-1-deficient mice
2. Beneficial effects of HO-1 overexpressed in NORMOXIC
endothelial cells
 Cell culture used is human microvascular endothelial cells
(HMEC-1)

 Nucleofection with plasmid encoding HO-1 (pCMV-HO-1)
 Western blot performed to make sure that transfection was
successful and HO-1 was indeed being overexpressed


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 Lecture 2: Gene Therapy
I. GENE THERAPY
 Change of patient‟s DNA to replace or compensate for a faulty
gene
 Replacement of a defective gene by a functional one would be
a true cure for a genetic disorder
 SIX possible approaches
 Normal gene inserted to compensate for a nonfunctional
gene.
 Abnormal gene traded for a normal gene
 Abnormal gene is repaired
 Change the regulation of the gene
 Mutating a normal gene
 Addition of a new gene
 How to define a gene: located on a chromosome, basic unit of
heredity, the open reading frame, encodes a protein, causes
dysfunction of a protein when altered
 Preliminary Requirements for Gene Therapy: identification of
the specific gene associated with the disease and mapping,
sequencing and study of the genE
 Genome Project: The human genome sequence is complete –
apprx 3.2 billion base pairs
 Bioinformatics is the use of computers to collect, analyze, and
interpret biological information at the molecular level
II. VIRUS
 A virus replicates by inserting its genes into the host cell’s
genome.
 This virus has three genes – A (allows virus to insert itself into
genome) B and C (cause the disease)
 In the context of gene therapy, you want to replace B and C
with a beneficial gene. Thus, the modified virus could introduce
the „good gene‟ into the host cell‟s genome without causing
any disease.
 How it works: A vector delivers the therapeutic gene into a
patient‟s target cell→ target becomes infected → functional
protein is created from therapeutic gene causing the cell to
return to normal state
 The Ideal Vector for Gene Transfer has high concentration
of virus, convenient, reproducible, ability to integrate into a
specific site, ability to target the desired cell type and does
not evoke an immune response
 4 Types of Viruses
1. Retrovirus- goes thru reverse transcription using reverse
transcriptase and RNA
o The double stranded viral genome integrates into the
human genome using integrase (can insert anywhere
thus can cause insertional mutagenesis)
o Vectors used are derived from HIV
2. Herpes Simplex viruses - Double stranded DNA viruses
that infect neurons
3. Adenoviruses- Have a double stranded DNA genome that
cause respiratory, intestinal, and eye infections in humans
o The inserted DNA is not incorporated into genome
o Not replicated though – has to be reinserted when more
cells divide, once stopped it won‟t exist no more.
4. Adeno-associated viruses- small, single stranded DNA
that insert genetic material at a specific point on
chromosome 19
o From parvovirus family – causes no known disease and
doesn‟t trigger patient immune response
o Low information capacity
o Gene is always “on” so the protein is always being
expressed
 Non-viral Options: Direct introduction of therapeutic DNA,
Creation of artificial lipid sphere with aqueous core, liposome ,
Chemically linking DNA to molecule that will bind to special cell
receptors, Introducing a 47 th chromosome
III. BIOLISTICS
 Gene gun- used to insert DNA into living tissue
 New design – uses high-pressure helium gas from a narrow
barrel to insert DNA attached to gold nanoparticle. High
pressure gas reduces recoil and prevent tissue damage
IV. TYPES OF GENE THERAPY
BASIC SCIENCE RESEARCH | Block #
1. Somatic-cell gene therapy- Repair or replace defective gene
in some or all body cells of an individual
THREE categories of somatic cell gene therapy
1) Ex vivo - cells removed from body, incubated with vector
and gene-engineered cells returned to the body
2) In situ – vector is placed directly into the affected tissue
3) In vivo – vector injected directly into the bloodstream; use
this method if you want a more systemic effect because it
goes into your bloodstream
2. Germ-line gene therapy- Repair or replace defective gene in
germ-line cells; repaired gene is inherited
 Problems with Gene Therapy: Short lived, can evoke an
immune response, May induce a tumor if integrated in a
tumor suppressor gene b/c of insertional mutagenesis
 Viral Vectors: Patient could have toxic, im mune,
inflammatory response, Also may cause disease once
inside
 Multigene Disorders such as Heart disease, high BP,
Alzheimer‟s, arthritis, and diabetes are harder to treat b/c
you need to introduce more than one gene
Classes of Mutations (I, II, III, and IV)
I. Cause defective protein production with a total loss of
functional CFTR‟s
II. Cause defective protein production leading to CFTR that
is in the improper location
III. Cause defective regulation of channel opening of CFTR
by changes in the nucleotide binding fold
IV. Cause defective ion conduction
V. CYSTIC FIBROSIS (CF)
 Autosomal recessive genetic disorder that affects most
critically the lungs, the pancreas, liver, and intestine
 Characterized by abnormal transport of chloride and sodium
across an epithelium, leading to thick, viscous secretions.
 Mutation for CF is on the 7 th chromosome
 Cystic fibrosis should be an ideal candidate for gene
therapy, for four main reasons: single gene defect,
recessive condition, accessible for treatment, progressive
disease offering a therapeutic window
VI. PERIPHERAL ARTERY DISEASE
 Form of atherosclerosis that most frequently affects the lower
limbs
 Risk factors for PAD include diabetes, smoking, sedentary life
style, bad diet and hereditary factors.
 Starts as leg pain and progresses to critical limb ischemia
(CLI)→ no pharmacological treatment
 Treatment Approach: Re-vascularize the ischemic limb and
d eliver vascular endothelial growth factor
 Pioneering Approach: Use of hypoxia-inducible promoter
and combine with stem cells
 Available Gene Therapies
 Gendicine – head and neck squamous cell carcinoma
(p53 adenoviral vector)
 Rexin-G – intractable metastatic cancers (cyclin-G1 in
retroviral vector)
 Collategene – critical limb ischemia (plasmid hepatocyte
growth factor)
 Advexin – head and neck cancer (p53 adenoviral vector)
 Cerepro – glioma (thymidine kinase adenoviral vector)
Lecture 3: Uncontrolled Cell Death
A. Two Different Types of Cell Death
Apoptosis Necrosis
“clean death” “dirty death”
Lack of growth factors, Anoxia
Causes Hormonal influences, Physical damage
Mild toxic influences Chemical change
First apparent
Shrinking, Swelling, content
cellular
Convolution leakage
changes
Condensation,
Nuclear
segmentation, DNA -
changes
fragmentation
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 Surface protrusions induce apoptosis in the cell
(blebbing), budding
Cell membrane F. Conditions associated with a defective apoptosis
changes in Smoothing, lysis
changes regulation
phosphatidylserine
distribution Excessive Apoptosis
Mitochondrial 1. Ischemia
- Swelling
changes 2. Heart failure
Active changes in gene 3. Neurodegeneration
Metabolic/ expression (e.g., BCL2, 4. Inflammation
Synthetic Bax); Active protein - 5. Osteoarthritis
changes synthesis; protease 6. Human immunodeficiency virus : depletion of T lymphocytes
activation 7. Bacterial Infection
Eliminate potentially Eliminate 8. Allograft rejection and graft versus host disease
Function damaging cells, for potentially 9. Type 1 diabetes : Immune-mediated destruction of the islets of
proper development damaging cells Langerhans
Insufficient Apoptosis
B. Studying Cell Death/Apoptosis in the Laboratory 1. Cancer
Important points to remember: 2. Autoimmunity
- When you grow cells in the lab, there is what we call as 3. Persistent Infections
“Minimum Seeding Density.” Cells are grown up to around
10,000 to 100,000 in quantity. They need to be in close contact G. IAPs: Inhibitor of Apoptosis Proteins
with each other in order to be stimulated and further replicate. - keep caspases in check
Cells would undergo apoptosis if they do not receive stimuli from - contain at least 1 copy of a Baculovirus IAP repeat (BIR)
other cells. domain
- Confluence is the term commonly used as an estimate of the - suppress apoptosis when overexpressed
number of adherent cells in a culture dish or a flask, referri ng to - several IAPs directly bind and inhibit caspases
the proportion of the surface which is covered by cells. - potential drug targets for cancer
- Contact inhibition is the cessation of cell motility that occurs - IAP genes could be used to treat neurological diseases
when a cell culture reaches confluence.

C. Apoptotic Parameters: Tools for Apoptosis Detection

1. Morphological features of apoptotic cells
2. DNA fragmentation: hallmark of apoptotic cell death
3. Activation of proteases
4. Cell membrane alterations
5. Mitochondrial changes

Distribution of DNA in a Norm al and in an Apoptotic Cell

Normal Cell Apoptotic Cell
compartment HMW LMW HMW LMW
Nucleus + -
Cytoplasm - - -

C. Methods for detecting apoptosis in cells

1. Morphology – microscope
2. DNA fragmentation – Gel Electrophoresis
3. Free ends from DNA fragmentation – TUNEL Method
4. Quantify Apoptotic cells – Flow Cytometry

D. Mechanisms of Apoptosis
What makes a cell decide to commit suicide?
 The withdrawal of positive signals; that is, signals needed
for continued survival (e.g., intrinsic factors, cell adhesion)
 The receipt of negative signals (e.g., death activators,
radiation, viral infection and cytotoxic T-cells)

2 general mechanisms by which a cell commits suicide by

apoptosis:
1. Intrinsic Pathway
- generated by signals arising within the cell (i.e., cell stress or
DNA damage)
Two Pathways:
a. Telomere Shortening
b. Mitochondrial Damage
2. Extrinsic Pathway
- triggered by death activators binding to the receptors at the cell
surface (i.e., Fas ligand, TNF-α, lymphotoxin)
- triggered by dangerous reactive oxygen species

E. Caspases – Cysteine Proteases

- function: induce apoptosis in the cell
- target site: cleave after Asp residues
Two Main Classes:
1. Initiator caspases (like caspase-9)
- the upstream activators of the effector
caspases (like caspase-3)
2. Effector caspases
- the executioners in the cell, they
cleave the proteins that actually

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