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Pulmonary Disease
Projects
Project: Cystic
Fibrosis
Terry
Flotte,
M.D.
Most
individuals
with
cystic fibrosis
(CF) suffer
from a particular
form
of chronic
obstructive
lung
disease characterized
by endobronchial
infection
with Pseudomonas
aeruginosa
and other
bacterial pathogens
and an exaggerated
inflammatory
response
to those pathogens,
that
leads over
time to bronchiectasis
and respiratory
failure.
At a molecular
level,
CF is due to
defects in
CFTR, a cAMP-regulated
chloride
channel. CFTR
likely plays
an important
role
in controlling
the bulk
flow and/or
ionic composition
of airway
surface liquid
(ASL). Changes
in the ASL
and in the
mucus layer
that sits right
above
it on the airway
surface
could predispose
CF patients
to airway
infection and/or
obstruction.
There
is increasing
evidence
to indicate,
however, that
patients with
CF also have
an exaggerated
inflammatory
response
as well, perhaps
as a
primary consequence
of CFTR
mutations.
A recent study
from
the Prince
laboratory
(7) has indicated
that
cells from
CF patients
have
increased NF-B
activation,
which leads
to increased
secretion of
pro-inflammatory
cytokines,
such as IL-8
and decreased
secretion of
anti-inflammatory
cytokines,
such as IL-10.
This in turn
results in
a massive influx
of neutrophils
into the airways
with subsequent
alteration
of airway function
and structure.
We hypothesize
that this pathway
plays a crucial
role both in
the pathogenesis
of CF lung
disease and
in creating
a barrier to
viral vector-mediated
CFTR gene transfer
in the airways
of affected
individuals.
We propose
to test these
hypotheses
by (1) evaluating
the role of
anti-inflammatory
and/or anti-protease
therapy as
a prelude to
AAV-CFTR gene
therapy and
(2) determining
whether CF
lung disease
can be ameliorated
by the controlled
expression
of anti-inflammatory
molecules from
rAAV vectors.
IL-10 expression
will be investigated
as a multi-functional
anti-inflammatory
in preclinical
models of CF
lung disease
as a potential
future anti-inflammatory
gene therapy.
This will be
accomplished
in the context
of the following
specific aims:
Specific Aim 1. The ongoing phase I trial of AAV-CFTR gene therapy in
adult CF patients will be modified to evaluate the role of anti-inflammatory
pretreatment as a means to improve gene transfer efficiency. Selected cohorts
of adult CF patients receiving the tgAAVCF vector via fiberoptic bronchoscopy
would either (1) undergo a pretreatment protocol consisting of intravenous antibiotics,
airway clearance maneuvers, and anti-inflammatory steroids, (2) undergo a similar
regimen with the addition of aerosolized AAT, or (3) receive the gene transfer
vector without any pretreatment
Specific Aim 2. As an initial means of developing anti-inflammatory gene
therapy, rAAV vectors expressing IL-10 under the control of a tetracycline-controlled
transciptional activator system will be constructed and tested in vitro.
The commercially available Tet-Off system (22) (Clontech, Palo Alto, CA) will
be evaluated in the context of rAAV-IL-10 vectors. In this system, IL-10 will
be under the control of a minimal promoter engineered to contain a series of
tetracycline responsive elements (TRE) in a rAAV backbone. A second gene expressing
the tetracycline transactivator (tTA) will also be engineered into rAAV. The
ability of the tTA protein to activate IL-10 transcription in the absence of
doxycycline will be evaluated.
Specific Aim 3. Repressible and constitutively active rAAV-IL10 vectors
will be evaluated in vivo in the lungs of IL-10 deficient mice. The safety, efficiency,
and stability of expression over a 1-year interval following intratracheal injection
will be examined. The optimal dosage of the rAAV vectors (including the tTA-expressing
vectors in the case of the TRE-constructs) will be evaluated in terms of the
safety and efficiency of expression. The ability of doxycycline to maintain repression
will also be assessed in the context of rAAV-TRE-IL-10/rAAV-tTA gene transfer.
And the ability of rAAV-IL10 to control the exaggerated inflammatory response
seen in Pseudomonas-infected IL-10 knock-out mice will also be investigated.
Specific Aim 4. Once the optimal dosages of rAAV are defined, we will
evaluate the ability of rAAV-derived IL-10 to ameliorate the lung disease phenotype
of the Pseudomonas-infected CFTR knockout mice. The effects of rAAV-IL-10 expression
will be tested with respect to the release of pro-inflammatory cytokines, the
degree of neutrophil influx, the stunting of weight gain, and the overall mortality
in this model of CF lung disease.
Our
ultimate goal
is to bring
into clinical
trials those anti-inflammatory therapies that appear to be the safest
and most effective.
It is difficult
to determine
at this time
whether this
type of anti-inflammatory
therapy would be used as a prelude to AAV-CFTR gene therapy in patients
with more advanced
CF lung disease
or as a stand-alone
therapy to
retard the
progression
of inflammatory lung damage.
Project: Alpha-1-antitrypsin
Deficiency
Terry
Flotte,
M.D.
Alpha
1-antitrypsin
(AAT) deficiency
(A1AD) is characterized
by a marked
decrease in
the circulating
levels of the
major serum
antiprotease,
AAT, with a
subsequent
compromise
of pulmonary
elastin resulting
in a susceptibility
to emphysema.
The most common
AAT mutation
associated
with this disease,
the PI*Z allele
(Glu342Lys),
accounts for
approximately
95% of cases.
A minority
of AAT deficient
individuals
will also develop
a liver disease
which is characterized
by polymerization
of the Z-form
of AAT (Z-AT)
within the
endoplasmic
reticulum (ER)
of hepatocytes,
which triggers
hepatocellular
injury, and
ultimately
cirrhosis.
While the pulmonary
disease associated
with AAT deficiency
can be treated
by replacing
serum levels
of the protein
up to a known
threshold (11 M),
the proximate
goals of therapy
for A1AD liver
disease are
less clear.
Since the pathology
appears to
relate to a
gain-of-function
by the abnormal
protein, down-regulation
of expression
of the mutant
endogenous
alleles might
be considered.
However, heterozygotes
appear much
less susceptible
to disease
than PI*Z homozygotes,
which may indicate
either a protective
effect of the
wild-type (PiM)
version of
the protein
or a gene dosage
effect. Futhermore,
recent reports
indicate that
chemical chaperones,
such as 4-phenylbutyric
acid (4-PBA),
can assist
the proper
folding of
Z-AT, perhaps
suggesting
that an alternate
therapeutic
approach by
augmenting
chaperone function
at a molecular
level. The
goal of this
application
is to sort
out these various
therapeutic
options using
recombinant
AAV vectors
that can stably
insert therapeutic
molecules without
significant
toxicity. This
will be accomplished
in the following
specific aims:
Aim
1: To utilize
gene transfer
vectors in
order to define
cellular endpoints
for therapy
of A1AD-liver
disease. Our
hypothesis
is that a 50%
decrease in
Z-AT by rAAV-ribozyme
vectors will
be sufficient
to prevent
cellular pathology
due to Z-AT
polymerization.
We will confirm
this by treating
stably transfected
CHO cell lines
that constitutively
express Z-AT
with transcriptionally-controlled
anti-AAT ribozymes
already in
use in our
laboratory.
The endpoints
for these studies
will be ER
polymerization
and accumulation
of Z-AT as
judged by EM
and immunofluorescent
staining combined
with confocal
microscopy.
The potential
need for augmentation
with PiM AAT
(M-AT) in facilitating
this correction
will also be
examined.
Aim
2: To define
the role of
augmenting
Hsp70 function
as therapy
for conformational
disease. Previous
studies with
chemical chaperones
suggest that
augmentation
of Hsp70 with
rAAV-Hsp70
vectors will
increase folding
of mutant AAT
and CFTR in
a native configuration
and facilitate
degradation
of the misfolded
protein. In
the case of
the Z-AT-expressing
CHO cell lines,
this should
result in an
increase in
secretion of
the mutant
protein into
the supernatant
media, as well
as correction
of the accumulation
of mutant protein
in the ER.
Aim
3: To evaluate
the potential
roles for rAAV-hAAT,
rAAV-Hsp70,
and rAAV-Rz,
vectors to
ameliorate
liver pathology
in mouse models.
In a companion
NIDDK-sponsored
program, our
laboratory
has been actively
working to
increase the
efficiency
of rAAV-mediated
transduction
of hepatocytes
in vivo as
a potential
means to deliver
therapeutic
molecules in
the context
of A1AD liver
disease. In
this final
aim, we will
combine those
molecular strategies
that appear
most fruitful
in the cell
culture models
with those
vector improvements
that result
in the greatest
enhancement
of rAAV-mediated
transduction.
The resultant
vectors will
be used in
one of two
kinds of models:
(1) a Z-AT
overexpressing
transgenic
mouse model
that has previously
been published;
or (2) mice
that are stably
expressing
human AAT from
previous portal
vein injection
of rAAV-hAAT
vectors. The
former model
will be useful
for all three
therapies,
while the latter
will only be
useful for
ribozyme experiments.
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