Endocrine
Disease Projects
Project: Ocular Applications
of Recombinant AAV
Mark
Atkinson, Ph.D.
The use
of recombinant viral techniques
to deliver and express genes
of therapeutic value to ocular
tissue is likely to be the
most important and direct interface
between basic science and clinical
ophthalmology in the next decade.
There are several reasons for
this: 1. A growing number of
genes responsible for presently
incurable ocular diseases or
for adverse side effects of
surgical procedures have been
or are being identified. They
provide a multitude of viable
genetic strategies for ocular
therapy; 2. The eye's accessibility,
immune and physical containment
and often rapid biological
response make it one of the
most likely targets for successful
gene therapy intervention in
the near future; 3. There are
multiple strains of animals
that either mimic or are the
exact genetic counterpart of
ocular disease in humans; 4.
Behavioral and physiological
assessment of visual function
(visual acuity tests, visual
field analysis, electroretinograms)
have been extremely well established
for many decades in literally
millions of patients. This
provides a robust set of criteria
for evaluating the success
of clinical trials. Three labs
at the University of Florida
are actively engaged in ocular
gene delivery experiments:
William Hauswirth-retina, John
Guy-optic nerve, and Gregory
Schultz-cornea. Two of the
laboratories will use adeno-associated
virus (AAV) as the vector for
gene delivery and this will
be done in collaboration with
Gene Therapy Vector Core Lab
and N. Muzyczka. Preliminary
experiments by W. Hauswirth
and N. Muzyczka in rats, guinea
pigs and mice have demonstrated
that AAV recombinant vectors
carrying the gene for beta
galactosidase under the control
of either a tissue specific
opsin promoter or the CMV promoter
can transduce the retinal epithelial
and sensory nerve cell layers
of the eye at high frequency.
Expression has been found to
continue for up to three months.
This result has suggested a
variety of applications for
hereditary retinopathies and
age related ocular degenerative
diseases.
Dr. Hauswirth
will be attempting rhodopsin
gene therapy in the pro23-his
transgenic mouse, an exact model
of one form of human retinitis
pigmentosa (RP). Dr. Hauswirth
is also collaborating with three
laboratories outside the University
that are interested in ocular
gene therapy. With Drs. Matt
Lavail and Roy Steinberg of UCSF,
recombinant AAV containing various
opsin promoter/ neurotrophin
constructs are being tested for
use as photoreceptor survival-promoting
agents in animal models of degenerative
retinal diseases. Using a rat
photoreceptor light damage model
which mimics human photoreceptor
degeneration, expression of protective
levels of human neurotrophins
could lead to human protocols
in 2-4 years. With Dr. John Flannery
at UC Berkeley, recombinant AAV
containing opsin promoter/b subunit
of cyclic GMP phosphodiesterase
constructs are being provided
for use in somatic gene therapy
of retinal degeneration in the
rd mouse, the major animal model
for recessive human RP. If successful,
human trials could begin in 3-5
years for human RP caused by
b-PDE genetic defects. Finally,
with Dr. Debra Faber at UCLA,
recombinant AAV containing opsin
promoter/reporter genes are being
provided for expression testing
in retinoblastoma cells in culture
as an initial assay of its utility
in treating ocular tumors. Potential
human trials with this intervention
are at least 5 years away. The
HAL facility on the GCRC will
be essential in producing clinical
grade vectors for each of these
protocols.
References
- Muzyczka
N. Use of AAV as a general
transduction vector for mammalian
cells. In: Curr. Top. Micro.
Imm. Viral Expression Vectors
(Muzyczka, N., ed), Springer
Verlag, Berlin, vol 158, pp.
97-129.
- DesJardin LE, Timmers
AM, Hauswirth WW. Transcription
of photoreceptor genes during
fetal retinal development:
Evidence for positive and
negative regulation. J Biol
Chem 268:6953-6960, 1993.
- Van
Ginkel PR, Hauswirth
WW.
Parallel regulation
of fetal
gene expression in different
photoreceptor cell types.
J Biol Chem 269:4986-4992,
1994
Project: Experimental Optic Neuritis:
Oxidative Injury
John Guy , Ph.D.
The
overall
aim of
this proposal
is to test
the hypothesis
that reactive
oxygen
species
play a major
role
in the
pathogenesis
of disruption
of the BBB and
demyelination
of the
optic
nerve.
We will
pursue
the following
specific
aims:
(1) Assessment
of the
response
of cell-specific
endogenous
antioxidant
and free
radical
scavenger
enzyme
defenses
in the
optic
nerve
to reactive
oxygen
species
in guinea
pigs
with
EAE.
The specific
cellular
expression
in endothelia,
axons,
astroglia
and oligodendroglia
of the
superoxide
dismutases
(Cu/Zn,
MnSOD
and ECSOD),
catalase
and glutathione
peroxidase
will
be visualized
by immunohistochemistry
and in
situ
mRNA
hybridization.
(2) Determination
of the
best
strategy
for the
suppression
of demyelination
and BBB
disruption
by genetic
amplification
of specific
free
radical
scavenger
defenses
in mice
transgenic
for the
superoxide
dismutases
(ECSOD
and MnSOD).
(3) Development
of a
clinically
useful
treatment
strategy
for prolonged
suppression
of demyelination
and BBB
disruption
by amplification
of beneficial
defenses
with
viral-mediated
transfer
of cDNAs
for the
superoxide
dismutases
(MnSOD,
Cu/Zn
and ECSOD)
and catalase.
Using
AAV mediated
gene
therapy
we hope
to markedly
increase
endothelial,
axonal
and oligodendroglial levels
of the SODs
and catalase. Continued
overexpression of these
enzyme defenses
may also prevent
the relapses of optic
neuritis and progression
to MS. Both
the HAL and GCRC
ward will
be essential for vector
production and delivery.
The OCI will
provide needed
research nurses
and patient registry
facilities.
References
- Guy
J,
McGorray
S,
Fitzsimmons
J,
Beck
B, Rao
MA.
Disruption
of the BBB in experimental optic neuritis: Immunocytochemical
co-localization
of H2O2 and extravesated serum albumin. Invest Ophthalmol Vis
Sci
35:1114-1123,
1994.
- Guy J, McGorray S, Qi X, Fitzsimmons J, Mancuso A, Rao NA. Conjugated
deferoxamine reduces blood-brain barrier disruption in
experimental optic neuritis. Ophthal Res. in press.
Project: Reduction
of Scarring
Following
Trabeculectomy
using Transiently
Transfected
Ribozymes
to TGF-b
Gregory
Schultz,
Ph.D.
The
hypothesis
is
that
the
scarring
which
occurs
following
trabeculectomy
is
primarily
controlled
by
transforming
growth
factor
beta
(TGF-b)
that
is
produced
by
cells
in
the
area
of
the
trabeculectomy.
Thus,
if
the
amount
of
TGF-b
protein
released
in
the
trabeculectomy
wound
area
can
be
limited,
the
amount
of
scarring
would
be
reduced
while
causing
minimal
side
effects.
To
accomplish
this
goal,
plasmids
will
be
synthesized
that
transcribe
ribozymes
which
will
selectively
destroy
TGF-b
mRNA.
The
plasmid
will
be
transiently
transfected
into
cells
of
the
trabeculectomy
wound
at
the
time
of
surgery
using
either
liposome
or
adenovirus
vectors.
Long-term
inhibition
of
TGF-b
action
would
not
be
required
to
prevent
bleb-scarring
since
bleb-failure
is
initiated
during
the
first
two-week
post-operative
period.
We have begun development of ribozymes specific for TGF-b1, TGF-b2 and the type
II TGF-b receptor, have synthesized plasmids which contain autocatalytic processing
hairpin ribozymes and are currently evaluating their ability to cleave TGF-b
and receptor mRNAs in vitro. We will continue to develop and refine the ribozymes
for TGF-b1, TGF-b2 and type II TGF-b receptor. This will involve construction
of plasmids which contain hairpin and hammerhead ribozymes. The ribozymes will
be tested for catalytic activity both in vitro and in vivo. After the ribozymes
have been optimized for maximum effectiveness, we would test them in patients
at high risk for trabeculectomy failure. Initially we expect to utilize an expression
plasmid with a strong promoter for human cells such as SV40 or CMV. Production
of GMP grade cationic liposome or adenovirus victors for humans would require
the use of the special gene therapy facilities of the HAL. Administration of
the ribozymes and monitoring of the patients would require the use of the OCI
and GCRC outpatient facilities.
References
-
PT
Khaw,
NL
Occleston,
G
Schultz,
I
Grierson,
MB
Sherwood,
G.
Larkin.
Activation
and
suppression
of
fibroblast
function.
Eye
8:188-195,
1994.
- PT Khaw, NL Occleston, K Sethl, RA Alexander, GS Schultz, I
Grierson. The effects of growth factors
(EGF,
bFGF,
TGF-b1,
and
IGF-1)
on
cellular
and molecular aspects of ocular fibroblast
wound
healing
behavior
In
Vitro.
European Tissue Repair Meeting, August
1994.
- R Tarnuzzer, R Stiff, GS Schultz. Development of hairpin ribozymes
for TGF-b1, TGF-b2, and type II receptor with defined 3'termini by autocatalytic
processing. J Cellular Biochem 19A:A6-319, 1995.
|
