High efficiency, long-term clinical expression of cottontail rabbit papillomavirus (CRPV) DNA in
rabbit skin following particle-mediated DNA transfer
High efficiency, long-term clinical expression of cottontail rabbit papillomavirus (CRPV) DNA in rabbit skin following particle-mediated DNA transfer <underline><i> </i></underline>
Wei
Xiao
1
and
Janet L.
Brandsma
1,2,3,
*
1
Section of Comparative Medicine and
2
Department of Epidemiology and Public Health,
3
Yale Skin Diseases Research Center, Yale University School of Medicine, PO Box
208016,
New Haven
, CT 06520-8016,
USA
Received February 21, 1996;
Revised and Accepted May 8, 1996
ABSTRACT
The ability of skin to support long lasting expression of genes delivered with a
particle-mediated system was evaluated in rabbits inoculated with cottontail rabbit
papillomavirus (CRPV) DNA. The optimal delivery force for maximal gene
expression in rabbit skin was determined in transient
[beta]
-galactosidase assays. Forty-five sites in four rabbits were then inoculated at 350-400 p.s.i. with CRPV DNA. All sites (100%) formed papillomas
with multiple papillomas at most sites. These results support the feasibility
of using a particle-mediated delivery system for gene therapy and suggest that some
papillomavirus features, such an origin of replication, may be well suited for
use in vectors to target long term expression to skin.
Skin is considered an ideal target for gene therapy (
1
). The ability of particle-mediated gene transfer
in vivo
to induce short term expression in skin has been demonstrated (
1
,
2
). Documentation of long lasting expression has been more difficult (
3
). In the cottontail rabbit papillomavirus (CRPV) DNA-rabbit system, the formation of cutaneous papillomas provides a phenotype
to document accurately the number of sites that retain and express a DNA
inoculum (
4
). Domestic rabbits do not replicate infectious CRPV virus. Papillomas persist
because the daughters of infected basal cells-that proliferate and replenish the epidermis-continue to express viral genes. Papillomavirus genomes contain an
origin of DNA replication and are maintained in keratinocytes as
extrachromosomal episomes (
5
).
In previous studies, we induced rabbit papillomas by inoculating skin with CRPV
DNA in an aqueous solution, under hydraulic pressure, through an intradermal
nozzle of a jet injector (
6
). Jet injectors were designed for delivering vaccines into human skin. Jet
injection of CRPV DNA was a significant advance over the older methods of
scarification and intradermal inoculation and puncture because it allowed
rabbits to be inoculated rapidly at >= 100 sites each. We used the method to study the papilloma-inducing activity of several CRPV mutant genomes, including some with
only one fourth the activity of wild-type CRPV DNA (
6
-
9
). The activity of mutants with much lower activity might be missed in this
assay, however, because the rates of papilloma formation in individual rabbits
vary widely: from <= 20 to >= 80% of wild-type CRPV DNA-injected sites. The genetic heterogeneity of rabbits
probably contributes to the observed variability, but so does the injection
device. Furthermore, jet injection requires substantial amounts of purified
DNA, e.g.
50 [mu]g per site.
Particle-mediated methods of DNA delivery
in vivo
are simple. Small quantities of DNA are precipitated onto inert particles of
gold and accelerated to high velocity, for example, by the instantaneous
release of high pressure helium, resulting in penetration of target tissues.
This approach has been used to express individual genes in skin
(
2
), and we also used it to show that human papillomavirus type 16 (HPV 16)
genomes could induce papillomas in human foreskin xenografts on severe combined
immunodeficient (
scid
) mice (
10
). The current study demonstrates that particle-mediated delivery of CRPV DNA can induce persistent papillomas in rabbit
skin with very high frequency. This will facilitate the study of CRPV genetics
in vivo
and also has implications for the use of papillomavirus-based vectors for gene therapy of skin.
Two plasmids were used. pCMV-[beta] (Clonetech, Palo Alto, CA) expressed the [beta]-galactosidase ([beta]-gal) gene from the cytomegalovirus (CMV)
promoter. CRPV-pLAII contained a full-length CRPV genome in plasmid pLAII. Supercoiled plasmids were
purified (
6
) and precipitated (
12
and Dennis McCabe, personal communication) onto gold particles (average
diameter 1-3 [mu]m), at a ratio of 1 [mu]g DNA:0.5 mg gold, in 0.1 M spermidine and 2.5 M CaCl
2
during a 10 min incubation at 20oC. The DNA-coated gold particles were pelleted at 12 000 r.p.m. for 30 s, washed three times with 100% ethanol, and resuspended at 2 [mu]g DNA/mg gold/ml ethanol. The DNA-gold-ethanol suspension was introduced into a 22I section of Tefzel tubing (1/8I outside diameter, 3/32I internal diameter) (McMaster-Carr, Elmhurst, IL). Particles were allowed
to settle onto the bottom of the tubing and the ethanol was then evacuated
using a peristaltic pump. The tubing was rotated at 20 r.p.m. for 30 s in a
device (Agracetus, Inc., Middletown, WI) designed to distribute the gold evenly
over the inner walls of the tubing. Rotation was continued as the DNA-gold was dried under a continuous stream of nitrogen gas delivered at 250
ml/min. The tubing was sliced into 1/2 I lengths to generate `shots' containing 1 [mu]g DNA/0.5 mg gold. The shots were loaded into a 12-shot barrels of a helium-driven Accelltm Gene Delivery Device (Agracetus, Inc., Middleton, WI) (
7
).
Two kilogram,
Pasteurella
-free female New Zealand white rabbits were anesthetized with ketamine (30
mg/kg) and xylazine (3 mg/kg). Hair was clipped from a 100-200 cm
2
area on the rabbit backs, and residual hair and superficial keratin were
treated with a commercial depilatory (Nair, Division of Carter-Wallace, Inc., New York, NY 10105). The Accelltm device was held against the skin, and up to 15 sites per rabbit
were inoculated.
Transient assays of pCMV-[beta] gal expression were used to evaluate the effectiveness of delivery
pressures from 300 to 700 p.s.i. A minimum of two skin sites per rabbit in at least two
experiments were evaluated for each pressure. Sites were collected 24 h after
inoculation and frozen in cryoembedding medium. Transverse frozen sections (5
or 10 [mu]m thick, ~15 mm long) were fixed in acetone-methanol (1:1) for 10 min at room temperature and stained in
standard X-gal buffer (44 mM HEPES-Na salt, 150 mM NaCl, 1.3 mM MgCl
2
, 3 mM K ferrocyanide, 3 mM ferricyanide, pH 7.4) containing 0.5 mg/ml X-gal at 37oC overnight. Sections were counterstained in hematoxylin and eosin
and examined by light microscopy. Sites that had been inoculated with pCMV-[beta] and previously shown to express [beta]-gal served as positive controls. Sites that had been
inoculated with gold particles and no DNA or not bombarded served as negative
controls. Positivity was quantified as the number of X-gal-expressing cells per field, examined with a 10* objective lens, as illustrated in Figure
1
.
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