HUMAN GENE THERAPY 12:1551–1558 (August 10, 2001)

Mary Ann Liebert, Inc.

Sustainable Systemic Delivery via a Single Injection of

Lentivirus into Human Skin Tissue

SEUNG-CHEOL BAEK,1 QUN LIN, PAUL B. ROBBINS, HONGRAN FAN, and PAUL A. KHAVARI

ABSTRACT

The skin offers a tissue site accessible for delivery of gene-based therapeutics. To develop the capability for
sustained systemic polypeptide delivery via cutaneous gene transfer, we generated and injected pseudotyped
HIV-1 lentiviral vectors intradermally at a range of doses into human skin grafted on immune-deficient mice.
Unlike Moloney murine leukemia virus (MLV)-based retrovectors, which failed to achieve detectable cuta-
neous gene transfer by this approach, lentivectors effectively targeted all major cell types within human skin
tissue, including fibroblasts, endothelial cells, keratinocytes, and macrophages. After a single injection, lentivec-
tors encoding human erythropoietin (EPO) produced dose-dependent increases in serum human EPO levels
and hematocrit that increased rapidly within one month and remained stable subsequently. Delivered gene
expression was confined locally at the injection site. Excision of engineered skin led to rapid and complete
loss of human EPO in the bloodstream, confirming that systemic EPO delivery was entirely due to lentiviral
targeting of cells within skin rather than via spread of the injected vector to visceral tissues. These findings
indicate that the skin can sustain dosed systemic delivery of therapeutic polypeptides via direct lentivector in-
jection and thus provide an accessible and reversible approach for gene-based delivery to the bloodstream.

OVERVIEW SUMMARY
Direct vector administration to skin avoids the costly,
painful, and scarring process of skin harvesting, in vitro
gene transfer, and regrafting. To develop an approach to
durable direct cutaneous gene transfer, we injected lentivec-
tors into human skin tissue on immune-deficient mice at a
range of doses. Lentivectors successfully targeted all major
cell types within the epidermis and dermis, supporting vec-
tor dose-dependent and stable delivery of erythropoietin to
the bloodstream. Gene delivery remained localized in the
skin and could be readily removed by punch biopsy of the
injected site. These data establish an approach to durable
systemic polypeptide delivery via a single injection of
lentivector into human skin tissue.
INTRODUCTION
sues, the skin is readily available for direct topical and injectable
gene transfer in a minimally invasive and uncomplicated fash-
ion. Direct gene delivery approaches to skin include topical ap-
plication (Li and Hoffman, 1995; Fan and Khavari, 1999; Do-
mashenko et al., 2000), intradermal injection (Hengge et al.,
1995; Choate and Khavari, 1997), bioplastic particle accelera-
tion (Wang et al., 1999; Davidson et al., 2000), and vector in-
fusion onto wounded skin ( Lu et al., 1997; Ghazizadeh et al.,
1999). In addition to ease of administration, the skin is easily
subjected to direct clinical inspection following gene transfer
with uncomplicated removal of genetically engineered tissue
achieved by simple excision if needed. These tissue character-
istics have facilitated progress in gene transfer for primary skin
disorders, including the recessive ichthyoses (Choate et al.,
1996a,b; Choate and Khavari, 1997; Freiberg et al., 1997a) and
epidermolysis bullosa subtypes (Seitz et al., 1999; Dellambra
et al., 2000; Robbins et al., 2001).
In addition to primary skin diseases, cutaneous gene trans-
fer offers promise for the treatment of systemic disorders re-

A S THE MOST ACCESSIBLE OF BODY ORGANS , the skin provides

an attractive tissue for therapeutic gene transfer (Cao et
al., 2000; Ghazizadeh and Taichman, 2000; Khavari, 2000;
Uitto and Pulkkinen, 2000; Vogel, 2000). Unlike visceral tis-
sponding to delivery of polypeptides to the circulation because
the skin is richly vascularized. A range of skin-produced pro-
teins have been demonstrated to reach the bloodstream follow-
ing a variety of approaches to gene insertion into cells of both

Veterans Affairs Palo Alto Healthcare System and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305.
1Present address: Department of Dermatology, Catholic University College of Medicine, Seoul, Korea.

1551
1552
the epidermis and the dermis or via addition of unmodified hu-
man skin elements. Such polypeptides include growth hormone
(Morgan et al., 1987; Teumer et al., 1990; Jensen et al., 1994;
Wang et al., 1997), apolipoprotein E (Fenjves et al., 1989), Fac-
tor IX (Gerrard et al., 1993), interleukin-10 (Meng et al., 1998),
and transferrin (Petersen et al., 1995). While convincingly
demonstrating the potential for cutaneous gene transfer to
achieve systemic delivery, these studies have predominantly re-
lied on ex vivo gene delivery to skin. Ex vivo gene transfer to
skin requires biopsy for tissue harvesting, then growth and gene
transfer in vitro followed by the painful and generally scarring
process of regrafting and is thus a costly, complicated and trau-
matic process. Widespread use of the skin for genetic treatment
of systemic disease will therefore require improved approaches.
To make skin gene transfer widely applicable to a range of
human systemic diseases, new capabilities for minimally inva-
sive direct cutaneous gene delivery are needed. These involve
sustainability, efficiency, and control of gene transfer to skin.
To date, no approach or vector yet studied has fully met these
criteria after direct administration to skin. Plasmid DNA, per-
haps the most well studied, displays very low efficiencies of
gene transfer when topically applied or injected and is subse-
quently lost within days due to lack of integration (Hengge et
al., 1996; Choate and Khavari, 1997; Fan and Khavari, 1999).
While vastly more efficient, adenoviral gene transfer also suf-
fers from a lack of durability due to a failure to integrate into
target cells and immunogenicity ( Lu et al., 1997; Ghazizadeh
et al., 1999). Moloney murine leukemia virus (MLV) retro-
viruses are also limited in that detectable levels of direct skin
gene transfer have only been achieved when the virus is in-
jected beneath hemorrhagic eschar after the skin has been sub-
jected to a tissue destructive wounding procedure to stimulate
cellular proliferation (Ghazizadeh et al., 1999). Adeno-associ-
ated virus (AAV) vectors are capable of infecting cells of
murine skin and achieving systemic protein delivery. However,
this is likely to be of limited clinical relevance because AAV
appears capable of sustaining delivery only via retention in the
mouse panniculus carnosus (Donahue et al., 1999), a muscular
structure that does not exist in human skin. Thus, prior ap-
proaches to direct cutaneous gene transfer have one or more
shortcomings in meeting criteria for practically effective direct
gene transfer to the skin.
Among the vector platforms available, HIV-1 lentiviral vec-
tors are attractive candidates because of their ability to initiate
and sustain gene delivery efficiently in both dividing and non-
dividing cells after injection into neural and muscle tissue (Nal-
dini et al., 1996; Zufferey et al., 1998; Kafri et al., 1999; Lever
et al., 1999). Among the systemic disorders potentially
amenable to cutaneous gene transfer are those characterized by
inadequate blood levels of specific polypeptides, such as is seen
in erythropoietin (EPO)-responsive anemia. Here we have de-
livered HIV-1-based lentivectors encoding human EPO along
with marker gene controls by a single intracutaneous injection
into full-thickness human skin tissue grafts on immune-defi-
cient mice. We have used human skin as a target tissue to en-
hance the practical value of this approach because it differs in
significant ways from mouse and other animal models, includ-
ing at the levels of anatomy and viral tropism. This gene de-
livery approach targeted all of the major cell types within skin
with achievement of sustained EPO delivery and increased
BAEK ET AL.
hematocrit in a vector dose-dependent manner. These findings
identify a promising approach to minimally invasive direct gene
transfer to skin for the treatment of systemic disease.
MATERIALS AND METHODS
Vectors
The self-inactivating lentiviral transfer vector backbones in
which an internal cytomegalovirus (CMV) promoter drives ex-
pression of either lacZ (Dull et al., 1998) or green fluorescent pro-
tein (GFP) (Naldini et al., 1996) have been described. The full-
length human EPO cDNA (gift of P. Aebischer) was digested with
BamHI and XhoI and subcloned into the BamHI and XhoI sites
of PHR-CMV-GFP (Naldini et al., 1996) to generate PHR-CMV-
EPO which contains 350 bp of gag as well as env sequences en-
compassing the Rev-responsive element (RRE). Lentivirus was
produced by transient co-transfection of human 293T cells with
three plasmids. A total of 15 mg of transfer vector, 10 mg of the
pCMVDR8.2 packaging plasmid, and 5 mg of pCI-VSV-G, which
encodes the vesicular stomatitis virus envelope G (VSV-G) gly-
coprotein, were transfected into 5 3 106 293T cells grown in
DMEM with 10% fetal bovine serum (FBS) in 10-cm dishes us-
ing FuGene 6 (Roche, Indianapolis, IN) according to the manu-
facturer’s instructions. Conditioned medium was replaced the day
following transfection and supernatant harvested 48 hr post-trans-
fection. Supernatant was passed through a 0.45-mm filter then con-
centrated by ultracentrifugation at 40,000 3 g for 2 hr. Pellets
were resuspended in media and then concentrated by precipita-
tion in a final concentration of 10% polyethylene glycol and pel-
leted by centrifugation for 20 minutes at 5000 3 g. The final vi-
ral pellet was resuspended for 4 hr at 4°C in phosphate-buffered
saline (PBS) to yield concentrations of 10- to 200-fold. Viral vec-
tor stocks were titered by infecting target cells with serial dilu-
tions in media supplemented with Polybrene at a concentration of
8 mg/ml. Expression of b-galactosidase was analyzed by staining
with X-gal (Sigma, St. Louis, MO.). Viral p24 antigen concen-
trations were determined by HIV-1 core profile enzyme-linked
immunosorbent assay (ELISA; NEN Life Science Products,
Boston, MA). In our studies, 1 mg of p24 equivalent corresponded
to approximately 2.5 3 106 infectious viral particles.
Cells and gene transfer
Primary human skin fibroblasts were isolated and maintained
in Dulbecco’s modified Eagle medium (DMEM) with 10% FBS
as described (Balcar et al., 1994; Freiberg et al., 1997b). Cells
were transduced in vitro in media supplemented with Polybrene
at 8 mg/ml by addition of vector followed immediately by 90
min of centrifugation at 1000 3 g at 32°C. Culture media of
EPO transduced cells was changed 2 hr before collection of su-
pernatants 48 hr following transduction.
Expression analysis
Human EPO was assayed by human species-specific ELISA
using the human EPO Quantikine IVD Kit (R&D Systems, Min-
neapolis, MN) according to the manufacturer’s recommenda-
tions. Blood obtained at various times before and after vector
administration was analyzed for hematocrit using an automated
SYSTEMIC EPO DELIVERY VIA HUMAN SKIN
hematology analyzer (Cell-Cyn 3500, Abbott Laboratories, Ab-
bott Park, IL). For immunohistochemistry, 5-mm skin cryosec-
tions were incubated with human species-specific antibodies at
1:50 dilution in PBS. Antibodies used recognized cell-surface
proteins expressed on human skin macrophages (F4/80, Serotec,
Oxford, UK), endothelial cells (Factor VIII, Dako, Carpenteria,
CA), and fibroblasts (a5 integrin, gift of J. Lie, Stanford, CA).
Tetramethylrhodamine isothiocyanate (TRITC)-labeled sec-
ondary antibodies (Sigma, St. Louis, MO) were used at 1:500
dilution in PBS. Co-staining was performed using fluorescein
isothiocyantate (FITC)-labeled antibodies to b-galactosidase at
a 1:50 dilution (Rockland, Gilbertsville, PA). Sections were
then mounted and visualized using dual-color fluorescence mi-
croscopy. For histochemical detection of lacZ gene expression,
grafted skin was biopsied and then frozen immediately on dry
ice. The 5-mm cryosections were fixed in 0.25% glutaraldehyde
and stained with X-Gal as described (Deng et al., 1998).
Animal studies
Adult CB.17 scid/scid mice were anesthetized with tribro-
moethanol by intraperitoneal (i.p.) injection. 2.0 3 2.0-cm
squares of full-thickness human skin were trimmed of subcuta-
neous fat and transplanted onto the backs of recipient mice at a
site in which overlying murine skin had been excised down to
fascia as described (Choate et al., 1996b). Nonhealing grafts,
which represented approximately 5% of the total, were excluded
from subsequent use. Lentiviral vector in a total volume of 50
ml was injected intradermally (i.d.) using a 30-gauge needle at
a single site in the center of the graft. Three months after graft-
ing, at a time when gene expression and cell division have re-
covered to normal levels seen in undisturbed normal human skin
(Medalie et al., 1996), mice were injected with either 1, 5 or 10
mg of p24 equivalent of lentivector encoding EPO (n 5 5 per
dose). Two mice were injected with lacZ control lentivector at
the 10 mg of p24 equivalent dose. Adult CB.17 scid/scid mice
were also used for injection studies using wholly murine skin.
RESULTS
Lentiviral gene transfer to human skin
To study the ability of lentiviral vectors to achieve cutaneous
gene transfer, we produced and injected VSV-G pseudotyped
HIV-1 lentiviral virus into full-thickness human skin grafted on
immune deficient CB.17 scid/scid mice. A self-inactivating
HIV-1 transfer vector (Naldini et al., 1996; Dull et al., 1998;)
was used; it contained a 400-bp deletion in the U3 long termi-
nal repeat (LTR), with transcription driven by internal CMV
promoter. This backbone transfer vector was used to deliver
lacZ and GFP marker genes directly to skin by injection into
the dermis, with five human tissue grafts injected with a 30-
gauge needle for each vector. Marker gene expression was ob-
served in all cases for both GFP and lacZ vectors after injec-
tion of 5 mg of p24 viral equivalent, with expression evident in
cells of both the dermis and epidermis (Fig. 1). Both lentivec-
tors produced equivalent levels of gene transfer when injected
into human skin at comparable doses. The IN-GFP MLV retro-
vector that we have previously shown can drive robust gene ex-
pression in skin tissue after ex vivo gene transfer (Deng et al.,
1553
1997), however, failed to produce detectable immunohisto-
chemical evidence of gene transfer when equivalent viral doses
were injected (data not shown). Lentivirus-mediated gene ex-
pression persisted for at least 6 months (the duration of analy-
sis) and was focally confined within the skin around the nee-
dle tract site of injection. To exclude the possibility that this
could be due to injection of protein present in the viral super-
natant, supernatants generated without Gag and Pol proteins
were also injected. These failed to show any evidence of marker
gene expression (data not shown), confirming that observed ex-
pression was due to viral infection of cells within skin.
In epidermal keratinocytes, marker gene expression was of-
ten evident in thin columns (Fig. 1), consistent with targeting
of long-lived progenitor cells at the basement membrane zone
with subsequent expression in their outwardly migrating prog-
eny. lacZ expression was also detected in major cell types
within the dermis, including endothelial cells, fibroblasts, and
macrophages using human species-specific antibodies to mark-
ers for these cell types (data not shown). Among transduced
cells, fibroblasts comprised approximately 50%, endothelial
cells 25%, and macrophages 10%; the remainder of lacZ[1]
cells did not appear to react with the cell-type-specific anti-
bodies used and thus may represent other dermal cell types.
Sustained systemic delivery of human erythropoeitin
After confirming the ability of HIV-1 lentivectors to effect
durable gene expression in significant numbers of cells within
human skin tissue, we wished to test the capacity of this ap-
proach to deliver therapeutic gene products to the bloodstream
sustainably. To do this, we constructed and produced a VSV-G
pseudotyped HIV-1-based vector encoding human EPO, a
polypeptide widely used in the treatment of anemia. First, we
confirmed the ability of this lentivector to produce high levels
of secreted EPO in target cells from human skin grown in vitro.
We infected primary skin fibroblasts and measured EPO se-
creted in the media 48 hr later. Gene transfer at multiplicities
of infection (MOI) of 15 was performed to achieve .99% ef-
ficiency lacZ marker gene transfer. In this setting, as few as
1 3 105 cells could produce EPO in a vector dose-dependent
manner at rates as high as 8.5 IU/hr (Fig. 2). This confirmed
the ability of lentivector to support EPO secretion in the pre-
dominant cellular target type within skin.
After confirming the effectiveness of this lentivector in pri-
mary cells from human skin in vitro, we wished to test its abil-
ity to achieve sustained systemic delivery via cutaneous gene
transfer. We injected 1, 5, and 10 mg of p24 equivalent intra-
dermally into human skin grafted on severe immunodeficient
(SCID) mice. This produced a prolonged delivery of human EPO
in the serum with a corresponding dose-dependentboost in hema-
tocrit (Fig. 3 and Table 1). The well-described mortality over
time characteristic of this grafting model was observed (Robbins
et al., 2001) and accounted for the animals noted as “not done”
at each time point; however, certain trends were clear in the data.
Although variability was seen between individual mice (Table
1), the average magnitude of this response corresponded to the
dose of vector initially administered. EPO delivery was stable for
the 6-month duration of the experiment, a time limit determined
primarily by the viability of both test and control grafted im-
mune-deficient mice in our hands. In the single mouse that sur-
1554 BAEK ET AL.

FIG. 1. Lentiviral vector gene delivery to human skin tissue. A total of 5 mg of p24 equivalent of lentiviral vector encoding
lacZ was injected into full-thickness human skin grafted on SCID mice. Localization of transduced cells by X-Gal staining. Note
that the first low-power panel demonstrates expression tightly localized to the needle tract, whereas the second demonstrates a
more loosely grouped distribution of expressing cells within the dermis. The third higher-power panel displays lacZ 1 cells ly-
ing among dermis, whereas the fourth panel shows a column of engineered keratinocytes in the overlying epidermis (arrows).

vived into the eleventh month post-injection (mouse 10-2, Table
1), both EPO levels and hematocrit were stable at 25 mIU/ml and
72%, respectively. To quantitate the numbers of cells in skin
transduced at the doses of lentivector producing these sustained
EPO levels, we performed immunohistochemical analysis of se-
rial 5-mm tissue sections spanning the entire region of injected
human skin tissue in multiple independently injected human skin
grafts. By this approach, we estimated that 3,343 6 1438 cells
in human skin are transduced per mg of p24 equivalent of
lentivector. This indicates that a relatively small number of sta-
bly engineered cells in skin can durably deliver systemically ac-
tive levels of proteins to the bloodstream.
Confinement of gene transfer to skin and reversibility
An ideal feature of cutaneous gene transfer would be reten-
tion of genetically engineered cells in a localized area of skin.
This would permit local control of gene delivery topically as
well as allow removal of all engineered cells in the event of a
hypersensitivity reaction to the delivered gene product. Did the
observed EPO expression meet this criteria? Intracutaneous in-
jection of 1–10 mg of p24 equivalent HIV-1 lacZ targeting the
well-vascularized dermis demonstrated lacZ expression local-
ized at the site of intradermal injection and not elsewhere in
skin away from the injection site or in visceral tissues (Fig. 1
and data not shown). Consistent with a lack of lentiviral dis-
semination, local punch biopsy excision of dermis and epider-
mis at the HIV-1 EPO skin injection site approximately 1 month
after injection rapidly led to complete loss of detectable human
EPO in the serum (Fig. 4). The fact that nonexcised skin tissue
could support stable EPO expression for as long as 10 months
more, in contrast to this drop to undetectable levels following
excision, confirms the constrained localization of lentiviral gene
transfer to the skin and indicates that the observed targeted skin
cells are responsible for EPO reaching the bloodstream.
DISCUSSION
We have demonstrated gene delivery with durable systemic
expression via a single lentivector injection into human skin tis-
sue. Advantages of this skin-targeted approach include the fact
that vector administration is minimally invasive and requires

A B

FIG. 2. Infection with a lentiviral vector encoding human EPO leads to dose-dependent EPO secretion in vitro. (A) Proviral
genome diagram of the PHR-CMV-EPO transfer vector. LTR, HIV-1 long terminal repeat; SD, splice donor; SA, splice accep-
tor; Ga, gag sequences; C, extended packaging sequences; CMV,799-bp cytomegalovirus immediate early gene promoter.
(B) Increasing amounts of EPO-encoding lentivector noted were used to infect primary human dermal fibroblasts in vitro. At
48 hr after infection, the presence and concentration of human EPO secreted into the media per hour was analyzed.
SYSTEMIC EPO DELIVERY VIA HUMAN SKIN 1555

A B

FIG. 3. Dose-dependent systemic delivery of human EPO and increase in hematocrit following a single intradermal lentivec-
tor injection in human skin tissue. Increasing amounts of EPO encoding lentivector were injected intradermally into full-thick-
ness human skin grafted onto SCID mice at time zero (n 5 5 independently grafted mice for each dose); lacZ lentivector at the
highest dose served as a control (n 5 2). (A) Levels of human EPO in serum as detected by human species-specific EPO ELISA.
(B) Hematocrit over time following injection.

only a single superficial cutaneous injection with a narrow-
gauge needle. In addition, the localized nature of gene delivery
was underscored by the fact that systemic gene product deliv-
ery could be completely terminated by removal of injected tis-
sue. This ability to achieve rapid and total cessation of gene de-
livery by skin biopsy excision of engineered cells offers
advantages over gene transfer to visceral organs where com-
plete removal of engineered cells either requires an invasive
procedure or, in the case of hematopoietic lineages dissemi-
nated in the circulation, may be nearly impossible. A further
advantage of this approach is its durability. Although human
skin/SCID mouse viability limited our studies to a maximum
of generally 6 months, but as long as 11 months in a single sur-
viving mouse, it is possible that cutaneous gene transfer of an
endogenous nonimmunogenic EPO gene to humans could re-
sult in much longer gene expression. This possibility is sup-
ported by the fact that we observed no evidence of local tissue
toxicity in skin after long-term lentiviral gene delivery as well
as by the fact that gene expression generally reached a plateau
within 1 month of injection that remained stable subsequently.

TABLE 1. EPO AND HEMATOCRIT V ALUES FOR INDIVIDUAL INJECTED MICE AT SPECIFIC TIME POINTS

Serum hEPO (mIU/ml) Hematocrit (%)

Dose Mouse Pre 2 wks 2 mo. 4 mo. 6 mo. Pre 2 wks 2 mo. 4 mo. 6 mo.
1 mg 1—1 ,2.5 ,2.5 ,2.5 ,2.5 ,2.5 42 44 44 41 40
1—2 ,2.5 ,2.5 ,2.5 ,2.5 ,2.5 44 47 43 44 43
1—3 ,2.5 3.3 2.8 ,2.5 ,2.5 41 50 49 47 49
1—4 ,2.5 4.9 3.7 2.8 3.4 42 53 51 48 48
1—5 ,2.5 5.3 6 4.2 5.5 45 54 55 51 50

5 mg 5—1 ,2.5 5 3.5 2.5 3.2 42 53 49 49 51

5—2 ,2.5 5.9 4.2 3.5 3.5 46 54 51 52 48
5—3 ,2.5 8.5 7.8 ND ND 44 55 56 ND ND
5—4 ,2.5 14.5 17.5 18.4 15.8 41 56 57 59 55
5—5 ,2.5 16.5 16 16 15 40 55 56 55 54

10 mg 10—1 ,2.5 17 16.5 ND ND 43 53 55 ND ND

10—2 ,2.5 24.3 30.5 25.5 26 42 58 64 70 68
10—3 ,2.5 25.5 28.5 25.5 27.7 45 57 60 70 65
10—4 ,2.5 34 40.4 36.8 39.5 44 60 72 76 72
10—5 ,2.5 49 55.8 ND ND 45 63 80 ND ND

LacZ C—1 ,2.5 ,2.5 ,2.5 ,2.5 ,2.5 42 40 42 42 40

C—2 ,2.5 ,2.5 ,2.5 ,2.5 ,2.5 43 39 40 39 41

These data represent the numbers used to generate the graphs in Fig. 3.
Serum hEPO and hematocrit levels in individual mice grafted with full-thickness human skin which received a single injec-
tion with the indicated HIV1 p24 equivalent doses of hEPO vector. These data are shown in graphical form in Fig. 3. ,2.5, Not
detected; ND, not done, due to the SCID mouse death commonly observed due to infection over time (Robbins et al).
1556
FIG. 4. Loss of serum human EPO after excision of injected
skin. Four weeks following a single EPO lentivector injection,
epidermis and dermis were removed by simple punch biopsy
excision of the engineered central portion of grafted skin and
human serum EPO levels were determined 10 days later.
Significantly, we failed to observe even short-term detectable
lentiviral gene transfer in wholly murine skin using immune-
deficient SCID animals for reasons that are unclear, pointing
out an important difference between this commonly used model
animal and the goal human tissue target. Thus, the potential
clinical utility of direct intracutaneous lentiviral injection for
systemic polypeptide delivery may be greater because we have
used human target tissue, verifying transfer to human cells us-
ing species-specific antibodies.
Systemically delivered gene products with significant bio-
medical impact have been delivered by a number of approaches
in visceral tissues and skin. EPO is one of the best-studied pro-
totype genes, and a variety of routes and vectors have been used
to achieve clinically significant delivery to the bloodstream. In
the case of ex vivo gene delivery, the primary cell types used
have been myoblasts engineered with plasmid (Dalle et al.,
1999) and retroviral vectors (Hamamori et al., 1995; Bohl et al.,
1997) as well as fibroblasts engineered with both (Serguera et
al., 1999). In the case of direct in vivo gene delivery, the pri-
mary tissue target has been the muscle, with plasmid (Abruzzese
et al., 2000; Lemieux et al., 2000), AAV (Yan et al., 2000), and
adenovirus (Svensson et al., 1997) delivered by direct injection.
The mouse has been the primary animal model used with sev-
eral studies in rats, cats and primates (Svensson et al., 1997;
Beall et al., 2000; Rudich et al., 2000). In several cases, the fo-
cus has been on regulated EPO delivery using either tetracy-
clines (Bohl et al., 1997), progesterone analogs (Abruzzese et
al., 2000) or rapamycin (Ye et al., 1999) for pharmacologic con-
trol of regulated promoters. In the single study in which EPO-en-
coding vectors were delivered directly to skin, an AAV vector
was delivered to entirely murine tissue. However, gene expres-
sion in this work could not be detected in cells of dermis or epi-
dermis, and only in the mouse panniculus carnosus (Donahue et
al., 1999), a muscular structure deep in the tissue that does not
exist in human skin. Consistent with this lack of expression in the
BAEK ET AL.
epidermis and dermis, we failed to observe any gene expression
after direct injection of AAV vector encoding lacZ into full-thick-
ness human skin tissue (data not shown). Thus assessment of in-
tracutaneous systemic gene delivery using AAV and other vec-
tors via human skin tissue awaits further development.
Dosing of serum EPO was achieved in a stable fashion that
correlated with the amount of vector administered. Such a sta-
ble and sustained level of gene expression may be useful in cer-
tain settings that do not require tight regulation, such as in de-
livery of Factor IX in hemophilia where there is a large
therapeutic window (Kay et al., 2000). Further refinements in
the ability to dose gene production via direct cutaneous trans-
fer are needed, however, for several reasons. First, there was
variability observed in delivered EPO, even among individual
mice receiving the same vector dose. Second, a number of other
applications will require the capacity for temporally regulated
dosing, such as for delivery of genes whose expression levels
must be more precisely regulated in time. Among the most
prominent of these is insulin for diabetes mellitus, where ex-
cessive amounts of delivered insulin could prove lethal in the
form of hypoglycemic shock. A number of other applications,
however, including EPO-responsive anemias, could also benefit
from the capability for exogenous regulation. In the case of the
latter, we have observed that excessive delivery via skin clearly
leads to dramatically elevated hematocrits that may pose a ma-
jor risk of thrombosis. Of the 3 mice that died by 4 months of
this study, one of these displayed a hematocrit of 80% and may
have died of this complication, although we could not confirm
this on autopsy. Thus, given the fact that stable systemic de-
livery is possible via intracutaneous lentiviral injection, a ma-
jor next step would be the capacity for regulated production. In
the case of the skin, such regulation may be approachable in a
convenient fashion by topical regulation via application of com-
mercially available gene regulatory ligands, such as glucocor-
ticoids, retinoids, and vitamin D analogs.
We have observed that pseudotyped HIV-1 lentivectors suc-
cessfully target all of the major cell types within human skin
tissue. Although the VSV-G envelope may not prove an ideal
choice for direct human administration (DePolo et al., 2000),
other envelopes with wide cellular tropism are available, in-
cluding alphaviruses (Sharkey et al., 2001), gibbon ape leuke-
mia virus (Stitz et al., 2000), and others. Which of the cell types
targeted contributed to EPO delivery to the bloodstream? While
skin fibroblasts have been clearly shown capable of systemic
delivery by ex vivo studies in which pure populations were en-
gineered then regrafted (Petersen et al., 1995), it is also of in-
terest that endothelial cells comprised approximately one-fourth
of the cells targeted. This raises the possibility that endothe-
lium may represent a key site of production-delivery by direct
secretion into the blood. Assessing the relative contributions of
each cell type in human tissue poses formidable technical chal-
lenges because absolutely cell type specific targeting vectors
for all of these cells in skin have yet to be developed.
These data indicate lentiviral vectors are capable of skin-lo-
calized gene transfer achieving systemic delivery of therapeu-
tic polypeptides after a single intradermal injection. The local-
ized nature of this gene transfer approach opens the way for
future development of direct lentiviral gene transfer for primary
skin disorders, as well as additional systemic diseases.
SYSTEMIC EPO DELIVERY VIA HUMAN SKIN
ACKNOWLEDGMENTS
This work was supported by the USVA Office of Research
and Development and by National Institutes of Health grants
AR44012, AR45192. and AR43799 to P.A.K. We thank L. Nal-
dini for the HIV-1 transfer vector backbones, P. Aebischer for
the human EPO cDNA, D. Kohn for helpful advice, and P.
Bernstein and N. Griffiths for expert administrative support.
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Address reprint requests to:
Dr. Paul A. Khavari
Program in Epithelial Biology
269 Campus Drive, Room 2145
Stanford, CA 94305
E-mail: khavari@CMGM.stanford.edu
Received for publication March 21, 2001; accepted after revi-
sion July 2, 2001.
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