Proc. Natl. Acad. Sci. USAVol. 90, pp. 10764-10768, November 1993Medical Sciences

Intraarticular expression of biologically active interleukin1-receptor-antagonist protein by ex vivo gene transfer

(arthritis/gene therapy/cell transplantation)

G. BANDARA*, G. M. MUELLERt, J. GALEA-LAURI*, M. H. TINDALt, H. I. GEORGESCU*, M. K. SUCHANEKt,G. L. HUNG*, J. C. GLORIOSOt, P. D. ROBBINSt, AND C. H. EVANS*tDepartments of *Orthopaedic Surgery and tMolecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; andtResearch Pharmaceuticals, Procter & Gamble USA, Cincinnati, OH 45239

Communicated by William S. Sly, August 9, 1993 (received for review June 15, 1993)

ABSTRACT Gene therapy offers a radical different ap-proach to the treatment of arthritis. Here we have demon-strated that two marker genes (lacZ and neo) and cDNA codingfor a potentially therapeutic protein (human interleukin 1-re-ceptor-antagonist protein; IRAP or IL-lra) can be delivered,by ex vivo techniques, to the synovial lining of joints; intraar-ticular expression ofIRAP inhibited intraarticular responses tointerleukin 1. To achieve this, lapine synoviocytes were firsttransduced in culture by retroviral infection. The geneticallymodified synovial cells were then transplanted by intraarticu-lar I jection into the knee joints of rabbits, where they effi-ciently colonized the synovium. Assay of joint lavages con-firmed the in vivo expression of biologically active humanIRAP. With allografted cells, IRAP expression was lost by 12days after transfer. In contrast, autografted synoviocytes con-tinued to express IRAP for -5 weeks. Knee joints expressinghuman IRAP were protected from the leukocytosis that oth-erwise follows the intraarticular injection of recombinant hu-man interleukin 1p3. Thus, we report the intraarticular expres-sion and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate thefeasibility of treating arthritis and other joint disorders withgene therapy.

Arthritis is a chronic, debilitating condition affecting over 30million Americans (1). Presently incurable, it remains theagent of considerable suffering and economic loss. Thera-peutic intervention in arthritis is hindered by a number offactors, including difficulties in targeting drugs to joints.Proteins are particularly vulnerable to this restriction, whichis of special concern, as many new agents with considerableantiarthritic potential are proteins. As an alternative to tra-ditional methods of drug delivery, we have suggested thetransfer oftherapeutic genes to the synovial lining ofjoints (2,3). Expression of these genes would overcome protein-delivery problems and lead to the intraarticular accumulationof the gene products at the site of disease, with reducedexposure of nontarget organs.Using the rabbit knee joint as a model system, we are

therefore developing in vivo and ex vivo methods for deliv-ering genes to joints. This model takes advantage of thesimilarity in size between the knee joint of the rabbit and thehuman proximal interphalangeal joint, a common site ofrheumatoid arthritis. Moreover, there exists a large body ofliterature on the biology of the rabbit's knee, includingwell-established methods for synovial cell culture (e.g., refs.4-6). Here we report the transfer to synovium of two markergenes and one potentially therapeutic gene by an ex vivoapproach. Intraarticular expression of the interleukin 1-re-

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

ceptor-antagonist protein (IL-lra, ref. 7, or IRAP, ref. 8) wassufficient to block the influx of leukocytes into the joint afterthe intraarticular injection of recombinant human interleukin1(3 (rhIL-1,8). These results demonstrate the feasibility oftreating arthritis and other connective tissue diseases by genetherapy.

MATERIALS AND METHODSCell Culture. For allografting experiments, synovia were

dissected from the knee joints of euthanized, young adultNew Zealand White rabbits (2.2-2.6 kg) and used as a sourceof synovial fibroblasts (type B synoviocytes) for cell cultureby methods described in detail elsewhere (6). As primarycultures provide relatively few cells, the present studies wereconsiderably expedited by using the HIG-82 cell line (6) as anadditional source of lapine synovial fibroblasts. In general,investigations were initiated and protocols were establishedwith the HIG-82 cells before conducting confirmatory andcomparative studies with primary cultures of synoviocytes.For autografting experiments, rabbits were anesthetized byinjection of 1.5 ml of Nembutal. One knee joint was shaved,and a surgical synovectomy was performed.

Cloning ofTRAP cDNA. A human monocyte cDNA library,in A gtlO, was purchased from Clontech (catalog no.HL1036a). The library was derived from the human mono-cyte cell line U937, which had been stimulated with 10 nMphorbol 12-myristate 13-acetate for 48 hr before isolation ofthe mRNA. The library was screened for IRAP clones viastandard plaque-lift assays from agar plates to nitrocellulosefilters. Two phage clones with EcoRI-flanking sites wereisolated and subcloned into the EcoRI site of pUC18 andshown to have identical restriction maps. One of the cloneswas then sequenced and amplified by PCR. The resultingIRAP insert had the following structure: a 5' HindIII sitefollowed immediately by bp 1, the entire IRAP protein-codingsequence, 3' flanking DNA from bp 543-577, followed im-mediately by a 3' HindIII site. This insert was subcloned intothe HindIII site of pSV2.

Production of Retroviral Vectors. The cDNA for humanIRAP was inserted into the retroviral vector MFG (9, 10)by first BamHI-linking the 3' HindIII site downstream fromthe stop codon in the IRAP-encoding gene, followed bydigestion with Pst I and BamHI. The Pst I-BamHI IRAPfragment was then inserted into MFG, digested with Nco I andBamHI, using the synthetic adapter CAIGAAATCTGCACTTTAGcontaining a 5' Nco I site and a 3' Pst I site; the initiation codonfor the IRAP protein is underlined. The resulting plasmid,MFG-IRAP, contains the entire coding sequence ofIRAP. To

Abbreviations: IL-1, interleukin 1; IRAP, IL-1-receptor-antagonistprotein; rhIL-1,f, recombinant human IL-1,B; BrdUrd, 5-bromode-oxyuridine.

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generate a high-titer, amphotropic producer of pMFG-IRAP,10 ug of the plasmid was cotransfected with 0.5 gg ofpSV2Neo into CRIP packaging cells, and G418-resistant cellswere selected and expanded. The individual clones werescreened for virus production by infecting NIH 3T3 cells orHIG-82 cells with 2 ml of supernatant. The media from theinfected cells were then assayed for IRAP production byELISA. An analogous method was used to generate a pCRIP-BAG producer (where BAG represents a retroviral vector)carrying both the neo and lacZ genes under control of thesimian virus 40 early promoter and the retroviral long terminalrepeat, respectively (11).

Transduction of Synoviocytes. Cells were grown to =75%confluence in a 25-cm2 flask containing 4 ml of Ham's F12medium/10%o fetal bovine serum. Medium was then re-moved, and the cells were infected by adding 1 ml of viralsuspension (titer of 106 per ml) in the presence of Polybreneat 8 ug/ml. After a 2-hr incubation with intermittent shaking,an additional 3 ml of medium was added, and the cultureswere returned to the incubator for 72 hr. Cultures were thentreated with trypsin and reseeded at a 1:5 split ratio. Whereappropriate, G418 (0.5 mg/ml) was added to select forneomycin-resistant cells.

Synovial Cell Transplantation. Cells were treated withtrypsin, washed, and resuspended in Gey's balanced saltsolution to a final concentration of 106-107 cells per ml. A1-ml sample of cell suspension was injected intraarticularlydirectly into the knee joints of recipient rabbits (12). To trackthe injected cells, certain cultures were prelabeled by theaddition of 10 ,uM bromodeoxyuridine (BrdUrd) 48 hr beforetreating with trypsin. After injection of these cells, synoviawere dissected from recipient kneejoints, and frozen sectionswere stained immunohistochemically for the presence ofnuclear BrdUrd by using a commercial kit (Boehringer Mann-heim).

Immunoblotting. Recombinant IRAP was obtained bycloning the cDNA into Escherichia coli; IRAP was thenpurified from these cells by liquid chromatographic tech-niques. Each of six rabbits was then inoculated with a totalof 11 mg of purified IRAP, and antiserum was generated bya commercial company (Babco, Richmond, CA). These an-tisera were used in immunoblotting done by standard proce-dures, using a chemiluminescent detection system (Amer-sham).In Vivo Effects of Interleukin 1 (IL-1). Just before use,

rhIL-1l3 (from Elizabeth Arner, DuPont Merck, Wilmington,DE) was diluted into 0.5 ml of sterile saline to produce thedoses indicated in the Results section. In each case, 0.5 ml ofsolution was injected intraarticularly into knee joints ofyoung male New Zealand White rabbits. Eighteen hourslater, each knee joint was washed with 1 ml of sterile saline.An aliquot of the washing solution was removed, and thenumber of leukocytes were counted with a hemocytometer.The remaining solution was subjected to cytospinning, andthe cells were observed microscopically after staining withDiff Quik (Baxter Scientific Products, McGaw Park, IL).Other Methods. LacZ+ cells were histochemically stained

with 5-bromo-4-chloroindolyl-,B3D-galactosidase by routinemethods (13). ELISA kits for measuring human IRAP con-centrations were purchased from R&D Systems and usedaccording to the manufacturer's instructions. Gelatinase in-duction in chondrocyte cultures was measured by describedmethods (14).

RESULTSSynoviocyte Transplantation. We have argued that the

intraarticular injection of suspensions of synoviocytes shouldlead to colonization of the recipient synovium by the injectedcells (12). Furthermore, the joint is commonly held to be an

immunologically privileged site, as allografted ligaments andosteochondral plugs are already in clinical use. Accordingly,initial transplantation experiments were conducted withHIG-82 cells and allografted primary cells. To identify thetransplanted cells in a way that does not rely upon geneexpression, cells were cultured with BrdUrd. Suspensions ofBrdUrd-labeled cells were then injected into the knee jointsof recipient rabbits. By use of an anti-BrdUrd monoclonalantibody, immunohistochemistry of synovia recovered 1week after injection confirmed that the transplanted cells hadcolonized both the synovial lining and subsynovial tissue ofthe recipient joints (Fig. 1).

In Vitro Transduction of Synoviocytes. To evaluate thesusceptibility of type B synoviocytes to gene transfer byretroviral vectors, lacZ and neo were chosen as a markergene and selectable marker gene, respectively. These twomarker genes were contained in the BAG retrovirus. Inaddition, a cDNA coding for human IRAP was used both asan additional marker and as an example of a gene withpossible future therapeutic use. IRAP is secreted, is easilymeasured, and has antiarthritic potential. This moleculeantagonizes the biological actions of IL-1, a cytokine presentin human synovial fluid (15) that provokes synovial inflam-mation and cartilage breakdown (16-18). IRAP protects theknee joints of rabbits from the intraarticular sequelae of aninjection ofIL-1 (19, 20) and shows antiarthritic activity in thestreptococcal cell-wall model of arthritis (21). The IRAPcDNA was inserted into the MFG retrovirus.

After infection with the amphotropic BAG retrovirus (11)carrying the lacZ and neo genes, =1-5% of the cells in theculture stained histochemically for ,B-galactosidase. Afterselective growth in G418 at 0.5 mg/ml, nearly all cells werelacZ+ (data not shown). The neo+, lacZ+ cells were theninfected with the MFG-IRAP retrovirus to produce culturesof HIG-82 cells, which were lacZ+, neo+, IRAP+. However,double infection in this manner was not possible for primarycultures, as they have a limited in vitro lifespan of 10-12population doublings (6), and selection in the presence of

FIG. 1. Immunohistochemical identification of transplanted sy-noviocytes. Frozen sections of recipient synovium were stained 7days after the injection of BrdUrd-labeled cells. Yellow fluorescentnuclei of the transplanted cells can be seen in the synovium; controlsynovia showed no fluorescence (data not shown).

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a

Retrieved

HIG-IRAP rIRAP

b I6d 3d '20ng

coJ

106-

80-

49.5-

32.5-

27.5-

18.5-

O*

0

(I,4 2-z

-iw 1-

CONTROL IL-1 IL-1 IL-I

cm IRAP-CM

FIG. 2. Retrieval of transplanted neomycin-resistant lac2

IRP synoviocytes from recipient synovia. (a) Flasks showing t]presence of neo+ lacZ+ cells grown from synovia recovered froknee joints injected with neo+ lacZ+, TRAP+ HTG-82 cells (flask 'A

or neo+ lacZ+ primary cells (flask 4). Synovia recovered from kn,

joints injected with naive HTG-82 (flask 1), primary cells (flask 3),

carrier solution (data not shown) contained no cells. (b) Tmmunob]

showing the production of human TRAP by HTG-82 cells aftinfection with pMFG-TRAP (lane 4). Uninfected cells (HIG-82, lai

5) produced no human TRAP. Cells retrieved from synovia 1 wei

after transplant (flask 2, a) continue to secrete TRAP in vitro at 3 da,

G418 had exhausted much of this lifespan. Thus, fresh

cultures of primary synoviocytes were infected with MFG-

TRAP independently of the cultures infected with the BAG

virus. ELISA measurements of human TRAP in medium

conditioned by IRAPI primary synoviocytes or HIG-82 cells

confi'rmed, in each case, the production of >500 ng per 106

cells during a 3-day incubation. Bioassays confirmed the

ability of the human TRAP synthesized by transduced lapine

synoviocytes to inhibit the responses of articular chondro-

cytes to rhIL-1/3 (data not shown).

Transplantation of Transduced Synoviocytes. Transplanta-tion of the genetically labeled, lacZ+, neo+, IRAP+ cells was

then undertaken. Recipient rabbits were euthanized 1 week

after transplant, and synovia were -dissected from their knee

joints. High background staining for (3-galactosidase pre-

cluded direct visual inspection of histochemically stained

intact tissue as an unambiguous means of assessing lacZ

expression. Thus, a more rigorous method was used, in

which cell cultures were established from recipient synoviaand G418 was added incrementally to a final concentration of

0.5 mg/ml to eliminate the host cells. Large numbers of

neomycin-resistant colonies were recovered from knees re-

ceiving transplants of transduced HIG-82 or primary cells

(Fig. 2a); these colonies also stained for the presence of

/3-galactosidase (Fig. 2a). In contrast, cells recovered from

naive knees (knees receiving transplants of nontransduced

synoviocytes) did not grow with G418 (Fig. 2a). Transplantsof neo+, 1acZ1 HIG-82 cells, which, as discussed above, also

carry the TRAP-encoding gene, continued to express highlevels of human TRAP in vitro after their retrieval from the

joint (Fig. 2b). IRAP produced by the retrieved, transduced

cells showed biological activity in inhibiting the induction of

gelatinase in chondrocytes exposed to rhIL-1p8 (Fig. 2c).

In Vivo Expression of Transgenes. Having validated the

transplantation technique, we evaluated the possible in vivo

expression of the transferred genes. Here we could take

advantage of the fact that, unlike j3galactosidase and neo-

mycin phosphotransferase, IRAP is a secreted protein. Thus,

despite its very rapid clearance from the joint, TRAP should

be detected in lavage of the joint were expression sufficiently

high. To evaluate intraarticular expression of TRAP, knee

joints were washed with 1 ml of saline solution at various

intervals after transplant. Washings were centrifuged, and

the TRAP content of the supernatants was assessed by ELISA

measurements. No transplanted cells were present in the

pellets, confirming that the injected cells had, indeed, been

removed from the joint space and had colonized the sy-

novium as the BrdUrd staining had indicated (Fig. 1).

Transplantation of 107 HTG-82 cells previously infected

with MFG-IRAP resulted in the accumulation of several

nanograms of human TRAP in the knee joint. However,

expression declined with time, and by 10-14 days after

transplant no TRAP could be detected in the washings (Fig.

3a). In vivo expression of TRAP from allografted IRAP+primary cells, in contrast, was stable for 10-12 days, after

which expression was rapidly lost (Fig. 3a). In both cases,

loss of neo+, lacZ+ cells from synovia paralleled the extinc-

tion of TRAP expression (data not shown). Possible reasons

for the extinction include repression of the transferred gene

and immune recognition of either the transplanted cells or

.1'their transgene product. Preliminary data using BrdUrd-

keeor

lot

terne

Lys

(3d, lane 2) and 6 days (6d, lane 1) after retrieval. Unglycosylatedhuman recombinant TRAP is shown in lane 3. (c) Medium condi-tioned by IRAP+ HTG-82 cells (IRAP-CM) inhibits the induction ofgelatinase in cultures of lapine articular chondrocytes exposed tohrIL-118 at 5 units/nil. Medium conditioned by untransduced HTG-82cells (CM) fails to do this. Instead, gelatinase induction is slightlyincreased, as these cells spontaneously secrete low levels of IL-1 andother chondrocyte-activating factors (22). U, units.

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Time (days)b 10000

c-a 1000

J-ICL< 100

10 4

FIG. 3. Intransplant. IRof recipient rassayed for hnaive, untranIRAP (lowercytes. (b) Aul

labeled sync14 days. Hirnovia indicarejection of

Thereforetografted IFnoviocytesinfected witcontralateraltions, IRAPthat the autoticularly forsion fell with5 weeks afte

Biologicalknees of ralinflux of leunomenon waHIG-82 celFduction of ]leukocytosissuppresseddoses of rhIlthrough whithat the IRAtransfer is biinflammatioi

x

-X

(DCL

0)

0.1

0 1 5 o

[IL-l ]ng/knes

FIG. 4. Antiinflammatory properties of IRAP-encoding gene.IRAP+ HIG-82 cells (107) or untransduced HIG-82 cells (107) weretransplanted to the knee joints of rabbits 3 days before intraarticularchallenge with the indicated amounts of rhIL-1p. Lavage of jointsoccurred 18 hr later, after which infi'ltrating leukocytes were counted.

DISCUSSIONDespite the existence of an extensive medical and surgicalarmamentarium, arthritis remains incurable. Contributing tothis state of affairs is the failure of traditional methods of drug

delivery to target drugs to joints. Gene therapy offers oneo way to obviate this limitation, so that antiarthritic proteins

are synthesized intraarticularly. The data presented heredemonstrate that exogenous genes can be transferred to

.________________________________ ..synoviocytes and expressed within the joint to prevent ar-

2 3 4 5 6 ticular inflammation. Such findings permit optimism thatTime (weeks) further development of these general approaches will lead to

a gene treatment for arthritis and other disorders of joints,vivo expression of IRAP as a function of time after such as injuries to the ligaments and cartilages (27).AP+synoviocytes were transplanted to the kneejoints Despite the temporal reduction in intraarticular IRAPrabbits that were washed at the indicated times and concentrations seen in the present work, this protein was

uman IRAP by ELISA. Joints receiving injections of produced in high enough quantities for a long enough dura-sduced synoviocytes contained no detectable humanlimit of detection was 6 pg). (a) Allografted synovio- tion to inhibit the leukocytosis that normally follows intraar-ltografted synoviocytes. ticular IL-1 injection. This effect is a noteworthy achieve-

ment, given the large molar excess of IRAP necessary toMviocytes suggested that loss of cells occurred at block the biological effects of IL-1 (23, 24). Indeed, the abilitystological examination of the corresponding sy- of -5 ng of IRAP, which accumulates within the geneticallyLted that this loss was associated with immune modified knee joints, to inhibit responses to 1 and 5 ng ofthe allografted cells (data not shown). injected hrIL-1,8 would not be predicted on the basis of data, these experiments were repeated with au- from experiments where IRAP protein has been coinjectedLAP+ synoviocytes. For this experiment, sy- into knees (20). This result suggests that gene delivery mayrecovered from one knee of the rabbit were be more effective than protein delivery as a way of blockingth pMFG-IRAP and then transplanted to the IL-1 in joints.I knee of the same rabbit. Under these condi- One of the major challenges to treating a chronic conditionexpression was greatly extended. Fig. 3b shows such as arthritis will be to achieve prolonged gene expres-igrafted cells continued to produce IRAP intraar- sion. Our data suggest that the relatively rapid loss of IRAPseveral weeks. Although the degree of expres- production by allografted cells reflects immune recognition oftime, IRAP at 100 pg/ml could still be detected the transplants. Thus, the joint may not be the immunolog-,r transplantation. ically privileged site commonly assumed. Loss of expressionActivity in Vivo. Injection of rhIL-1f3 into the by autografted synoviocytes could reflect a slower immunebbits provokes, among other things, a marked reaction to specific epitopes created by treating the cells withikocytes into the joint space (16-18). This phe- trypsin, or to the human protein being synthesized in theis unaffected by prior transplant of untransduced rabbit knee (25). Alternatively, nonimmune mechanisms mays into the knees (Fig. 4). However, after intro- be involved. The decline in expression, noted here, parallels[RAP+ HIG-82 cells into the knee joints, the other reports that gene expression driven by viral promotersproduced by injection of rhIL-lp was strongly in fibroblastic cells in vivo is temporary (26). This impediment

(Fig. 4). Inhibition was greatest at the lowest may be overcome in the future by the use of nonviralL-1ip, as expected by the competitive mechanism promoters or more permissive types of cells as vehicles. It isch IRAP antagonizes IL-1. This result confirms also important to note that the present work was conductedkP produced intraarticularly as a result of gene with normal knee joints. The success of the transplantationiologically active and can protect the joint from procedure and the longevity of expression may differ in then provoked by IL-1. rheumatoid joint, where the synovium is both hypertrophic

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and hyperplastic and where a rich cytokine environmentexists.Although it is not yet clear whether IRAP will prove to

possess the antiarthritic properties expected, the gene-transfer technologies under development should be able todeliver any gene or combination ofgenes to joints. They can,thus, be applied to the transfer of additional genes withtherapeutic potential as these become available. In additionto ex vivo methods of the type described here, direct in vivomethods for delivering genes to joints may be successful (2,3); liposomes and adenovirus show promise in this regard(unpublished work).Our demonstration that genes can be transferred to joints

and expressed intraarticularly brings another site of applica-tion into the arena of gene therapy.

We thank Lorraine R. McKenzie and Cathy L. Oppenheimer fortechnical assistance, Dr. Richard C. Mulligan of the WhiteheadInstitute for supplying the MFG virus, Elizabeth Arner of DuPontMerck for providing the hrIL-1,8, and Mrs. Lou Duerring for typingthe manuscript. This work was supported, in part, by a UniversityExploratory Research Project grant from Procter & Gamble and byRO1 DK446640 from the National Institute ofDiabetes and Digestiveand Kidney Diseases.

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