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Molecular and Cellular Neuroscience 17, 706–716 (2001)MCN

Sustained Effects of Gene-Activated Matricesafter CNS Injury

Martin Berry,* Ana Maria Gonzalez,† Wendy Clarke,‡

Lydia Greenlees,† Lee Barrett,§ Wailin Tsang,*eonard Seymour,§ Jeffrey Bonadio,† Ann Logan,‡

and Andrew Baird†,1

*Centre for Neuroscience, Neural Damage and Repair, GKT (Guy’s Campus), HodgkinBuilding, London Bridge, London SE1 1UL, United Kingdom; †Selective Genetics Inc.,1035 Roselle Street, San Diego, California 92121; and ‡Department of Medicine and

§Department of Cancer Studies (LS), University of Birmingham,irmingham B15 2TT, United Kingdom

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We show that when gene-activated matrices (GAM) areplaced between the proximal and distal stumps of severedrat optic nerves, DNA is retained within the GAM, promot-ing sustained transgene expression in the optic nerve, inthe GAM itself, and, more importantly, in axotomized ret-inal ganglion cells (RGC). Plasmids that encode basicfibroblast growth factor (FGF2), brain-derived neurotro-phic factor (BDNF), and neurotrophin-3 (NT3) promotesustained survival of RGC for over 3 months after theinitial injury. These findings suggest that immobilized DNAimplanted into a CNS lesion will be delivered by axonterminal uptake and retrograde transport to axotomizedneurons. GAM may therefore be a useful agent for pro-moting sustained neuron survival and axon regeneration.Whether further optimization of the matrices, plasmids,promoters, and genes present in the GAM will promoteeven more survival or, alternatively, axon regenerationremains to be determined.

INTRODUCTION

There are a number of significant hurdles precludingrecovery after CNS trauma: physical barriers formed byglial scarring, damage from proinflammatory cyto-kines, free radicals released during inflammation, thepresence of growth inhibitory molecules, inefficient re-moval of cellular debris, the limiting supply of neuro-trophic factors, and the programmed cell death that

p1 To whom correspondence and reprint requests should be ad-ressed. Fax: 1 858 625 0222. E-mail: abaird@selectivegenetics.com.

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naturally follows trauma (Windle et al., 1952; Zhang etal., 1997; Liu et al., 1997; Fitch et al., 1999; Crowe et al.,997; Conti et al., 1998; Logan et al., 1998; Berry et al.,998, 1999). Yet, numerous neurotrophic, anti-inflam-atory, antiscarring, and antiapoptotic factors have

een identified. They are presumed to be capable ofltering the natural course of injury in the CNS if theppropriate modes for their delivery could be identifiedDi Polo et al., 1996; Mansour-Robaey et al., 1994; Sieverst al., 1987). Unfortunately, it has been difficult to de-ne, let alone meet, the spatiotemporal parameters re-uired for effective delivery of proteins that can modifyhe injury response.

For example, neurotrophic factors have generallyailed to promote sustained regeneration of axons be-ond the lesion even when placed directly at sites of

njury in vivo. While their powerful neuroregenerativectivity stimulates axons to grow into the site of deliv-ry, axons become entrapped and form disorganizedundles (Sawai et al., 1996; Clarke et al., 1998; Tuszynskind Kordower, 1999). Moreover, the pharmacokineticsf many proteins are simply not amenable to therapy.ntiapoptotic factors act intracellularly and must beelivered inside cell bodies to induce a response. Anti-carring and anti-inflammatory strategies require pro-onged delivery to achieve effective concentrations inhe local milieu without causing systemic toxicity. Fi-ally, the systemic administration of neurotrophic fac-

ors leads to unacceptable toxicity or, alternatively,

oor efficacy.Because gene-activated matrices (GAM) repair bone

1044-7431/01 $35.00Copyright © 2001 by Academic Press

All rights of reproduction in any form reserved.

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707Sustained Effect of GAM after CNS Injury

fractures (Fang et al., 1996; Bonadio et al., 1999), weeasoned that this technique might offer an unantici-ated way of delivering therapeutic agents to the CNSfter trauma. As with bone fractures, a CNS injury sites invaded by repair cells which would endocytose themmobilized DNA and then express the transgene lo-ally at a low, but persistent, level. Moreover, if regen-rating axons penetrate the GAM, they could retro-radely transport DNA to neuronal cell bodies distalrom the site of injury. Continuous transfection of in-ading cells (and neuronal processes) would result inustained transduction with local, low-level protein ex-ression.We tested the efficacy of GAM to promote the sur-

ival of retinal ganglion cells (RGC) after transection ofxons in the rat optic nerve. In this model, most RGCie within the first week and the remainder are lostver a period of several months (Allcott et al., 1984;illegas-Perez et al., 1993). We show here that GAM,

mplanted into the site of optic nerve transection, wouldransduce injury repair cells in the CNS and, throughetrograde axonal transport, deliver neurotrophic factorenes to axotomized RGC.

RESULTS

GAM Placed at the Site of Injury Delivers DNA tothe Retina

The trafficking of DNA contained in a GAM im-planted between the cut ends of the optic nerve wasmonitored throughout the course of injury using PCR.Transcription into mRNA was evaluated by nested RT-PCR. These techniques are nonquantitative and detecteither the presence or the absence of DNA or RNA,respectively. To this end, we selected a plasmid con-taining the herpes thymidine kinase (TK) gene undercontrol of the cytomegalovirus (cmv) promoter becauseof the availability of highly specific primers for TK, thelow background, and the ease of simultaneously mea-suring mRNA and DNA. Rat optic nerves weretransected 2 mm from the eye, and either collagen alone(control) or GAM containing the condensed plasmidwas implanted into the lesion. Six and 40 days later(Fig. 1), the presence of DNA (Figs. 1A and 1C) andmRNA (Figs. 1B and 1D) was evaluated using nonquan-titative PCR and RT-PCR, respectively.

On day 6 after grafting, no DNA signal was detectedin retinal extracts of control animals that received the

collagen matrix alone (Fig. 1A, lane 2). Similarly, noDNA signal was detected in the retinae of control ani-

mals at day 40 (Fig. 1C, lanes 2 and 3). By contrast, theforeign DNA was readily detected in animals im-planted with a GAM containing condensed plasmid. Atdays 6 and 40, extracts of retinae had detectable DNA(Fig. 1A, lanes 3 and 4; Fig. 1C, lanes 4 and 5). In the6-day analysis, the GAM was also collected and evalu-ated. As shown in Fig. 1A (lane 5), DNA was detected.

A similar profile was observed when samples wereprocessed to evaluate mRNA (Figs. 1B and 1D). No signalwas detected in animals receiving collagen alone (Figs. 1B

FIG. 1. Retrograde transport of DNA and mRNA expression afterinjury. Six days (A and B) and 40 days (C and D) following injury andplacement of GAM, DNA persistence (A and C) and mRNA expres-sion (B and D) were analyzed using nonquantitative PCR and RT-PCR, respectively. (A) PCR was performed on individual retinae fromcontrol (lane 2) and GAM-treated (lanes 3 and 4) animals along withthe corresponding GAM implants (lane 5) 6 days after injury. (B)RT-PCR was performed on individual retinae (lane 2), proximal opticnerve stump (lane 3), and implant (lane 4) from control and GAM-treated (lanes 5, 6, and 7) animals. (C) PCR was performed on indi-vidual retinae from control (lanes 2 and 3) and GAM-treated (lanes 4and 5) animals 40 days after injury. (D) RT-PCR was performed onindividual retinae (lane 2), proximal optic nerve stump (lane 3), andimplant (lane 4) from control and GAM-treated (lanes 5, 6, and 7)animals. In all cases the DNA ladder is shown in lane 1.

and 1D, lanes 2, 3, and 4). In the animals receiving GAM,mRNA was detected at both 6 (Fig. 1B) and 40 days (Fig.

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1D) in the retina (lane 5), the proximal nerve stump (lane6), and the remaining implant (lane 7).

Gene Expression after DNA Deliverythrough a GAM

If the results above pointed to the ability of a GAM totransfect cells after optic nerve injury, it became impor-tant to determine whether enough protein was beingproduced to elicit a biological response. We thereforedesigned a GAM to deliver the human basic fibroblastgrowth factor FGF2 gene (Florkiewicz and Summer,1989). Because the expression vector also contains theNPTII gene (a selectable marker), the presence of DNAwas monitored in tissue extracts with primers amplify-ing NPTII. As shown in Fig. 2, NPTII DNA was presentin retinal extracts collected 34 days after GAM implan-tation at the site of nerve transfection (Fig. 2A, lanes 2and 3). No DNA was detected in retinal extracts ofcontrol animals that received collagen alone (Fig. 2A,lane 4). Thus the plasmid was transported to the retina.

We were able to exploit the availability of highlyspecific antibodies that distinguish the transgene prod-uct, human FGF2, from endogenous rat FGF2 and es-tablish that the GAM implanted into the severed opticnerve resulted in gene expression in the retina (Figs. 2Band 2C). Retinae of GAM-treated animals were ex-tracted and proteins precipitated with heparin–Sepha-rose beads. After the precipitated proteins were re-solved on SDS–polyacrylamide gels, they wereimmunoblotted with antibodies that recognize humanbut not rat FGF2. All four isoforms of human FGF2were detected in retinal extracts of GAM-treated ani-mals (Fig. 2B, lanes 3 and 4). The antibodies recognizedrecombinant human FGF2 (lane 1) but not rat FGF2(lane 2), and when blots were probed with antibodies torat FGF2 (Fig. 2C), the human FGF2 was not recognized(lane 1), but all the forms of endogenous rat FGF2 weredetected (lane 2). On the basis of these specificity stud-ies, we concluded that the immunoreactivity detectedin the retinal extracts of GAM-treated animals was thatof human FGF2. Because the human gene encodes high-molecular-weight forms of FGF2 that are different insize from those in rat tissues (Florkiewicz and Summer,1989; Shimasaki et al., 1988), the protein detected clearlyoriginates from the transgene. Most importantly, thesehigh-molecular-weight forms of human FGF2 areknown to localize to the nucleus of transfected cells(Florkiewicz et al., 1991). Therefore, the human FGF2

etected in retinal extracts is due to gene expression inGC and not to axonal uptake of human FGFs ex-

pressed and released by glial and inflammatory cellsinfiltrating the GAM at the site of injury.

Biological Response to NTF GAM

In order to evaluate the biological response to GAM,two kinds of plasmids were prepared: one condensedwith polylysine and another with recombinant FGF2chemically conjugated to polylysine (Sosnowski et al.,1996). The inclusion of recombinant FGF2 in the con-

FIG. 2. Transgene expression of human FGF2 in the rat retina.Thirty-four to 40 days after implantation of GAM containing plasmidsencoding human FGF2, retinae were collected and processed for PCR(A) or immunoblotting (B and C). (A) PCR for NPTII (encoded on theFGF2 expression vector) in the retinae from control (lane 4) andGAM-treated (lanes 2 and 3) animals generated a signal in bothuntargeted (lane 2) and FGF-targeted (lane 3) GAM but none fromcontrols (lane 4). (B) The specific human FGF2 antibody immunoblots50 ng of human recombinant FGF2 (lane 1) but not rat brain FGF2(lane 2). It also immunoblots the human FGF2 expressed in the ratretina in GAM-treated animals (lanes 3 and 4). (C) The specific ratFGF2 antibody does not immunoblot 50 ng of human recombinantFGF2 (lane 1) but recognizes the expected molecular forms of ratFGF2 in a rat brain extract (lane 2).

densate served two purposes. First, it allowed us toevaluate the targeting of plasmids into cells via the FGF

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709Sustained Effect of GAM after CNS Injury

high-affinity receptor. Second, it allowed us to comparethe effects of the growth factor protein and gene, im-mobilized in a matrix.

To test these plasmids in vivo, DNA encoding GFP,FGF2, or NTFs was condensed and placed at the site ofthe optic nerve transection (Fig. 3). Activity was thenevaluated 40 and 100 days later by counting the totalnumber of RGC labeled by the retrograde transport oflysinated rhodamine–dextran (LRD). Previous studiesshowed that DNA condensed with recombinant FGF2–

FIG. 3. Functional effects of delivering plasmids that encode neurotretinae. (A) Retina from an intact, untreated control. (B) Retina 40 da

Retina 40 days after implantation of a GAM containing a combinationmm. The relative numbers of surviving RGC are visualized by the labEffects of condensed plasmids in GAM. Lysinated rhodamine–dextrafter placing the GAM. Control and FGF2 GAMs contained 7.5 mg of pNT3, and BDNF plasmids (total 7.5 mg). (E) Effects of FGF2-targeted pcounted in whole mounts of retinae 40 days after placing the FGF2-taothers contained a mixture of 2.5 mg each of condensed FGF2, NT3, anhe percentages shown are compared to the number of cells measure

polylysine transduced cells in vitro (not shown). In fact,cells treated with combinations of targeted plasmids

encoding FGF2/BDNF/NT3 (NTFs) simultaneously ex-pressed more than one transgene.

Retinal whole mounts showed labeled RGC and theiraxons were seen converging onto the optic disc (Figs.3A–3C). There was a clear and recognizable decrease inlabeling of the retina in intact (Fig. 3A) versus treated(Figs. 3B and 3C) animals, reflecting the cell loss thatoccurs in the 40-day experiment (Fig. 3A vs Figs. 3B and3C). Yet, even a visual inspection of retinal mountsshowed a clear difference between GAM containing

factors. (A–C) LRD-filled RGC somata and axons in whole-mountedter implantation of a GAM containing a plasmid encoding GFP. (C)lasmids encoding FGF2/BDNF/NT-3 (NTFs). Magnification bar, 250somata and associated projections converging on the optic disc. (D)

RD)-labeled RGC were counted in whole mounts of retinae 40 daysd while others contained a mixture of 2.5 mg each of condensed FGF2,ids in GAM. Lysinated rhodamine–dextran (LRD)-labeled RGC wered GAM. Control and FGF2 GAMs contained 7.5 mg of plasmid whileNF plasmids (total 7.5 mg). Results are means 6 SEM (N 5 4–7) andwhole mounts of untreated control (80,202 6 2773).

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GFP plasmids (Fig. 3B) and those containing NTF plas-mids (Fig. 3C). The distribution of surviving RGC was

710 Berry et al.

uniform and all size classes of RGC were represented.Most importantly, the cells could be counted and theamount of cell loss and cell survival could be deter-mined (Figs. 3D and 3E).

As shown in Figs. 3D and 3E, GAM at 40 days werebiologically active and the greatest neuroprotection wasobserved with the NTF genes. The number of labeledRGC counted after retrograde LRD labeling was notdifferent when condensed plasmids delivered eitherGFP or FGF2 (Fig. 3D). However, when a combinationof untargeted (Fig. 3D) or FGF2-targeted (Fig. 3E) plas-mids encoding NTFs was delivered in a GAM, therewas a significant increase in the number of LRD-labeledRGC in the retinae, compared to that in animals receiv-ing GAM containing GFP-encoding plasmids (P ,0.005). In this instance, there was no significant differ-ence attributable to the presence or absence of the re-combinant FGF2 in the condensate, suggesting thatwith the mixed NTF, the effect of recombinant FGF2was negligible.

It is noteworthy that when the GFP plasmid wascondensed with FGF2–polylysine (Fig. 3E), a clear neu-roprotective effect was measured (P , 0.005), reflect-ing the intrinsic activity of recombinant FGF2. This ispresumably due to the recombinant FGF2–polylysineused to condense DNA which has full intrinsic activityof FGF2 (Sosnowski et al., 1996). At this same time point(40 days), there was little neuroprotective effect of GAMcontaining the FGF2 gene without the inclusion of theprotein (Fig. 3D). Unlike later time points (see below),the neuroprotective activity observed is ascribed to theintrinsic activity of the targeting agent, recombinantFGF2, and the fact that the GAM sequesters the proteinat the site of injury. However, this effect is not sustained(see below).

Histological Response to GAM

GAM integration between optic nerve stumps wasanalyzed in longitudinal sections for several parametersof cellular invasion, regeneration, and inflammation(Fig. 4). For example, 40 days after surgery, GAP43,expressed by regenerating axons, clearly showed inva-sion of the GAM by growing axon terminals, therebyproviding the rationale for transgene uptake and retro-grade transport to RGC (Fig. 4A). CAII (Fig. 4B) showedthe presence of oligodendrocytes approaching the ma-trix and demonstrated the viability of the proximalstump. Moreover, ED1-positive (Fig. 4C) macrophagesand microglia suggested that the GAM was not proin-

flammatory. GFAP-positive astrocytes (Fig. 4D) in-vaded the matrix, while laminin, a component of basal

lamina (Fig. 4E), demonstrated the high degree of neo-vascularization at the lesion/graft interface.

Sustained Responses to GAM Are Gene,Not Targeting, Dependent

In an effort to assess the duration of gene delivery byGAM, we evaluated animals 100 days after the injury(Fig. 5). At this time, no neurotrophic effects have everbeen described. Yet, the benefit of DNA delivery be-came significantly more apparent since DNA remaineddetectable in the retinae of GAM-implanted animals(Fig. 5A, lanes 3 and 4). No signal was detected incontrols (Fig. 5A, lane 2). The number of LRD-labeledRGC was significantly greater in NTF-treated animalsthan in controls receiving the GFP gene. Although thecell number was decreased in all groups when com-pared to 40 days (see Fig. 3), the presence of the FGF2gene significantly prolonged the survival of LRD-la-beled RGC at 100 days. This demonstrates the funda-mental difference between protein and plasmid deliv-ery. The sustained effect of the FGF2 gene wasincreased even further when a combination of plasmidsencoding NTFs was placed in the GAM (Figs. 5B and5C). There were no long-lasting effects of the recombi-nant FGF2 used for targeting that were initially ob-served at 40 days. At 100 days (not shown), no signifi-cant cellular differences from those described at 40 dayswere seen.

DISCUSSION

Normally, over 99% of the RGC population die 40 daysafter optic nerve transection. The results presented hereestablish that during this time, surviving RGC can projectregenerating fibers into a NTF gene-laden GAM that isplaced at the lesion site and that a combination of NTFgenes can elicit significant survival. The 2.60 and 0.96%increases in RGC survival at 40 and 100 days, respectively,may not appear physiologically important but it is note-worthy in the sustained nature of the response and thefact that even more optimal responses may be observedwith other combinations of genes and matrices.

The mechanism of sustained activity may be attribut-able to several different factors. First, the GAM environ-ment enables DNA to live much longer than a simplebolus application of DNA. Second, the uptake and expres-sion of foreign DNA by invading repair cells that remodelthe GAM/lesion result in local sustained production of

NTFs where they are needed. With a bolus delivery ofprotein, elevated levels are achieved over short periods of

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711Sustained Effect of GAM after CNS Injury

time. The repeated transfection of repair cells as theyinvade the GAM results in low but sustained local pro-duction of protein extended over time.

The axon-mediated transfection of RGC differs fromthat previously reported (Garcia-Valenzuela andSharma, 1998; Kugler et al., 1999) in several importantrespects. First, the uptake of DNA into RGC is mediatedby transected nerve terminals sprouting into the GAM,where the DNA is immobilized. This is in contrast tothe findings of Garcia-Valenzuela and Sharma (1998),who delivered genes to RGC through uptake from in-tact synaptic arbors within the superior colliculus. Sec-ond, we exploited the prolonged availability of DNAimmobilized in the GAM to deliver genes to repair cells.The efficacy of GAM delivery also relies on cell invasionof the matrix for DNA uptake. Finally, we observed

FIG. 4. Cellular response to GAM 40 days after implantation. Forty dayFGF2, the implant with proximal and distal nerve stumps was dissected43 kDa), a marker for growing axons; (B) CA II (carbonic anhydrase),marker); (D) GFAP (glia fibrillar acidic protein), a marker for astrocytes

sustained RGC transgene expression and survival 3months after surgical placement of the GAM. Like Gar-

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cia-Valenzuela and Sharma (1998), we found no mor-phological evidence that transfected RGC suffered celldamage over the course of the experiment.

The DNA-delivering properties of GAM are best il-lustrated by the detection of DNA in retinae 3 monthsafter implanting of the GAM into transected opticnerves. This is considerably longer than times reportedby groups like Kugler et al. (1999) using adenoviral

ector systems. In our studies, we also established thatransgene expression occurred in the neuronalerikarya, presumably as a result of retrograde trans-ort of DNA from the site of optic nerve transection.his conclusion is supported by the fact that the high-olecular-weight forms of human FGF2 detected in rat

etinal extracts are nuclear (Florkiewicz et al., 1991),uggesting that their presence is due to local produc-

r placing a GAM containing an FGF2-targeted plasmid encoding humantained for different cell markers. (A) GAP43 (growth-associated proteinrker for oligodendrocytes; (C) ED1 (a pan macrophage and microglia(E) laminin (LAM, basal lamina marker). Bar, 10 mm.

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ion, not to transport from the site of injury.Interestingly, we failed to observe differences between

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712 Berry et al.

polylysine- and recombinant FGF2–polylysine-targetedcondensates. This suggests that the effect of GAM on RGCwas not mediated by receptor endocytosis. Instead, theeffectiveness of GAM is likely attributed to nonspecificendocytosis of DNA during remodeling of injured tissuesand regenerating transected axons.

The observation that transgene mRNA was detect-able in extracts of GAM suggests that invading repaircells were also transfected during these experiments.Whether targeting will enhance transgene expression in

FIG. 5. Sustained responses after 3 months. (A) PCR for NPTII was) animals 100 days after injury. (B) Long-term effects of condensed pounted in whole mounts of retinae 100 days after placing the GA

contained a mixture of 2.5 mg each of condensed FGF2, NT3, and BDNn GAM. Lysinated rhodamine–dextran (LRD)-labeled RGC were couAM. GAM contained 7.5 mg of human FGF2 plasmid while otherslasmids (total 7.5 mg). Results are means 6 SEM (N 5 4–7) and the p

mounts of untreated control (80,202 6 2773).

repair cells that invade the matrix remains to be deter-mined. Unless controlled, this phenomenon could ab-

rogate access of RGC to the GAM, generate a neurotro-phic “sink,” and preclude successful regeneration.Given this finding, a GAM might be readily engineeredto include cell-specific promoters to confine gene ex-pression to RGC, for example, and thereby excludeglial/inflammatory cell transfection in the wound toavoid axonal entrapment within the GAM-integratedtissue. The inclusion of plasmids with exogenously in-ducible genes and cell-specific promoters could providea powerful means of controlling the spatiotemporal

rmed on retinae from control (lane 2) and GAM-treated (lanes 3 andids in GAM. Lysinated rhodamine–dextran (LRD)-labeled RGC wereontrol and FGF2 GAMs contained 7.5 mg of plasmid while otherssmids (total 7.5 mg). (C) Long-term effects of FGF2-targeted plasmidsin whole mounts of retinae 40 days after placing the FGF2-targetedined a mixture of 2.5 mg each of condensed FGF2, NT3, and BDNFtages shown are compared to the number of cells measured in whole

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expression of neurotrophic, scar-inhibitory, antiapop-totic, anti-inflammatory, and regenerative proteins.

713Sustained Effect of GAM after CNS Injury

At present, it is not known whether sustained RGCsurvival reflects sustained expression either from a sin-gle transfection event or from repeated transfectionsfrom the depot store of DNA in the GAM. The numbersof viable RGC continued to decline over the final 90days of the experiments described here, possibly reflect-ing a declining vigor of either the axonal growth re-sponse or the availability of DNA to the survivingneurons. Consequently, a reduction in the number offibers accessing stores of DNA in the GAM wouldreduce levels of transgene uptake and expression inRGC, thereby providing less effective neuroprotection.There was no evidence of vigorous axonal regenerationthrough the GAM and into the distal optic nerve seg-ment similar to that described by Berry et al. (1996, 1999)after vitreal NTF delivery. We predict that the deliveryof appropriate combinations of DNA encoding otherNTFs would promote more robust regenerationthrough the GAM and also facilitate a continued supplyof DNA from the GAM. Furthermore, deployment ofGAM more compatible with axon growth (e.g., a matrixcontaining laminin) would provide bridging supportfor axons traversing the wound.

After significant optimization, GAM-mediated trans-gene delivery may have wide therapeutic uses to promoteneuron survival and could be applied to deliver intracel-lular proteins encoded by antiapoptotic genes (Crowe etal., 1997; Conti et al., 1998) as well as secreted proteins. Theimplantation of GAM in the site of CNS injury (1) pro-vides a nonimmunogenic depot for DNA uptake, (2) im-mobilizes therapeutic genes, (3) protects the DNA fromdegradation at the lesion site, (4) controls DNA uptake byaxons growing into the matrix over protracted periods oftime after injury, and (5) provides a loosely woven scaf-fold supporting astrocyte immigration, axonal penetra-tion, and bridging. Moreover, since only injured axonssprout into the matrix and access the plasmid, there isspecific delivery by retrograde axonal transport to neu-rons axotomized by the lesion. All of the above character-istics have the potential to increase neuronal survival,promote functional axonal regeneration, and targetwidely dispersed neurons.

EXPERIMENTAL METHODS

Expression Vectors

The expression vectors for green fluorescent protein(GFP) (pEGFP-N1, Clontech, Palo Alto, CA) and FGF2

(pcDNA3, Invitrogen, San Diego, CA) cDNA containeda neomycin resistance gene (neomycin phosphotrans-

dA

ferase–II; NPTII) driven by the SV40 early promoter andwas used as the marker in the 100-day PCR studiesdescribed below. The plasmid encoding herpes thymi-dine kinase (TK) cDNA that was used for the 6- and40-day PCR and RT-PCR studies and the plasmids forGFP, FGF2, BDNF, and NT3 cDNA that were used toassess biological activity were subcloned into thepNGVL-3 vector described below. The original mam-malian expression vector encoding TK cDNA was ob-tained from Inder Verma (The Salk Institute, La Jolla,CA) and the original mammalian expression plasmidencoding human FGF2 was a generous gift of R. Z.Florkiewicz (San Diego, CA). The FGF2 cDNA includedthe coding sequences for all three CUG and the AUGtranslation start sites (Florkiewicz and Summer, 1989)and produced four isoforms of FGF2. Mammalian ex-pression plasmids for NT3 and BDNF cDNA were ob-tained by PCR from human genomic DNA. Each cDNAinsert was subcloned into the pNGVL-3 vector usingstandard recombinant DNA procedures. The plasmidvector backbone (pNGVL-3) was obtained from theUniversity of Michigan National Gene Vector Labora-tory. pNGVL-3 is a 4005-bp eukaryotic expression plas-mid that utilizes the 1073-bp cytomegalovirus (cmv)immediate early promoter/enhancer/intron A elementto drive gene expression. Intron A was not intact, butrather was a XhoI–HindIII genomic fragment derivedfrom intron A. This promoter/enhancer/intron elementhas been used to constitutively express reporter genesin a variety of cell types. Downstream from the sub-cloning site is the rabbit b-globin polyadenylation se-quence. Even further downstream is the gene encodingkanamycin resistance.

Preparation of Condensed DNA and GAM

Condensed DNA encoding FGF2, GFP, and TK wasprepared according to Sosnowski et al. (1996) with somemodifications. Briefly, DNA condensates were pre-pared in 20 mM glycine buffer, pH 6.0, containing 0.5%sucrose and 0.5% mannitol, at a ratio of poly-d-lysine:DNA of 2:1. The FGF2-targeted poly-d-lysine used forcondensation retained full biological activity of FGF2(Sosnowski et al., 1996). For the preparation of com-bined plasmids, 2.5 mg of DNAs encoding FGF2, BDNF,and NT3 was mixed prior to condensation. The conden-sates were lyophilized in vials, each vial containing asingle animal dose. Batches of lyophilized condensateswere tested for the ability to transfect baby hamsterkidney (BHK) cells in vitro prior to use, because they

id not express any of the NTF used here (see below).t the time of surgery, recombinant FGF2-targeted and

714 Berry et al.

nontargeted DNA were prepared by mixing the lyoph-ilized condensates with 1–2 mg of bovine collagen typeI paste (Cohesion Technologies, Palo Alto, CA). Eachdose contained 2.5–7.5 mg of plasmid DNA. The cDNAsencoding TK and NPTII were used as reporter genes forthe detection of DNA and RNA in the tissues. DNAencoding GFP was used as a nonrelevant gene.

In Vitro Studies

BHK cells were treated with FGF2-targeted (and con-densed) DNA encoding FGF2, BDNF, and/or NT3 pre-pared as described above. Immunolocalization studieswere performed using a guinea pig antibody againstthe high-molecular-weight form of human FGF2 thatdoes not recognize the 18-kDa isoform of FGF2 (gift ofR. Z. Florkiewicz). As such, it only recognizes the prod-uct of the human transgene. A chicken anti-BDNF anda chicken anti-NT3 (Promega, Madison, WI) were usedto detect immunoreactive BDNF and NT3. Texas red-labeled anti-guinea pig secondary antibodies (VectorLaboratories, Burlingame, CA) were used to detect theimmunoreactive FGF, and fluorescein-labeled anti-chicken secondary antibodies (Jackson Immunore-search, Westgrove, PA) were used to detect immunore-active BDNF and NT3.

Optic Nerve Injury

Groups of four to six female adult Wistar (200–250 g)rats were anesthetized by intraperitoneal injection ofphysiological saline solution containing ketamine (40mg/kg), acepromazine (1.2 mg/kg), and xylazine (8mg/kg). Both optic nerves were accessed intraorbitallyby a dorsal approach. After the dural sheath wasopened, the nerve was transected 2 mm distal to thelamina cribosa without damage to the central retinalartery. In this model, all axons in the optic nerve weresevered under direct vision as described by Berry et al.(1999). Both optic nerves were injured and either thecondensed plasmid DNA in collagen (GAM) or a pelletof collagen alone was implanted between the opticnerve stumps. Following GAM implantation, the lesionsite was closed by standard surgical procedures. Ani-mals recovered from surgery with little or no morbidityand were maintained in accordance with British HomeOffice guidelines.

Analysis of Transgene Expression

For PCR analyses, two to three animals were killed at6 and 40 days after injury with an overdose of sodium

pentobarbital (60 mg/kg, iv), and retinae, optic nerves,GAM, and collagen implants were collected and snapfrozen in liquid nitrogen. DNA was extracted in urealysis buffer and quantified by spectrophotometry. PCRamplification was performed using 1 mg DNA templatein the presence of 200 mM each dNTP, 1.5 mM MgCl2, 1ml primers, and 2.5 units AmpliTaq Gold (Perkin–Elmer, Branchburg, NJ) per 25-ml reaction volume. Am-plification was carried out by denaturation at 95°C (10min) and 40 cycles of denaturation at 95°C (30 s), an-nealing at 65°C (30 s), elongation at 72°C (30 s), andextension at 72°C (7 min). A total of 20 ml of amplifiedproduct was analyzed on a 2% agarose gel in 13 TBEbuffer. Oligonucleotide primer sequences (59 3 39)were NPT sense, gac tgg gca caa cag aca atc; NPTantisense, cgg cca cag tcg atg aat cc; TK sense, cgc ctcgac cag ggt gag a; and TK antisense, acc cgc cgc act gcagat ac.

For RT-PCR analyses, RNA was prepared by homog-enization of frozen tissues in TRIzol (GIBCO/BRL,Gaithersburg, MD) using the FastPrep system (BIO101,Vista, CA). Following RNA quantitation and DNase I(GIBCO/BRL) treatment, 0.2–1 mg of heat-denaturedRNA was used as a template for RT-PCR analysis using“Ready-To-Go” RT-PCR beads (Amersham/Pharmacia,Piscataway, NJ) containing 200 mM each dNTP, 1.5 mMMgCl2, Moloney murine leukemia virus (M-MuLV) re-verse transcriptase, and 2 units of Taq DNA polymer-ase. Random hexamers (GIBCO/BRL) were added to500 ng/reaction and PCR primers to a concentration of0.25 mM. RT-PCR was performed by reverse transcrip-tion at 42°C (30 min), heat denaturation at 95°C (10min), and 40 cycles of denaturation at 95°C (1 min),annealing at 65°C (1 min), elongation at 72°C (1 min),and final extension at 72°C (7 min). Oligonucleotideprimer sequences were as described above and 20 ml ofamplified product was analyzed on a 2% agarose gel in13 TBE buffer.

In the event that product was not readily detected byRT-PCR, nested PCR was performed using 20% (10 ml)of RT-PCR product as template. PCR amplification wasperformed in the presence of 200 mM each dNTP, 1.5mM MgCl2, 0.25 mM primers, and 5 units AmpliTaqGold (Perkin–Elmer) per 50-ml reaction volume. Ampli-fication was carried out by denaturation at 95°C (10min) and 40 cycles of denaturation at 95°C (1 min),annealing at 65°C (1 min), elongation at 72°C (1 min),and final extension at 72°C (7 min), and 20 ml of ampli-fied product was analyzed on a 2% agarose gel in 13TBE buffer: TK nested sense, cga cca ggg tga gat atc gg;

TK nested antisense, ctg cag ata ccg cac cgt att g.

In order to evaluate the in vivo expression of the trans-

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715Sustained Effect of GAM after CNS Injury

gene, retinae were collected at 34 days after injury andGAM implantation. Retinae and brain were dissected,frozen in liquid nitrogen, and stored at 270°C. Frozenamples were homogenized in cold extraction buffer [20M Tris, pH 7.5, containing 0.33 M sucrose, 2 mM EDTA,

.5 mM EGTA, 2 mM PMSF (Calbiochem, San Diego, CA),mM pepstatin A (ICN Biochemical, Costa Mesa, CA),

0.3% aprotinin (v/v) (Sigma, St. Louis MO), 20 mM leu-peptin (ICN Biochemical), 1% Triton X-100 (Sigma), and0.5% NP-40 (Calbiochem)]. Protein concentrations weredetermined using the BCA protein assay reagent (PierceChemical, Rockford, IL). Equal amounts of total proteinfrom each sample were incubated with prewashed hepa-rin–Sepharose CL-6B (Pharmacia, Uppsala, Sweden)beads for 2 h at 4°C. Proteins were eluted directly into

DS–PAGE sample buffer for 12% SDS–PAGE, trans-erred to a PVDF membrane (Immobilon P, Millipore,ertfodshire, UK), and blocked in TBS buffer containing

.05% Tween 20 (TBS-T) and 5% nonfat milk for 1 h at RT.embranes were treated with rabbit anti-rat FGF2

Ab106) or rabbit anti-human FGF2 (Ab937) overnight at°C, rinsed, and incubated with horseradish peroxidase-abeled anti-rabbit IgG (1/10,000, Santa Cruz Biotechnol-gy, Santa Cruz, CA). Signal was visualized by generationf light via the HRP-catalyzed breakdown of luminolECL detection reagents, Amersham Pharmacia BiotechK, Buckinghamshire, UK).

mmunohistochemical Analysis

Some of the animals treated with GAM were anes-hetized with a lethal dose of pentobarbital, followed byranscardiac perfusion with physiological saline for 1

in and then with 4% paraformaldehyde in 0.1 Mhosphate buffer, pH 7.2, for 5 min. After perfusion,oth optic nerves and retinae were dissected (4–6 tissueamples), dehydrated through a graded alcohol series,mbedded in a low-melting-point polyester wax, andtored at 4°C. Seven-micrometer-thick longitudinal sec-ions of the optic nerve were cut with a cooled chuck,oated onto a 1% gelatin solution on slides, and air-ried.Immunohistochemical staining of optic nerve sec-

ions was performed according to established tech-iques. Primary antibodies included rabbit polyclonalnti-GAP43 (1:5000, G. Wilkin, Imperial College, Lon-on, UK), rabbit anti-bovine glial fibrillar acidic protein

astrocytic marker) (1:1000, Sigma, St. Louis, MO), rab-it anti-mouse sarcoma laminin (1:100 Sigma), rabbitnti-rat carbonic anhydrase-II (oligodendrocyte

arker) (1:5000, N. Gregson, GKT, London), and mouse

nti-rat monocyte marker ED1 (1:200, Serotec Ltd., Ox- B

ord, UK). Briefly, antibodies were diluted as indicatedbove in PBS containing 1% (w/v) bovine serum albu-in, and 60 ml was applied to sections and incubated

overnight at 4°C. Slides were immersion washed twicein PBS and then incubated for 1 h in appropriate FITC-labeled (CAII, LAM, GAP43) or TRITC-labeled (ED1,GFAP) anti-IgG secondary antibodies (Vector Labora-tories, Burlingame, CA). Slides were evaluated usingimmunofluorescence microscopy.

Tracing Studies

Retrograde tracing was performed in both eyes ofthree animals by injection of 2 ml of 20% lysinatedrhodamine–dextran (LRD, Molecular Probes, Eugene,OR) into the proximal optic nerve segment at 39 and 99days postlesion according to the method of Berry et al.1996). After 24 h, the retinae were collected, whole-

ounted, and the total number of LRD-filled RGC inach retina counted.

The total number of RGC in the retinae of unlesionedats was estimated by serial alignment of a squareounting grid along the radius of each retinal quadrant.he side length of each grid defined a series of concen-

ric rings centered on the optic disc. The total number ofGC was calculated from the sum of the total numberf RGC in each concentric ring (the product of the meanensity of RGC in the four quadrant grids and theolume of each concentric ring). Volumes were esti-ated by weighing the paper cutouts of the projected

utlines of each concentric ring.

tatistical Analysis

Total counts of LDR-labeled RGC were statisticallynalyzed using ANOVA and the Bonferroni/Dunn postoc test. Data were evaluated as means 6 SEM.

ACKNOWLEDGMENTS

This work was supported in part by grants from the WellcomeTrust, the International Spinal Research Trust, and the Shelley and theSteven Einhorn Optic Nerve Research Fund of the Glaucoma Foun-dation.

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Received October 6, 2000Revised February 7, 2001

Accepted February 16, 2001