Proc. Natl. Acad. Sci. USAVol. 92, pp. 5592-5596, June 1995Pharmacology

Vector-mediated delivery of a polyamide ("peptide") nucleic acidanalogue through the blood-brain barrier in vivoWILLIAM M. PARDRIDGE*, RUBEN J. BOADO, and YOUNG-SOOK KANGDepartment of Medicine and Brain Research Institute, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90024

Communicated by Avram Goldstein, Stanford, CA, March 10, 1995 (received for review December 17, 1994)

ABSTRACT Polyamide ("peptide") nucleic acids (PNAs)are molecules with antigene and antisense effects that mayprove to be effective neuropharmaceuticals if these moleculesare enabled to undergo transport through the brain capillaryendothelial wall, which makes up the blood-brain barrier invivo. The model PNA used in the present studies is an 18-merthat is antisense to the rev gene of human immunodeficiencyvirus type 1 and is biotinylated at the amino terminus andiodinated at a tyrosine residue near the carboxyl terminus.The biotinylated PNA was linked to a conjugate of streptavidin(SA) and the 0X26 murine monoclonal antibody to the rattransferrin receptor. The blood-brain barrier is endowed withhigh transferrin receptor concentrations, enabling the 0X26-SA conjugate to deliver the biotinylated PNA to the brain.Although the brain uptake of the free PNA was negligiblefollowing intravenous administration, the brain uptake of thePNA was increased at least 28-fold when the PNA was boundto the 0X26-SA vector. The brain uptake of the PNA bound tothe 0X26-SA vector was 0.1% of the injected dose per gram ofbrain at 60 min after an intravenous injection, approximatingthe brain uptake ofintravenously injected morphine. The PNAbound to the 0X26-SA vector retained the ability to bind tosynthetic rev mRNA as shown by RNase protection assays. Insummary, the present studies show that while the transport ofPNAs across the blood-brain barrier is negligible, delivery ofthese potential neuropharmaceutical drugs to the brain maybe achieved by coupling them to vector-mediated peptide-drugdelivery systems.

Antisense oligodeoxynucleotides (ODNs) are potential neu-ropharmaceutical agents. Metabolically stable ODNs includephosphorothioate ODNs (PS-ODNs), and recent studies haveshown pharmacologic effects in brain following direct intra-cerebral injection of ODNs (1-4). These molecules are notpharmacologically active in brain following systemic adminis-tration, owing to the negligible transport of such polar com-pounds through the brain capillary endothelial wall, whichmakes up the blood-brain barrier (BBB) in vivo. However,PS-ODNs may be neurotoxic at therapeutic concentrations (5,6). Therefore, it is desirable to develop other antigene orantisense molecules that have chemical properties differentfrom those of the highly charged ODN molecules. One suchclass of compounds is the polyamide ("peptide") nucleic acids(PNAs), which have a neutral polyamide backbone (7, 8).However, PNAs undergo negligible transport across cell mem-branes and pharmacologic effects in cells in tissue culturerequire direct injection ofPNAs into the cytoplasm (9). On thebasis of these findings it is expected that PNAs undergonegligible transport through the BBB in vivo. Thus, if PNAsare to be effective pharmaceuticals for brain, or other organs,it will be necessary to conjugate these molecules to transcel-lular drug delivery systems.

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

One strategy for peptide delivery through the BBB isvector-mediated drug delivery wherein a biotinylated thera-peutic compound is coupled to a conjugate of avidin and abrain drug delivery vector (10). The latter consists of proteinsthat undergo absorptive mediated or receptor-mediated trans-cytosis through the BBB in vivo, including proteins such ascationized albumin or the OX26 murine monoclonal antibodyto the rat transferrin receptor (11). Linkage of a biotinylatedvasoactive intestinal peptide analogue to a conjugate of avidinand the OX26 antibody resulted in a pharmacologic effect onthe central nervous system in vivo-namely an increase incerebral blood flow (10). In the absence of the drug deliverysystem, no pharmacologic effects on the central nervoussystem were observed following the systemic administration ofthe peptide therapeutic (10). Therefore, the present studiesexamine the feasibility of coupling a biotinylated PNA to aconjugate of streptavidin (SA) and the OX26 monoclonalantibody and then examine the extent to which the BBBtransport of the PNA is enhanced with the use of this deliverysystem. The model PNA used in the present studies is an18-mer that corresponds to nt 5980-5997 of the genome ofhuman immunodeficiency virus type 1 (HIV-1) and is anti-sense to a region of the rev mRNA around the methionineinitiation codon (12). The brain is a shelter for HIV, andanti-HIV PNAs may prove to be effective neuropharmaceu-ticals for the treatment of cerebral acquired immune deficiencysyndrome (AIDS). The retention of biologic activity of thePNA bound to the OX26-SA vector is demonstrated withRNase protection assays using synthetic rev mRNA.

MATERIALS AND METHODS

Materials. PNAs are analogues of DNA in which thephosphate backbone is replaced with a polyamide backbonehaving the repeating structure H-[NH(CH2)2N(COCH2B)-CH2CO]n-NH2, where B is one of the four bases (A, adenine; G,guanine; C, cytosine; or T, thymine) (7, 8). The PNA was customsynthesized by Millipore with the following sequence: biotin-CTCCGCTTCTTCCTGCCA-Tyr-Lys-CONH2, wherein theamino terminus was biotinylated, and tyrosine (Tyr) and lysine(Lys) were placed at the amidated carboxyl terminus. The iden-tical PNA was also synthesized without the biotin moiety. Thetyrosine residue near the carboxyl terminus allows for radiola-beling with 125I, and the carboxyl-terminal lysine inhibits PNAself-aggregation without affecting hybridization (13). The com-pound had a calculated mass of 5542 Da and an observedmolecular weight of 5541, as determined by mass spectrometry

Abbreviations: BBB, blood-brain barrier; HIV, human immunodefi-ciency virus; PNA, polyamide ("peptide") nucleic acid; bio-PNA,biotinylated PNA; ODN, oligodeoxynucleotide; PS-ODN, phospho-rothioate ODN; PO-ODN, phosphodiester ODN; SA, streptavidin;TCA, trichloroacetic acid; PS, permeability-surface area; Vss, steady-state volume of distribution; MRT, mean residence time; AUC, areaunder the plasma concentration curve; %ID, percent of injected dose.*To whom reprint requests should be addressed at: Department ofMedicine, University of California, Los Angeles, School of Medicine,Los Angeles, CA 90024.

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analysis by the manufacturer. The ultraviolet absorption spec-trum of the PNA showed the absorption maximum at 268 nm inphosphate buffer at 23°C. The PNAwas purified by reverse-phasehigh-performance liquid chromatography (HPLC). [125I]Iodinewas obtained from DuPont/NEN. Male Sprague-Dawley rats(265-310 g) were purchased from Harlan-Sprague-Dawley. Re-combinant (SA) and all other reagents were obtained fromSigma.

Preparation of 0X26-SA Conjugate. The OX26-SA conju-gate was prepared in a manner similar to that recently de-scribed for an OX26-neutral avidin conjugate (14). OX26 wasthiolated with a 10:1 ratio of Traut's reagent. This ratiogenerated, on average, two thiol groups per OX26 molecule,as determined with 5,5'-dithiobis(2-nitrobenzoic acid) (Ell-man's reagent; Pierce). The recombinant SA was activated witha 20-fold molar ratio of m-maleimidobenzoyl N-hydroxysuccin-imide ester. Following conjugation of the thiolated 0X26 and theactivated SA, the 0X26-SA conjugate was purified by chroma-tography on a column (1.6 cm x 94 cm) of Sephacryl S300HR(14). The number of biotin binding sites per 0X26-SA conjugatewas determined to be 3.3 ± 0.3 by a [3H]biotin binding assay (14).

lodination of PNA. Biotinylated PNA (bio-PNA, M1.8nmol), [125I]iodine [1.0 nmol, 2 mCi (74 Bq)], and chloramineT (10 nmol) were mixed at a total volume of 40 ,lI of phosphatebuffer (pH 7.4). After 60 sec at room temperature, the reactionwas terminated with the addition of 62 nmol of sodiummetabisulfite, 1% trifluoroacetic acid (TFA) was added, andthe mixture was passed over a C18 Sep-Pak extraction cartridge(Waters). The cartridge was then washed with 10 ml of 0.1% TFAand 5 ml of 5% acetonitrile in 0.1% TFA; the 125I-labeledbio-PNA was eluted with 40% acetonitrile in 0.1% TFA and wasstored at 4°C after evaporation of acetonitrile. The final specificactivity was 42 ,uCi/,ug (0.23 mCi/nmol) with a trichloroaceticacid (TCA) precipitability of 98%. Although the cytosine bases ofthe PNA are potentially subject to iodination (15), we assume thatthe PNA is labeled nearly exclusively at the tyrosine residue,because iodination of nucleic acids requires much higher molarratios of iodine and chloramine T, longer reaction times, andlower pH than those used in the present studies to iodinate thetyrosine residue (15).

Gel Filtration HPLC. Binding of the 12'I-bio-PNA to the0X26-SA conjugate was confirmed by gel filtration HPLC witha TSK-gel G2000 SWxL column (7.8 mm x 300 mm; Tosohaas,Montgomeryville, PA) and isocratic elution in PBST buffer (0.01M Na2HPO4, pH 7.4/0.15 M NaCl/0.05% Tween 20) at a rate of0.5 ml/min. The 125I-bio-PNA was injected onto the columneither alone or bound to the 0X26-SA conjugate.

Pharmacokinetics and Brain Uptake. The pharmacokinet-ics and brain delivery of 1251-bio-PNA, either alone or boundto the OX26-SA vector, were determined following singleintravenous injection into rats anesthetized with ketamine (100mg/kg) and xylazine (2 mg/kg) given intraperitoneally. Thefemoral vein injection solution contained 0.20 ml of PBTbuffer (0.01 M Na2HPO4, pH 6.0/0.1% Tween 20 with ratalbumin at 1 mg/ml), 5 ,tCi of 1251-bio-PNA, and 0 or 20 ,ugof OX26-SA. Blood samples were collected from a femoralartery, as described (14). One hour after injection, the animalswere decapitated, the brains were removed and weighed, andbrain radioactivity was measured. Pharmacokinetic parame-ters were determined by fitting plasma TCA-precipitable-radioactivity data to a biexponential equation (14). The areaunder the plasma concentration curve (AUC), the steady-statevolume of distribution (Vss), plasma clearance, and the meanresidence time (MRT) were calculated fromA1,A2, Kl, and K2,as described by Gibaldi and Perrier (16), and the body weight(kg) of the rat. The brain volume of distribution (VD) of1251-bio-PNA was determined from the ratio of dpm/g of braindivided by dpm/,tl of corresponding terminal plasma at 1 hrafter injection.

The organ permeability-surface area (PS) product of the15Ibio-PNA was calculated as

= [VD - Vo]Cp(T)- t

Cp(t)dt

where Cp(T) is the terminal plasma concentration and V. is theorgan plasma volume, which has been measured previously(14). The organ delivery of 1251-bio-PNA was determined as(14)

t t

%ID/g = PS x AUC |,0 0

where PS and AUC correspond to the 60-min time period and%ID/g is the percent injected dose taken up per gram oforgan.The BBB transcytosis of the 1251-bio-PNA bound to the

OX26-SA conjugate was assayed with a capillary depletiontechnique (17). In these experiments, 20 ,Ci of 1251-bio-PNAand 30 ,tg of OX26-SA conjugate were injected per rat.The metabolic stability in plasma of the 1251-bio-PNA bound

to the OX26-SA conjugate was. examined by gel filtrationHPLC. In these studies, blood was removed at 60 min followingintravenous injection, and 20 ,lI of serum from each of threerats was pooled, diluted to a total volume of 250 Al with PBSTbuffer, and injected onto the gel filtration HPLC column, asdescribed above. Column fractions (0.5 ml) were assayed for1251 radioactivity. The void and salt volumes of the columnwere 6 and 11 ml, respectively.

Synthesis of 32P-Labeled Sense and Antisense rev RNA andRNase Protection Assay. The pCV1 plasmid, containing therev gene of HIV-1, was obtained through the AIDS Researchand Reference Reagent Program, Division of AIDS, NationalInstitute of Allergy and Infectious Diseases, from FlossieWong-Staal. To synthesize 32P-labeled sense and antisense revRNA, the rev coding region was subcloned in the plasmidp-Bluescript (Stratagene) as described previously for the tatcDNA (18). A 727-nt DNA fragment containing the rev gene(19) was released from pCV1 by digestion with Bsu36I and theends of the fragment were filled in with Klenow DNA poly-merase. This rev cDNA was subcloned in p-Bluescript whichwas linearized with Sma I and treated with alkaline phos-phatase to prevent religation of the vector. The new plasmid,named BK-rev, was sequenced in both directions by using T7DNA polymerase and the dideoxy chain termination method(20); sequencing confirmed the presence of the target DNAsequence, and the rev cDNA insert was found to be in theforward orientation. After linearization of BK-rev with Pst IandXba I, sense and antisense RNAs were synthesized with T7and T3 RNA polymerase, respectively, using [a-32P]ATP (18).The specific activity was 10.7 and 7.7 ,uCi/pmol for the senseand antisense RNAs, respectively.For the RNase protection assay, 0.5 pmol of either biotinylated

or nonbiotinylated PNA was incubated with or without 10 pmolof OX26-SA for 5 min on ice in 19 Al of reaction buffer (0.3 MNaCl/10 mM Tris, pH 7.4/4mM EDTA/0.02% tRNA); 105 cpmof 32P-labeled sense or antisense rev RNA (4.2 or 5.9 fmol,respectively) was added in 1 ,ul of reaction buffer and annealedfor 30 min at 42°C. Twenty units of RNase Ti and 2.5 ,tg ofRNase A were added to the samples in 10 pI1 of reaction buffercontaining 1.5 ,tg of bovine serum albumin, and digestion ofunprotected RNA was carried out for 30 min at 37°C. RNAfragments were analyzed by 7 M urea/20% polyacrylamide gelelectrophoresis and autoradiography as described (18). A 5'-amino, 3'-biotinyl PS-ODN (Keystone Laboratories, Menlo Park,

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15

10-

5

0

CO

x 3E

2-

1-

0 20

Fraction

4.5

03.0 c,"

x

E

11.5 a-C-

FIG. 1. (A) Gel filtration HPLC of free 1251-bio-PNA (a) and125I-bio-PNA bound to the OX26-SA vector (0), before injection ofeither tracer into rats. (B) Elution of pooled rat plasma obtained 1 hrafter a single intravenous injection of 125I-bio-PNA bound to theOX26-SA vector. Fraction size was 0.5 ml.

CA) with the same nucleotide sequence as the PNA was used as

a positive control in the RNase protection assay. Labeled RNA,PS-ODN, and PNA were heat denatured for 2 min at 95°C andthen incubated on ice for 1 min immediately before the experi-ment or conjugation to OX26-SA.

RESULTS

The binding of the 125I-bio-PNA to the OX26-SA conjugatewas confirmed by gel filtration HPLC (Fig. 1A). Free radio-labeled bio-PNA was eluted at 12 ml, whereas the 125I-bio-PNAbound to OX26-SA was eluted at 7 ml (Fig. 1A).The free 125I-bio-PNA was rapidly removed from the plasma

compartment following intravenous injection (Fig. 2 Left),with a distribution ty12 of 1.3 0.1 min, an elimination til2 of 29+ 3 min, and a MRT of 33 3 min (Table 1). The Vs,, 278 ±

22 ml/kg, was severalfold above the plasma volume, 71 ± 3ml/kg (21). The systemic clearance, 8.6 ± 0.3 ml/(min.kg), was

a 1.1

Plasma clearance Metabolism

(a

a

'I-

Time, min

FIG. 2. Clearance from plasma (Left) and serum TCA precipita-bility (Right) of 125I-bio-PNA in its free form (0) or bound to theOX26-SA vector (-) is shown for up to 60 min after single intravenousinjection. Data are mean ± SE (n = 3 rats); some SE bars are notvisible because the SE is smaller than the symbol.

Table 1. Pharmacokinetic parameters

Parameter bio-PNA bio-PNA/OX26-SA

K1, min-1 0.54 ± 0.04 0.49 ± 0.11K2, min-1 0.024 ± 0.003 0.013 ± 0.002A1, %ID/ml 4.9 ± 0.04 2.9 ± 0.1A2, %ID/ml 0.70 ± 0.05 3.3 ± 0.3AUC, (%ID/ml)-min 38 ± 2 265 ± 34VS,, ml/kg 278 ± 22 96 ± 7Clearance, ml/(min.kg) 8.6 ± 0.3 1.3 ± 0.1MRT, min 33 ± 3 77 ± 9t1½,, min 1.3 ± 0.1 1.6 ± 0.4t2½,min 29 ± 3 55 ± 7

Values were computed from data in Fig. 2 Left.

high and approximated that for sucrose, 10.8 ± 0.4 ml/(min-kg) (22), suggesting the bio-PNA was removed principallyby glomerular filtration and renal clearance. This was con-firmed in two ways. First, the PS product of the free bio-PNAin kidney was very high, 320 ± 25 ,ul/(min g), 150-fold greaterthan the corresponding parameter for liver (Table 2). Second,cannulation of the urinary bladder with a PE50 cannulashowed that 40 ± 6% of the injected dose was cleared into theurine within the first 60 min after intravenous injection. Whenthe urine radioactivity was included in the computation of therenal uptake, expressed as %ID/g (Table 2), then the renaluptake of the unconjugated bio-PNA was increased from 9.8± 0.6%ID/g (Table 2) to 27.1 ± 1.8%ID/g, or 58 ± 4% of thedose, given a total kidney weight of 2.14 ± 0.02 g (mean ± SE,n = 3 rats).The brain uptake of the unconjugated bio-PNA was negli-

gible, with a PS product of 0.10 ± 0.01 ,ul/(min.g) and anuptake of 0.0031 ± 0.0002%ID/g (Fig. 2). The pharmacoki-netic and organ uptake data for the 125I-labeled PNA shown inFigs. 2 and 3 and Tables 1 and 2 pertain to the biotinylatedPNA. However, the identical PNA was also synthesized with-out the amino-terminal biotin moiety and was radiolabeledwith 1251 and chloramine T, and a pharmacokinetic study wasperformed. These data were essentially identical to that ob-tained for the biotinylated analogue, indicating that biotiny-lation per se did not alter the pharmacokinetics or organdistribution of the unconjugated PNA.

Binding of the 1251-bio-PNA to the OX26-SA vector con-siderably retarded the rate of removal of the PNA from theplasma compartment (Fig. 2) and resulted in a 7-fold reductionin the systemic clearance (Table 1) and a 2.3-fold increase inMRT (Table 1). The increased blood level of bio-PNA boundto the OX26-SA vector was due to a >10-fold reduction inrenal clearance rate (Table 2). The clearance of the bio-PNAby other peripheral tissues, such as lung or heart, was alsoreduced for bio-PNA bound to the OX26-SA vector (Table 2).In contrast, the PS product for the bio-PNA in liver wasincreased 8-fold by conjugation to the OX26-SA vector, con-sistent with high levels of transferrin receptor on liver cells(23). Brain uptake was increased 28-fold when the bio-PNAwas bound to the OX26-SA vector (Fig. 3), due to the

Table 2. Organ clearance of bio-PNA and bio-PNA/OX26-SAOrgan PS product,

p.l/(min.g) Uptake, %ID/gPNA/ PNA/

Organ PNA OX26-SA PNA OX26-SA

Liver 2.1 ± 0.1 18 ± 2 0.065 ± 0.005 2.5 ± 0.03Kidney 320 ± 25 31 ± 2 9.8 ± 0.6 4.5 ± 0.5Lung 8.8 ± 2.0 0.74 ± 0.21 0.27 ± 0.06 0.11 ± 0.03Heart 0.86 ± 0.11 0 0.026 ± 0.003 0

Values are mean + S.E. (n = 3 rats). Measurements were made 60min after intravenous injection.

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FIG. 3. The 60-min plasma AUC [(%ID/ml).min], the BBB PSproduct [,ul/(min g)], and the brain %ID/g for free bio-PNA (openbars) and bio-PNA bound to the OX26-SA vector (solid bars). Dataare mean ± SE (n = 3 rat).

combined effects of a 6-fold increase in the BBB PS productfor the bio-PNA bound to the OX26-SA vector, and a 5-foldincrease in the 60-min plasma AUC of the bio-PNA bound tothe OX26-SA vector, in comparison to the free bio-PNA (Fig.3). A capillary depletion analysis showed that the bio-PNAbound to the OX26-SA vector was transcytosed through theBBB; the VD in the postcapillary supernatant, 45 + 8 ,ul/g, was3-fold greater than the VD in the vascular pellet, 15 ± 2 ,ll/g(mean ± SE, n = 3).The 1251-bio-PNA bound to the OX26-SA vector was met-

abolically stable in the circulation in rats. Virtually all of theplasma radioactivity in plasma obtained 60 min after intrave-nous injection migrated in a peak that was coeluted with thebio-PNA linked to the OX26-SA conjugate, and no radioac-tivity was eluted from the column in the volume corresponding

A S ASW

B 1 2 3

Sense Antisense

1 2 3 4 5 1 2 3 4 5

RRF--t

BPB_ .

.. i, * *, *

..

FIG. 4. RNase protection assay with rev 32P-labeled mRNA and revbio-PNA. (A) Both sense (S) and antisense (AS) synthetic 32P-labeledRNAs showed no small fragments corresponding to incomplete syn-thesis or degradation of transcripts. The migration of xylene cyanol(XC) is indicated at left. (B) RNase protection assay with sense rev32P-labeled RNA incubated without oligomer (lane 1), with rev

bio-PNA (lane 2), or with rev bio-PS-ODN (lane 3). Identical RNase-resistant fragments (RRF) were observed with incubation of eitherbio-PNA or bio-PS-ODN. (C) Effect of OX26-SA on the RNase

protection of either sense or antisense rev 32P-labeled RNA with PNA.Lanes: 1, 32P-labeled RNA alone; 2, plus nonbiotinylated PNA; 3, plusbio-PNA; 4, plus nonbiotinylated PNA and OX26-SA; 5, plus bio-PNAand OX26-SA. Migration of RRF and bromophenol blue (BPB) areindicated. Incubation of bio-PNA or PNA with sense RNA producedsimilar RRF, and the formation of this fragment was not modified byOX26-SA (see lanes 3 and 4 for sense RNA). On the contrary, no RRFwas seen with antisense RNA incubated with or without PNA, or withsense RNA incubated without PNA (lane 1), indicating that the RNaseprotection was exerted through a sequence-specific mechanism. Au-

toradiograms were exposed for 16 hr (A) or 3 days (B and C) at -70°C.

to the free bio-PNA (Fig. 1B). Binding of the 125I-bio-PNA tothe OX26-SA vector also retarded the systemic degradation ofthe PNA as judged by the plasma TCA-precipitable radioac-tivity (Fig. 2 Right).The ability of the PNA to hybridize to target mRNA

following biotinylation and binding to OX26-SA was demon-strated by RNase A/Ti protection assay. Both the free andOX26-SA-bound rev PNA hybridized to the rev sense (but notantisense) RNA and resulted in protection of the same sizeoligoribonucleotide as that found by hybridization with aPS-ODN of identical sequence (Fig. 4).

DISCUSSIONPNAs contain an electrically neutral polyamide backbone inlieu of an anionic phosphodiester (PO)-ODN or PS-ODNbackbone, and all three classes of compounds are potentialantisense or antigene therapeutics. PS-ODNs are resistant tonucleases that rapidly degrade PO-ODNs (24). However,PS-ODNs are neurotoxic when administered in large doseseither systemically (5) or intracerebroventricularly (6), and thistoxicity may be related to the anionic backbone of the meta-bolically stable PS-ODN. Therefore, it may be desirable todevelop antisense or antigene compounds with a neutralbackbone, and PNAs are such compounds (7, 8). The difficultyin achieving pharmacologic effects with PNAs, however, is thenegligible transcellular delivery of these molecules (8, 9). Thepresent study examines the potential of a vector-mediated drugdelivery system to enhance brain uptake of systemically ad-ministered bio-PNAs.Our results are consistent with the following conclusions. (i)

Free PNAs have very low rates of clearance by brain and otherorgans (except kidney) and are largely excreted into the urinewithin 60 min after an intravenous injection. (ii) Binding ofbio-PNA to the OX26-SA vector redirects PNA delivery fromkidney to organs with abundant transferrin receptors, such asliver or the BBB. (iii) The bio-PNA bound to OX26-SA ismetabolically stable, as shown by measurement of serumTCA-precipitable radioactivity (Fig. 2 Right) and serum HPLCanalysis (Fig. 1B). (iv) Conjugation of bio-PNA to OX26-SAdoes not interfere with PNA hybridization to target mRNA(Fig. 4).With the exception of the kidney, the organ PS product for

the free bio-PNA is low, indicating negligible organ uptakefollowing systemic administration. For example, the PS prod-uct in liver, 2.1 ± 0.1 ,ul/(min g) (Table 2), is -0.1% of the rateof blood flow through this organ. Similarly, there is negligibletransport of the free bio-PNA through the BBB, as the brainPS product is less than that of sucrose (22). Actually, the PSproduct for the free bio-PNA shown in Fig. 3 is an upper limitestimate and may reflect artifactual uptake of iodotyrosinereleased by systemic degradation of the bio-PNA that occursin parallel with the increase in plasma TCA-soluble metabo-lites (Fig. 2 Right).

Binding of the bio-PNA to the OX26-SA vector increases theeffective molecular size of the PNA, reduces glomerularfiltration, and redirects PNA clearance from the kidney toorgans with high levels of transferrin receptors, such as liver(20) or the BBB (25, 26). The high concentration of transferrinreceptor on the BBB allows the OX26-SA conjugate to targetthis membrane, resulting in a brain delivery of the bio-PNAvector complex on the order of 0.1%ID/g of brain (Fig. 3). Thislevel of brain uptake approximates that for morphine, which isalso 0.05-0.10%ID/g'brain following intravenous administra-tion (27). The brain uptake of the bio-PNA bound to theOX26-SA vector is at least 28-fold greater than the brainuptake of the free bio-PNA at 60 min after injection (Fig. 3).The enhanced brain uptake is due to dual effects of the vectorthat (i) raise the plasma AUC and (ii) increase the BBB PSproduct (Fig. 3). The plasma AUC is increased by inhibition of

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glomerular filtration of the bio-PNA bound to the high mo-lecular weight transport vector. The enrichment of the brainuptake of the bio-PNA by the vector will be even greater attime periods beyond 1 hr, since the plasma AUC of thebio-PNA bound to OX26-SA is 7-fold greater than the plasmaAUC of the free bio-PNA (Table 1).The bio-PNA bound to the OX26-SA vector is metabolically

stable. For example, the 60-min serum radioactivity migratesat an elution volume consistent with a sustained association ofthe bio-PNA to the OX26-SA vector (Fig. 1B). In addition,measurements of serum TCA-soluble radioactivity show thatthe metabolic stability of the bio-PNA is increased when it isbound to the OX26-SA vector (Fig. 2 Right). The metabolicstability of the bio-PNA is attributed to blockade of both theamino and carboxyl termini of the polyamide linkage. Thepolyamide backbone also appears to be relatively resistant toendopeptidase activity when the bio-PNA is bound to theOX26-SA vector. The metabolic stability of the bio-PNAbound to the OX26-SA vector is not observed with biotinylatedPO-ODN bound to an 0X26-neutral avidin vector, which israpidly degraded in vivo by widely distributed nucleases, evenwith protection of the 3' terminus (28).The biotin linker used in the present studies is a noncleav-

able amide linker as opposed to the cleavable disulfide biotinlinker used in previous studies to bind an analogue of vaso-active intestinal polypeptide to 0X26-avidin (10). The non-cleavable biotin linker is expected to increase the metabolicstability of the overall conjugate, providing the noncleavablelinker does not compromise interaction of the PNA with itstarget nucleic acid. The RNase protection assays (Fig. 4) showthat the PNA effectively hybridizes to the target mRNAfollowing biotinylation and binding to OX26-SA. These resultsparallel recent findings that a PO-ODN bound to a BBBtransport vector via noncleavable biotinylation linkers alsoeffectively hybridize to target mRNA (18).

In summary, the present studies show that in the absence ofthe transcellular delivery system, the BBB transport of PNAsis negligible. However, biotinylated PNAs may be targeted tobrain and transported through the BBB in vivo by couplingthese potential therapeutic drugs to a peptide drug deliveryvector. The use of such delivery systems will allow in vivo testingof the pharmacologic activity of PNAs in animal models.

Emily Yu skillfully prepared the manuscript, and Jody Buciakprovided expert technical assistance. This work was supported byNational Institute of Health Grant R01-AI28760.

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