In Vivo and In Vitro Target Validation with Nucleic Acid Aptamers as Pharmacological Probes

P. Shannon Pendergrast and David M. Epstein

11.1

Introduction

Aptamers are small nucleic acid molecules that function as direct protein inhibitors. Over the last several years, research and development has led to the preclinical evaluation of serum-stabilized aptamers in a variety of in vitro and in vivo systems. Not only have aptamers now demonstrated their broad utility as an exciting class of compounds for use as pharmacological tools for target validation within drug discovery, they have also demonstrated broad potential as therapeutic candidates in drug development.

11.2

Target Validation with Aptamers as Pharmacological Probes

Aptamers are synthetically derived oligonucleotides that bind with high affinity and specificity to protein targets. Serum-stabilized aptamers suitable for in vivo studies can be developed rapidly, usually within 9-14 months (Fig. 11.1). The ap-tamer generation process is divided into four stages (Fig. 11.1). Identifying a biologically functional and potent aptamer "hit" is a rapid process, taking from five to six months. This target-to-hit step is made highly efficient because the selection of target binders is coupled iteratively to function-based screening (biochemical or cellular assay), such that aptamers identified throughout the selection process are readily characterized for desired potency and functional properties (e.g. specificity) and mechanism-of-action. Over two to three months (stage 2), the targetto-hit aptamer is minimized to elucidate its smallest functional unit, usually from 15 to 35 nucleotides. Over several months medicinal chemistry efforts are aimed at incorporating site-specific backbone modifications to stabilize the minimized aptamer against serum nucleases. Finally, the lead aptamer is readied for use in vivo proof-of-concept studies through conjugation to give the final in vivo aptamer lead its desired pharmacokinetic properties. The combination of back-

Target Partially stabilized nucleic acid pool

Fig. 11.1 Overview of the timeline and the process of discovery and development of aptamers for use as in vivo pharmacological probes.

bone modifications within the aptamer core and the final conjugation (e.g. 20 kDa, 30kDa, 40kDa polyethylene glycol (PEG)) provide control of and the ability to tune the pharmacokinetic and distribution properties of the in vivo lead molecule (Fig. 11.2).

Aptamers as pharmacological probes have proven to be versatile reagents for in vivo target validation and proof-of-concept studies in that they provide direct evidence for the effect of target inhibition in vivo. We describe the use of aptamers as both intracellular (in vitro) and extracellular (in vitro and in vivo) pharmacological probes. Importantly, these same pharmacological probes are also being developed into therapeutic agents for the treatment of acute, subacute, and chronic indications (Longman, 2004).

In vivo target validation determines whether a drug target is involved in disease pathology, and therefore, is a point of intervention for new therapeutics. For most of the pharmaceutical industry's targets, which are now genomically derived, as well as for many targets that are biochemically and genetically validated, there

100 O

0.01

| primate

| rodent

Fullj" 2" oneirnojifr}

.-T

irj-PDGF (rpojis]

t

AKC183 / i /

Fig. 11.2 Overview of the combined effects of apta-mer backbone composition and aptamer 5'- or 3'-ter-minal conjugation on in vivo pharmacokinetic halflife (measured in rodents and primates).

DNA DNA/RNA/OMe OMe

Composition

Fig. 11.2 Overview of the combined effects of apta-mer backbone composition and aptamer 5'- or 3'-ter-minal conjugation on in vivo pharmacokinetic halflife (measured in rodents and primates).

may be little, if any, in vivo biological data that can be used to guide drug discovery and development decisions. Traditionally, target validation in preclinical discovery research has relied on biochemical (in vitro) or genetic (in vivo) means for linking a targets biological mechanism to disease pathology. In this review we discuss the merits of biochemical, or pharmacological, based approaches to target validation that utilize nucleic acid aptamers as in vivo agents to probe pharmacology and mechanism- of-action in animal models of disease. We contrast the aptamer-based target validation approach to gene or message-directed methods for assessing a target's biological function. Aptamers are highly specific and target-selective pharmacological probes, and as such, we argue that aptamer-based methods have the advantage of allowing the researcher to directly interrogate the pharmacological effect of target blockade in vivo, in a dose-dependent manner. Hence, an apta-mer-based in vivo validation approach provides direct information on target biology, while also providing two distinct routes to drug discovery: one route utilizes the aptamer itself as the drug development candidate and the second route uses the aptamer as the pharmacological probe against which small molecule or biologic agents are screened and subsequently developed.

11.3

Limitations of Target Validation by Gene or mRNA Knockout

A biological approach for determining the relevance of a protein to a particular disease is to block expression of the gene in a model organism. As such, target validation methods have been devised that attack gene expression at the DNA and mRNA level (Fig. 11.3). Blocking gene expression at the genetic level is accomplished by constructing a knockout animal. The gene knockout approach completely abolishes gene expression such that the function of a drug target can be examined in every system of the mammalian species simultaneously. In our view, however, one is forced to make relatively risky drug discovery decisions if target validation is based solely on a gene knockout approach, because the results observed in the model organism often do not translate to understanding human disease. It is worrying that transgenic knockouts, which otherwise have been well-characterized in cellular assays, often exhibit phenotypes that are inconsistent with those observed in previous work (Pich and Epping-Jordan, 1998). These conflicting phenotypic effects can manifest either as a very mild phenotype, possibly as a result of an additional redundant gene, or as an animal that has developmental defects resulting in lethality (Ihle, 2000). More importantly, however, since knockout approaches result in the complete removal of a protein target of interest, it is likely impossible to predict target-specific pharmacological effects from the phenotypic effects of the knockout. Alternatively, in vivo aptamer-based target validation studies afford a direct link between dose level and efficacy.

r

W

Protein

__ mRNA

mRNA

\\N \\\ V\\ \\V "NA

L

The levels of gene expression and target validation techniques that target each.

The levels of gene expression and target validation techniques that target each.

Gene-level target validation approaches include techniques that knock out gene expression at the mRNA level. RNA interference (RNAi) (Hannon, 2002) and antisense (Taylor et al., 1999) are two such approaches. RNAi uses double-stranded RNA to induce homology-dependent degradation of the cognate mRNA, and therefore block the expression of the desired protein. Initially, a major drawback of RNAi was that when applied to mammalian cells it triggered non-specific responses which obscured sequence-specific silencing. One of these non-specific responses was the activation of the RNA-dependent protein kinase (PKR) pathway, which phosphorylates EIF-2a and non-specifically arrests translation. Due to their small size, short inhibitory RNAs (siRNAs) can effectively block protein expression, without inducing PKR (Elbashir et al., 2001). Although some non-specific effects via the interferon response (Sledz et al., 2002; Bridge et al., 2003) and possibly other pathways (Persengiev et al., 2004) have been reported more recently. Furthermore, there still remain significant technical and scientific hurdles to the broad, systemic use of functional siRNA molecules in vivo in animal models of disease. Indeed, the delivery and uptake of functional siRNA molecules into mammalian model organisms has been achieved by either local injection (intra-vitreal) or injection at high pressure and volume. So far, for instance, there have only been a few examples of their use in mouse models for disease (Buckingham et al., 2004; Tompkins et al., 2004; Xia et al., 2004).

In the case of antisense, base pairing of an antisense strand of RNA (or DNA) with its corresponding mRNA blocks translation of the mRNA by marking the RNA for degradation by RNase or by blocking the protein translation machinery. Antisense reagents have a number of disadvantages for use in in vivo target validation studies, including poor pharmacokinetic properties and concerns about specificity. Advances in the chemical modification of antisense RNA backbones have alleviated some of these concerns (Gewirtz, 1999; Bennett, 2002), but do not address fundamental issues of intracellular uptake and cellular targeting of a oligonucleotide agent, nor do they address the broad utility of antisense or siRNA as pharmacological probes of target function. In addition to validating the role of the protein in disease pathology, it is also important to validate the protein as a target for therapeutic intervention. Since proteins sometimes have more than one function and are part of more than one multiprotein complex, deletion of the protein through altering mRNA levels can lead to the disruption of numerous pathways and regulatory cascades, some of which may not be relevant to the disease model in question. Finally, from the aptamer-centric view, validation techniques which function at the gene level do not afford predictable pharmacology, such as that based on dose-dependent inhibition of protein function.

Validating drug targets at the protein level with aptamers yields direct evidence on pharmacology and mechanism-of-action (Shaw et al., 1995; Ruckman et al., 1998), and may lead to predictions of what may be expected of a small molecule or biological drug directed at the same target. Antibodies, which bind proteins with high affinity and specificity, are also a powerful way to inactivate protein targets (Lichtlen et al., 2002). Some progress has been made towards the use of single-chain antibody fragments (scFvs) for intracellular applications, however even for these molecules the reducing conditions within the cytoplasm lead to misfold-ing and aggregation (Lichten et al., 2002).

11.4

Target Validation Using Nucleic Acid Aptamers

The interrogation of target function in vivo using nucleic acid aptamers, probes the mechanism-of-action of target blockade and the pharmacology related to target inhibition. Aptamers have many attributes that make them suitable for use as pharmacological probes in in vivo proof-of-concept studies and, more broadly as reagents for in vivo target validation. They bind with high affinity (James, 2000) and exhibit a degree of specificity that allows them to distinguish between closely related molecules and includes the ability to discriminate between the phosphorylated forms of a MAP kinase (Fig. 11.4). Like small-molecule therapeutics, their dosage can be easily adjusted and they exhibit a dose response (Reyder-man and Stavchansky, 1998). Importantly, an aptamer can inhibit target function in vivo by blocking or knocking out a single domain of a protein while leaving the remainder of the protein functional. Small-molecule drugs are also well known for this capability (Aramburu et al., 1999). Small molecules and aptamers share another characteristic in that they both, potentially, can act as agonists.

Along with the above conceptual advantages, there are also practical advantages to working with aptamers. Being nucleic acids, aptamers are easy to produce, store and modify. Large amounts of aptamers can be produced in vitro either en-zymatically or synthetically. They can be easily delivered intracellularly by standard transfection techniques (Chan et al., 2006) or produced in vivo with the appropriate expression vectors. Lyophilized aptamers can be stored for years without loss of activity and once they are reconstituted, they can be boiled or subjected to numerous freeze-thaw cycles. Their stability both in vitro and in vivo can be further enhanced by various chemical modifications such as 2'-fluoro and 2'-O-methyl substitution (Brody and Gold, 2000).

Fig. 11.4 Aptamer specificity is demonstrated through binding to only the phosphorylated form a MAP kinase, pERK.

Positive and negative

selection methods were used to drive drives preferential recognition of pERK (squares) over that

protein conc.

1000

10000 of native ERK (circles).

In Vitro Target Validation with Aptamers against Intracellular Targets

A number of studies have demonstrated the use of aptamers as intracellular target validation tools. Aptamers have been selected against HIV-1 Rev and Tat, two RNA-binding proteins that are necessary for HIV-1 replication. Rev interacts with the Rev binding element (RBE) and functions to transport the viral RNA from the nucleus to the cytoplasm. Tat is an RNA-binding transcription factor that is required for viral replication. When expressed intracellularly anti-Rev or anti-Tat aptamers have inhibited HIV-1 production in cell culture (Lee et al., 1995; Symens-ma et al., 1996).

Further studies have demonstrated that aptamers can recognize proteins that mediate the mammalian signal transduction processes. For example, Blind et al. (1999) selected aptamers that recognize the cytoplasmic domain of aLß2-integrin. ß2-Integrins have been demonstrated to play a critical role in leukocyte cell adhesion. Anti-ß2 aptamers were demonstrated to inhibit aLß2-integrin activity, as measured by a decrease in cell adhesion (Blind et al., 1999). Mayer et al. (2001) have extended these observations by knocking out cytohesin 1, a signaling protein downstream of aLß2-integrin. The anti-cytohesin 1 aptamer was demonstrated to inhibit the guanine nucleotide exchange factor (GEF) activity of cytohesin 1. Moreover, cytohesin 1 inhibition translated into a biological effect as measured by cell adhesion and cytoskeletal rearrangement.

Another consequence of working at the protein level is that aptamers can compliment gene-level validation approaches. Recently, we have reduced expression of another signal transduction protein, NFkB, by overexpression of an aptamer specific for its p50 subunit (Chan et al., 2006). NFkB activity was best inhibited by transfecting a vector that expressed a transcript consisting of the aptamer and

GCGCGUGCcfJ ^UCCC* Cu

QUeGCGCOGG AflQ^t C G c g g c U

ogggucc caccucaqglkac coada

5' G*vaGCLGSA AUCeCL> U aglkcagg a ^cligg^uguaglig cgcuftu AUCC AAACUa U

AGAGCGGAC

UUJCGCCUG C

Terminator

7SL-NF-kB

Fig. 11.5 Diagram of theoretical transcript generated from aptamer expression constructs. Transcript generated from 7SL-NFkB. 7SL sequences are in black, aptamer sequence in red and terminator sequence in blue.

CG" a-p50

Fig. 11.6 NFkB activity is most significantly plasmid (siRNA2 + 7SL), p50-specific aptamer inhibited in the presence of both p50-specific + siRNA control plasmid (siRNA CON + 7SL-a-siRNA and p50-specific aptamer. Bar graph of p50), and plasmids expressing both p50-spe-

results of NFkB-dependent luciferase assay of cific siRNA and aptamer (siRNA 2 + 7SL-a-

cells transfected with NFkB-dependent reporter p50). Shown experiment is representative of plasmid along with control plasmids for both multiple experiments. Error bars generated by siRNA and aptamer expressors (siRNA CON + Microsoft Excel. 7SL), p50-speciflc siRNA and aptamer control

Fig. 11.6 NFkB activity is most significantly plasmid (siRNA2 + 7SL), p50-specific aptamer inhibited in the presence of both p50-specific + siRNA control plasmid (siRNA CON + 7SL-a-siRNA and p50-specific aptamer. Bar graph of p50), and plasmids expressing both p50-spe-

results of NFkB-dependent luciferase assay of cific siRNA and aptamer (siRNA 2 + 7SL-a-

cells transfected with NFkB-dependent reporter p50). Shown experiment is representative of plasmid along with control plasmids for both multiple experiments. Error bars generated by siRNA and aptamer expressors (siRNA CON + Microsoft Excel. 7SL), p50-speciflc siRNA and aptamer control the stabilizing natural RNA, 7SL (Fig. 11.5). The aptamer reduced NFkB activity by 64%, about the same as could be achieved using siRNA (Fig. 11.6). Interestingly, cotransfection of vectors expressing the NFkB-specific aptamer with vectors expressing the NFkB-specific siRNA resulted in nearly complete inhibition NFkB (Fig. 11.3). This result is consistent with the idea that attacking gene expression at multiple levels (in this case at the mRNA and the protein levels) might be the most effective way to reduce activity. Given that both siRNA and aptamers can be delivered via an RNA transcript, it may be possible to express them simultaneously from one vector.

In Vivo Target Validation with Aptamers against Intracellular and Extracellular Targets

Transgenic aptamer-expressing Drosophila have been used to demonstrate the efficacy of aptamers as target validation tools in whole animals. Shi et al. (1999) showed that an anti-B52 aptamer could function in vivo to inhibit B52 function. B52 is a member of the Drosophila SR protein family, a group of nuclear proteins that are essential for pre-mRNA splicing. Shi et al. (1999) developed an RNA aptamer that specifically bound to B52 with high affinity and configured a multiva-

lent aptamer for expression in cells and in flies. Previous work has shown that the level of B52 expression is critical for normal Drosophiia development. B52 deletion results in death (Ring and Lis, 1994), and overexpression of B52 is associated with lethality and morphological defects including missing bristles and the absence of salivary glands (Kraus and Lis, 1994). Shi et al. (1999) used anti-B52 aptamers to suppress the effects of B52 overexpression in Drosophiia. They observed the reversal of abnormal bristle, wing, abdominal sternite, and salivary gland developmental associated with B52 overexpression. Moreover, an increase in the survival rate was observed for flies coexpressing B52 and the anti-B52 aptamer as compared with B52 expressers alone. These results demonstrate that aptamers can be used to inhibit intracellular target function in a non-mammalian, whole animal systems.

More relevant to drug discovery and development is the ability to use serum-stabilized aptamers in vivo as pharmacological probes. Over the last several years, research and technology enhancements, including facile synthesis and conjugation chemistries, have led to the preclinical evaluation of serum-stabilized aptamers in a variety of in vitro and in vivo systems, as summarized in Table 11.1. Thus, aptamers have now demonstrated their broad utility as a novel class of compounds for use as pharmacological tools for in vivo target validation and as unique agents for drug discovery. The ability to synthesize appropriate quantities of stabilized lead aptamers and to subsequently couple such aptamers with high molecular weight PEG polymers (termed "PEGylation," see below) without abrogating biological function allows serum-stabilized aptamers to survive and remain pharmacologically active in vivo, rendering them exceptionally specific tools for extracellular target validation (Fig. 11.1). A nice illustration of the use of such molecules for extracellular target validation is a series of experiments from Pietras et al., utilizing an anti-PDGF-B aptamer (Pietras et al., 2001, 2002). Platelet-derived growth factor (PDGF) belongs to the cysteine-knot growth factor family and was originally isolated from platelets for promoting cellular mitogenic and migratory activity. The binding of PDGF isoforms to their cognate receptors induces the phosphorylation of specific residues in the intracellular tyrosine kinase domain of the receptors and activation of the signaling pathway. In general, PDGF isoforms are potent mitogens and thus are targeted for proliferative diseases such as cancer, diabetic retinopathy, glomerulonephritis, and restenosis.

PDGF-B has been implicated in the regulation of interstitial fluid pressure (IFP) in human tumors. Elevated IFP is one of the physiologically distinctive properties of solid tumors that differ from healthy connective tissue and is considered to be a primary obstacle limiting free diffusion of therapeutic agents into solid tumors. IFP increases as a function of tumor size and malignancy. In general, high IFP in cancer patients is associated with poor prognosis. Notably, solid tumors, including those that are treated with standard chemotherapy regimens, exhibit high IFP. In addition, the stromal tissue into which the tumor epithelial cells proliferate contains PDGF-B responsive fibroblasts. Current data indicate that tensile strength and mechanical stiffness of connective tissue are regulated by a complex interaction between cells such as fibroblasts with extracel-

Table 11.1 Overview of in vitro and in vivo pharmacological properties of select functional aptamers:

The protein target of interest is listed along with key results on the in vitro mechanism and in vivo proof-of-concept studies

Table 11.1 Overview of in vitro and in vivo pharmacological properties of select functional aptamers:

The protein target of interest is listed along with key results on the in vitro mechanism and in vivo proof-of-concept studies

Target

Biochemical

In Vitro cell-based activity

In Vivo disease model

Angiopoietin-2

Kd

= 10 nmol/L

Aptamer blocks Angl, Ang2-mediated inhibi

Aptamer inhibits neovascularization in a bFGF-mediated corneal

(White et al., 2003)

tion of apoptosis in TNF-a treated HUVEC cells

nicropocket angiogenesis assay in rat

VEGF-A

Kd

= 300 pmol/L

Aptamer blocks VEGF165-mediated

Aptamer blocks Wilms' tumor growth in mouse xenograft model

(Ruckman et al., 1998;

proliferation

Aptamer blocks A673 rhabdomyosarcoma growth

Drolek et al., 2000;

Aptamer blocks neovascularization in corneal angigenesis and

Ishida et al., 2003)

ROP models

PDGF-B

Kd

= 100 pmol/L

Aptamer blocks PDGF-BB-mediated

Aptamer reduces tumor interstitial fluid pressure and increases

(Pietras et al., 2003)

proliferation

taxol uptake and efficacy

Aptamer blocks experimental glomerulonephritis in rat model

Aptamer blocks restenosis in rat model

CTLA-4

Kd

= 30 nmol/L

Aptamer blocks CTLA-4 mediated negative

Aptamer enhances tumor immunity in a B16 melanoma mouse

(S antulli-Marotto

regulation of stimulated T-cellsleading to

tumor model

et al., 2003)

enhanced proliferation

Tenascin C

kd

= 5 nmol/L

Aptamer binds equivalently to cellsurface target

91>Tc-labeled aptamer specifically localizes to tumors

(Hiche et al., 2001)

Thrombin

Kd

= 10 nmol/L

Aptamer increases clotting times (ACT, aPTT

Aptamer increases clotting times (ACT, aPTT and PT) during

(Bock et al., 1992;

and PT) in plasma from rat, primate, human

intravenous infusion and reverses rapidly without antidote in dog

Griffin et al., 1993;

cardiopulmonary bypass, sheep hemodialysis and, primate

DeAnda et al., 1994)

infusion models

Factor IXa

Kd

= 650 pmol/L

Aptamer increases clotting times aPTT but

Aptamer increases clotting times ACT and aPTT but not PT,

(Rusconi et al., 2004)

not PT, in human plasma. In vitro anti-clotting

when administered by infusion in pig and murine models. In vivo

activity of the aptamer can be reversed by

anticlotting activity of the aptamer can be reversed by addition of

addition of antidote sequence

the antidote sequence

GnRH

Kd

= 20 nmol/L

Aptamer GnRH-mediate calcium flux in

In a castrated rat model, GnRH-mediated luteinizing hormone

(Wlotzka et al., 2002)

Chinese hamster ovaiy cells expressing the

(LH) levels are reduced, upon aptame treatment, to those

GnRH receptor

observed in intact animals

VEGF, vascular endothelial growth factor PDGF, platelet-derived growth factor;

CTLA, cytotoxic T lymphocyte antigen; GnRH, gonadotropin-releasing hormone; TNF, tumor neurosis factor; ACT, activated clotting time; aPTT, activated partial thromboplastin time; PT, prothrombin time.

lular matrix components such as collagen and hyaluronan. PDGF is known to up-regulate synthesis of collagen and to mediate interactions of anchor proteins such as integrins with extracellular matrix components. Thus, it has been hypothesized that tumor cell-derived PDGF-B secretion leads to proliferation of tumor stromal fibroblasts and deposition of collagen, thereby leading to reduced fluid flow within the tumor and elevated IFP levels.

Pietras et al. sought to test the hypothesis that local PDGF-B secretion within the tumor microenvironment leads to high IFP levels in human tumors. To test this hypothesis Pietras examined the ability of the PDGF-B aptamer to block PDGF-B function in vivo, and whether PDGF-B inhibition was specific for PDGF-B receptors within the tumor stroma, and finally whether the PDGF-B inhibition leads to decreased IFP levels and concomitantly increased levels of chemotherapeutic agents within the tumor (Pietras et al., 2001). Using a KAT-4 thyroid carcinoma xenograft model these researchers demonstrated that the KAT-4 tumor cells in vivo do not express PDGF p-receptors and thus are not responsive to PDGF-B inhibition. Further, they demonstrated that PDGF p-receptor expression is localized to the tumor stroma in this xenograft model and that labeled PDGF-B bound only to the stromal component of the KAT-4 xenograft. To examine the relationship between PDGF-B signaling and IFP in the KAT-4 model, STI571, a small-molecule tyrosine kinase inhibitor was administered by oral gavage once daily at a dose of 100mg/kg per day. Daily STI571 treatment significantly decreased tumor IFP in vivo (Fig. 11.7a), leading to increased uptake of Taxol. Most significantly, STI571 treatment also enhanced the antiproliferative effect of Taxol (Fig. 11.8a). These data, along with previously published data, indicates that PDGF-B is likely a good target for lowering IFP and concomitantly increasing chemotherapeutic efficacy. However, STI571, like all small-molecule

control PDGF aptamer aptamer

Fig. 11.7 Treatment with platelet-derived PDGF receptor inhibitors were administered growth factor (PDGF) receptor antagonists for a total of4 days. (a) mice were treated with lowers interstitial fluid pressure (IFP) in KAT-4 phosphate-buffered saline (PBS) (n = 8) or tumors. Tumor IFP was measured 1-2 h after STI571 (n = 9). (b) Mice were treated with last administration of PDGF receptor inhibitor control aptamer (n = 8) or PDGF aptamer in KAT-4 tumors grown s.c. in SCID mice. (n = 8). From Pietras et al. (2002).

Time after start of treatment (days) Fig. 11.8 Treatment with platelet-derived growth factor (PDGF) receptor antagonists enhances the effect ofTaxol on KAT-4 tumors in vivo. Growth curves of KAT-4 tumors grown s.c. in SCID mice. (a) Mice received no treatment (solid square, n = 8), STI571 (solid circle, n = 6), Taxol (solid triangle, n = 4), or STI571 and Taxol (cross, n = 8). (b) Mice received polyethylene glycol (PEG) (solid square, n = 8),

Time after start of treatment (days)

PEG-conjugated PDGFaptamer (solid circle, n = 8), PEG and Taxol (solid triangle, n = 8), or PDGFaptamer and Taxol (cross, n = 8). *P < 0.05, PDGF receptor antagonist + Taxol versus Taxol alone, Student's t-test, **P < 0.01 PDGF receptor antagonist + Taxol versus Taxol alone, Student's t-test. From Pietras et al. (2002).

Time after start of treatment (days) Fig. 11.8 Treatment with platelet-derived growth factor (PDGF) receptor antagonists enhances the effect ofTaxol on KAT-4 tumors in vivo. Growth curves of KAT-4 tumors grown s.c. in SCID mice. (a) Mice received no treatment (solid square, n = 8), STI571 (solid circle, n = 6), Taxol (solid triangle, n = 4), or STI571 and Taxol (cross, n = 8). (b) Mice received polyethylene glycol (PEG) (solid square, n = 8),

Time after start of treatment (days)

PEG-conjugated PDGFaptamer (solid circle, n = 8), PEG and Taxol (solid triangle, n = 8), or PDGFaptamer and Taxol (cross, n = 8). *P < 0.05, PDGF receptor antagonist + Taxol versus Taxol alone, Student's t-test, **P < 0.01 PDGF receptor antagonist + Taxol versus Taxol alone, Student's t-test. From Pietras et al. (2002).

kinase inhibitors, is known to affect other tyrosine kinases, it was impossible to know if the effect was due to PDGF-B blockade alone.

This dilemma was solved by using a highly specific aptamer to block PDGF-B in similar experiments. The PDGF-B aptamer (Fig. 11.9) was isolated through single-stranded DNA SELEX (Green et al., 1996). The aptamer has an affinity of 100pmol/L for PDGF-B and no appreciable affinity for the PDGF-A isoform. As with STI571, treatment of KAT-4 xenograft mice with PEG-conjugated PDGF-B aptamer lowered IFP (Fig. 11.7b) and significantly increased tumor uptake ofTaxol. Most importantly, aptamer treatment strongly enhanced Taxol's ability to inhibit tumor growth (Fig. 11.8b). Given the high specificity of aptamers, these experiments further validate the concept of blocking PDGF-B as a means of enhancing the uptake and efficacy of chemotherapeutics. The rapid generation and utilization of the anti-PDGF-B aptamer to rapidly obtain in vivo proof-of-con-cept data illustrates the utility of aptamers for target validation. Furthermore, because aptamers in general and the anti-PDGF-B aptamer in particular, already have many of the attributes required for a therapeutic, the anti-PDGF-B aptamer can directly enter into a therapeutic development program.

Fig. 11.9 Anti-PDGF-B specific aptamer, ARC127.

11.5

Summary

High-affinity, target-specific aptamers for use as both in vitro and in vivo pharmacological probes can be generated within one year. As such, aptamers provide versatile tools for the validation of intracellular and extracellular targets. In the case of extracellular targets, such as vascular endothelial growth factor (VEGF), thrombin, and PDGF discussed here, aptamer-based validation affords a direct path to therapeutic development. Therapeutic aptamer leads can be readily stabilized or shielded from renal filtration by chemical or compositional modification for evaluation in in vivo preclinical discovery programs. Aptamers appear poised to make a significant contribution to the treatment of acute and chronic diseases.

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