Standard heparin, low molecular weight heparin, low molecular weight heparinoid, and recombinant hirudin differ in their ability to inhibit transduction by recombinant adeno-associated virus type 2 vectors

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Abstract

Recombinant adeno-associated virus type 2 (rAAV) is a promising vector for in vivo gene therapy. Transduction by rAAV requires binding to heparan sulfate proteoglycan on the cell surface, and heparin can block this binding. Because heparin is administered to most patients undergoing cardiovascular gene transfer in order to prevent thrombotic events, it is important to identify anticoagulants which do not interfere with rAAV transduction. Therefore, we examined the influence of different anticoagulants on rAAV transduction in vitro. rAAV transduction was inhibited by 40.5 ± 7.9% at heparin concentrations of 0.1 U/ml, and by 81.7 ± 3.6% at 1.0 U/ml. The low molecular weight (LMW) heparin tinzaparin inhibited rAAV transduction by 20.2 ± 3.8% at 0.1 U/ml and 37.1 ± 1.8% at 1.0 U/ml. The inhibitory effect was significantly weaker compared with heparin at 1.0 U/ml, (P < 0.01). The LMW heparinoid danaparoid inhibited rAAV transduction by 8.8 ± 3.5% at 0.1 U/ml (P < 0.01 compared with heparin). In contrast, recombinant hirudin did not interfere at all with rAAV transduction. In summary, the results demonstrate that inhibition of rAAV transduction by heparin occurs rapidly and at therapeutically used concentrations. LMW heparinoids and above all recombinant hirudin might be alternatives for heparin when vascular gene transfer with rAAV requires transient anticoagulation.

Main

Recombinant adeno-associated virus type 2 (rAAV) is a viral vector system which allows long-term transgene expression after in vivo application in various tissues.1,2,3,4,5 rAAV has the potential to be used for the treatment of genetic diseases,4,6,7 cancer gene therapy8,9 and cardiovascular diseases.10,11,12 Results demonstrating that rAAV can mediate efficient gene transfer into rat arteries10 and into mouse hearts12 in vivo underline that rAAV might be a promising vector for cardiovascular gene therapy. Recently, heparan sulfate proteoglycan has been proposed to be an important attachment molecule13 or receptor14 for rAAV. Transduction efficiency of rAAV is decreased in a dose-dependent manner in the presence of heparin by abolishing rAAV binding to, and transduction of, target cells.14 Because heparin is administered to most patients undergoing cardiovascular gene transfer in order to prevent the incidence of thrombotic events,15,16,17 it is important to identify anticoagulants which do not interfere with rAAV transduction. Therefore, we tested the influence of four anticoagulants, heparin, tinzaparin, danaparoid and recombinant hirudin, on the transduction efficiency of rAAV in vitro. Heparin exerts its antithrombotic effect through anti-thrombin III (AT III) mediated inhibition of coagulation factor Xa.18 Tinzaparin sodium is a low molecular weight (LMW) heparin, produced by enzymatic depolymerization of unfractionated heparin.19 It is a heterogeneous mixture of sulfated polysaccharide glycosaminoglycan chains with a mass ranging between 5500 and 7500 daltons. Danaparoid is a LMW heparinoid consisting of heparan sulfate, dermatan sulfate and chondroitin sulfate. Binding to AT III leads to an accelerated inhibition of factor Xa resulting in the antithrombotic effect of danaparoid.20 Hirudin is a potent thrombin inhibitor originally derived from the medicinal leech which can now be produced using recombinant technology. Unlike heparin, hirudin acts directly on thrombin, rather than through other clotting factors.21

It has recently been demonstrated that heparin can inhibit rAAV-mediated gene transfer in a dose-dependent manner by binding to rAAV and thus blocking viral attachment to target cells.14 During vascular interventions and cardiovascular surgery heparin is routinely used to prevent the incidence of thrombotic events.15,18 In order to identify the effects of different clinically used anticoagulants on rAAV-mediated HeLa cell transduction, we examined the inhibitory effects of heparin, LMW heparin, LMW heparinoid and recombinant hirudin at equipotent concentrations. When using heparin at concentrations from 0.1 to 1000 anti-factor-Xa U/ml, rAAV-mediated target cell transduction was blocked up to 100% in a dose-dependent manner (Figure 1a). At concentrations of 0.1 U/ml, heparin inhibited viral transduction by 40.5 ± 7.9%, and 1.0 U/ml heparin caused 81.7 ± 3.6% inhibition after a 10 min incubation time. Prolongation of the incubation time from 10 min to 2 h did not significantly enhance the inhibition of transduction (Figure 1a).

Figure 1
figure1

For the generation of rAAV virions, 293 cells (a gift from M Lose, Max-Planck-Institute of Biochemistry, Martinsried, Germany) were cotransfected by calcium phosphate precipitation with the plasmids pRC, containing the AAV rep and cap genes, pGFP, containing the EGFP cDNA (Clontech, Palo Alto, CA, USA) within the adeno-associated virus ITR-sequences (both kindly provided by Anne Girod, Gene Center, Munich, Germany), and pXX6, containing the adenovirus helper genes (kindly provided by RJ Samulski22 University of North Carolina at Chapel Hill, NC, USA). After 48 h, the cells were lysed and fractioned ammonium sulfate precipitation was performed. rAAV virions were further purified and concentrated by ultracentrifugation on a CsCl gradient (P = 1.37 g/ml) and column chromatography. Genomic titers were determined by dot blot as described.23 For determination of the infectious titers, HeLa cells were plated in 12-well plates at 7 × 104 cells per well. Twenty-four hours later the cells were irradiated with 100 Gy and serial dilutions of the recombinant virus were added to the cells. After 48 h, the number of GFP-expressing cells was quantified by flow cytometry. Using these methods, titers up to 8 × 1011 genomic particles/ml and infectious titers of up to 8 × 108 infectious particles/ml were detected. For the inhibition experiments HeLa cells were pretreated as described above. rAAV at a concentration of 3.2 × 104 infectious particles/ml was incubated with serial dilutions of anticoagulants for 10 min or 2 h at room temperature in serum-free DMEM (Biochrom, Berlin, Germany). 250 μl of these solutions were then added to the HeLa cells, corresponding to an MOI of 0.1. After a 2 h incubation time, the cells were washed twice with PBS, and DMEM/10% FCS was added. Forty-eight hours later the number of GFP-expressing HeLa cells was quantified by flow cytometry. All experiments were performed in duplicate. Standard error of the mean (s.e.m.) was calculated from three independent experiments (n = 6). After performing an F-test to confirm heterogeneity of variances, Student's t test was calculated using Excel software (Microsoft, Seattle, WA, USA). The level of significance was set at P < 0.05. The following anticoagulants were used: heparin sodium (B Braun, Melsungen, Germany), tinzaparin sodium (Innohep, B Braun), danaparoid sodium (Orgaran, Thiemann, Germany) and the recombinant hirudin lepirudin (Refludan, Aventis, Frankfurt, Germany). HeLa cells were purchased from ATCC (Manassas, VA, USA). Inhibition of rAAV target cell transduction in the presence of increasing concentrations of anticoagulants (a) heparin, (b) tinzaparin, (c) danaparoid and (d) lepirudin, after a 10 min incubation time of the anticoagulant with the virus (solid line) or a 2 h incubation time (dashed line) before the addition to HeLa cells is shown. Heparin, tinzaparin and danaparoid concentrations are given as anti-factor-Xa U/ml, lepirudin concentrations are given as anti-factor-II U/ml. Standard error of the mean is indicated by error bars.

When using a 10-fold concentrated virus a significantly lower inhibition of transduction of 18.1 ± 1.2% was detected at low heparin concentrations of 0.1 U/ml, compared with a one-fold concentrated virus preparation (P = 0.029). However, at higher heparin concentrations (1.0–1000 U/ml) no significant differences could by identified (Figure 2). Taken together, these data confirm the results of Summerford and Samulski,14 and demonstrated that heparin decreased rAAV-mediated target cell transduction. Furthermore, they indicate that the inhibition of rAAV transduction efficiency by heparin may be a relevant problem in the clinical setting when rAAV is used as a vector system for cardiovascular gene transfer because (1) it occurs at therapeutical heparin concentrations of 0.1–1 U/ml, (2) the binding of heparin to rAAV is a rapid event, and (3) increasing the viral titer by a factor of 10 can reduce the inhibitory effect of heparin only at a low concentration of 0.1 U/ml.

Figure 2
figure2

Inhibition of rAAV transduction by heparin in the presence of a one-fold concentrated rAAV-stock (solid line) and a 10-fold concentrated rAAV-stock (dashed line) after a 10 min incubation time of the anticoagulant with the virus before the addition to HeLa cells. *Indicates P < 0.05.

In the presence of the LMW heparin tinzaparin, inhibition of target cell transduction was 20.2 ± 3.8% at 0.1 anti-factor-Xa U/ml and 37.1 ± 1.8% at 1.0 U/ml. Nearly complete inhibition occurred only at a high concentration of 1000 U/ml, a dose which is not used clinically. A prolongation of the incubation time (2 h versus 10 min) had no influence on the results (Figure 1b). Compared with heparin, the inhibitory properties of tinzaparin were significantly weaker at a concentration of 1.0 U/ml (P < 0.01).

Danaparoid consists of a mixture of the glycosaminoglycans heparan sulfate, dermatan sulfate and chondroitin sulfate. It is a low molecular weight heparinoid. Therapeutic concentrations of 0.1 U/ml inhibited HeLa cell transduction by 8.8 ± 3.5%, (P < 0.01) compared with heparin (Figure 1c). Full inhibition was obtained at a concentration of 1000 U/ml which is not utilized in in vivo conditions. The incubation time did not significantly alter the inhibitory effect of danaparoid (Figure 1c). The differences in the inhibitory properties between the compounds can be explained by differences in their chemical composition. Danaparoid is a mixture of heparan sulfate, dermatan sulfate and chondroitin sulfate.20 It has been demonstrated previously that dermatan sulfate and chondroitin sulfate exert only weak inhibitory effects on rAAV-mediated target cell transduction.14

Recombinant hirudin is a peptide that directly inhibits the action of coagulation factor II.21 Recombinant hirudin does not show structural similarities to heparin, LMW heparin or the heparinoids. At anti-factor-II concentrations ranging from 1.0 U/ml to 10 000 U/ml, no inhibitory effects of recombinant hirudin on rAAV-mediated transduction of HeLa cells were detected (Figure 1d). Moreover, recombinant hirudin had full anti-factor-II activity in the presence of rAAV (data not shown).

In summary, our data indicate that inhibition of rAAV transduction by heparin occurs rapidly and at therapeutic concentrations. Using increasing virus concentrations, this effect was reduced only in part and only at low heparin concentrations. Therefore, the use of heparin at the time of rAAV-mediated gene transfer may be a problem for cardiovascular gene therapy. Inhibitory effects of LMW heparins and heparinoids were weaker compared to standard heparin. Since these data are derived from in vitro studies, further in vivo studies in animal models are necessary before extrapolating these results to in vivo cardiovascular gene therapy in humans. Finally, recombinant hirudin might be the most suitable anticoagulant during rAAV-mediated in vivo cardiovascular gene transfer, because it did not interfere with rAAV transduction.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft grant number RE 1424/1–1 to HR and MH, by the Deutsche Forschungsgemeinschaft grant number SFB 455 and Bayerische Forschungsstiftung grant number VV5 to MH and by Thiemann Arzneimittel, Germany. We thank Dr RJ Samulski for providing the plasmid pXX6, Dr S King for most helpful discussions, and Karin Messerer for excellent technical assistance.

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Correspondence to M Hallek.

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Hacker, U., Gerner, F., Büning, H. et al. Standard heparin, low molecular weight heparin, low molecular weight heparinoid, and recombinant hirudin differ in their ability to inhibit transduction by recombinant adeno-associated virus type 2 vectors. Gene Ther 8, 966–968 (2001) doi:10.1038/sj.gt.3301466

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Keywords

  • adeno-associated virus type 2
  • anticoagulants
  • heparin
  • gene transfer
  • inhibition

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