AAV-mediated expression of an ADAMTS13 variant prevents shigatoxin-induced thrombotic thrombocytopenic purpura

The metalloproteinase ADAMTS13 cleaves ultralarge von Willebrand factor (UL-VWF) multimers on endothelial cell surfaces, in blood, and in growing thrombi; this process is essential for normal hemostasis.¹  Inhibition or deficiency of plasma ADAMTS13 activity causes thrombotic thrombocytopenic purpura (TTP),²  a syndrome characterized by thrombocytopenia and microangiopathic hemolytic anemia.^3,4  Some patients have end-organ damage including neurologic signs and symptoms and renal failure.^3,4  As much as 5% of cases of TTP are hereditary, caused by mutations in the ADAMTS13 gene^5,6  that result in deficiency of plasma ADAMTS13 activity. The current treatment of hereditary TTP is plasma infusion to maintain trough levels of ADAMTS13 activity >5% of normal values.⁷  However, plasma therapy is inconvenient, can result in complications such as allergic reactions, and carries the potential risk for blood-borne infections.⁸ 

Gene transfer may provide a superior alternative to plasma therapy. The therapeutic levels of ADAMTS13 activity have been reported in animal models after gene transfer with lentivirus⁹  and adenovirus¹⁰  vectors, as well as after transplantation of hematopoietic progenitor cells after lentiviral transduction.¹¹  However, approaches based on these vectors are limited by safety concerns. Recent clinical studies using adeno-associated virus (AAV)-based gene delivery vectors have documented safety and long-term expression of therapeutic transgenes for two human genetic diseases.^12,13  In this study, we report the safety and efficacy of AAV8-mediated expression of a C-terminal truncated ADAMTS13 variant in a murine model of hereditary TTP, providing proof-of-concept to support further development of an AAV8-based clinical gene therapy approach for hereditary TTP.

The animal protocol was approved by IACUC of the Children’s Hospital of Philadelphia.

Recombinant AAV8 encoding a murine ADAMTS13 variant (aa 1-2055) (AAV8-hAAT-mdtcs) or a β-galactosidase (lacZ) (AAV8-hAAT-lacZ) under a human α-1 antitrypsin promoter (hAAT) was prepared according to the method described previously.¹⁴  Purified vector was dialyzed in phosphate-buffered saline (PBS) and then supplemented with Lutrol F68 to a final concentration of 0.001%. The purified vector was quantified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with Coomassie blue staining, comparing VP1, VP2, and VP3 staining intensity by scanning densitometry with an established AAV reference vector. The titer was further confirmed by UV spectrophotometry.¹⁵ 

Adamts13^−/− mice (CAST/Ei) aged 3 to 4 weeks were injected via a tail vein with AAV8-hAAT-mdtcs (2.6 × 10¹⁰, 2.6 × 10¹¹, and 1.3 × 10¹² vg/kg) or with AAV8-hAAT-lacZ (1.3 × 10¹² vg/kg) as a control. Five mice in each group were injected. Blood (100 μL) was collected from the retinal orbital sinus perplex and anticoagulated with 0.19% sodium citrate before vector administration and at 2, 4, 6, 8, 12, 16, and 20 weeks after injection. Plasma was prepared after centrifugation for 10 minutes at 10 000 rpm and stored in aliquots at −80°C.

Total RNA was isolated from tissues using Trizol (Invitrogen). The mRNA was converted to cDNA by reverse transcription, followed by polymerase chain reaction amplification of the mdtcs fragment (0.25 kb) using a primer pair (forward: 5′-AGGAATCCTCACTGGTGC-CATCTT-3′ and reverse: 5′-TAGTGAGCACGTAGCGTTC-TGTGT-3′) and the β-actin (0.54 kb) using a primer pair (forward: 5′-TGT TAC CAA CTG GGA CGA CAT CG-3′ and reverse: 5′-TCG GAA CCG CTC GTT GCC-3′).

Plasma ADAMTS13 activity was determined by the cleavage of a murine-specific rFRTES-mVWF73, as previously described.¹¹  Normal plasma pooled from 10 wild-type mice was used for calibration (1 U/mL).

ADAMTS13 antigen was quantified by a murine-specific enzyme-linked immunosorbent assay (ELISA), as described previously, with modifications.^9,16  A rabbit IgG against a murine ADAMTS13-MDTCS fragment (Open Biosystem, Huntsville AL), unlabeled and biotinylated, was used for antigen capture and detection. Purified murine MDTCS fragment was used for calibration.

VWF multimers were determined by the method described previously.^9,11 

Two weeks after AAV vector administration, Adamts13^−/− mice (CAST/Ei) were injected via tail vein with 20 pg/g of Shigatoxin-2 (Stx2) (Toxin Technology, Sarasota, FL). The effective dosage was determined by a pilot experiment as the minimal dose of Stx2 necessary to consistently induce thrombocytopenia (>30% drop of platelet count from the baseline) in Adamts13^−/− mice (data not shown). Blood (50 µL) was collected through the retinal orbital sinus perplex before and daily for 6 days after Stx2 injection. Complete blood count was performed using a Hemavet M2950HV analyzer (Drew Scientific, Waterbury, CT).

Blood smears were stained with Wright stain. The tissues were embedded in paraffin, sectioned, and stained with hematoxylin-eosin or immune-peroxidase as described previously.^17,18 

Plasma alanine aminotransferase levels were determined by a colorimetric assay (Teco Diagnostics, Anaheim, CA).

IgGs against transduced ADAMTS13 variant were determined by ELISA using a purified recombinant mdtcs (2 µg/mL) for capture and a peroxidase-conjugated rabbit anti-mouse IgG (1:5000) (Dako) for detection, respectively.

To date, plasma infusion is the only effective prophylaxis or treatment available for hereditary TTP. Here, we report success in correction of a murine model of hereditary TTP by a single intravenous administration of an AAV8 vector encoding a C-terminal truncated murine ADAMTS13 variant (mdtcs) under the control of a liver-specific promoter. AAV8 vectors were produced and highly purified (Figure 1A-B). After intravenous infusion of vector, liver-specific expression of the transgene product was demonstrated by reverse transcriptase–polymerase chain reaction (Figure 1C) and immunohistochemistry (Figure 1D). Control mice that were treated with PBS or AAV8-hAAT-lacZ were negative for ADAMTS13 expression (Figure 1C,E). Plasma ADAMTS13 activity and antigen increased with vector dosage, reaching a comparable plateau of activity (∼0.7 U/mL) (Figure 2A) and antigen level (∼0.8 μg/mL) (Figure 2B) after the medium to high doses. The lack of a dose response at the 2 higher doses suggests possible saturation of vector uptake and/or transgene synthetic capacity by the transduced hepatocytes. The AAV8 doses (>2.6 × 10¹¹ vg/kg) required to achieve therapeutic levels of plasma ADAMTS13 activity and antigen in this study are comparable with those reported for hemophilia B in animal models^19,20  and in patients.¹³ 

The efficacy of the ADAMTS13 variant expressed in this animal model was evaluated by the reduction in the size of plasma VWF multimers and protection against shigatoxin-induced “TTP-like” syndrome. Previous studies have shown that Adamts13^−/− mice are prothrombotic with increased formation of “string-like” UL-VWF polymers on the endothelial cell surface after stimulation^21,22  and increased ratios of high-molecular-weight to low-molecular-weight VWF multimers in circulating blood,^9,11,23  which contribute to the accelerated rate of thrombus formation after oxidative injury.^16,23,24  An infusion or expression of human (or murine) full-length ADAMTS13 or the C-terminal truncated variants via a lentiviral vector or transplantation of reconstituted hematopoietic progenitor cells in these mice can correct prothrombotic phenotypes.^{9,11,16,21,22}  In this study, we demonstrate that AAV8-mediated expression of a murine MDTCS fragment at vector doses ≥2.6 × 10¹¹ vg/kg is sufficient to markedly reduce circulating UL-VWF or large VWF, thereby reducing ratios of high to low molecular weight multimers in plasma (supplemental Figure 1). When challenged with Stx2, known to trigger “TTP-like” syndrome in Adamts13^−/− CAST/Ei mice,¹⁷  a significant drop (40%-60%) in platelet counts was observed after 24 to 48 hours (Figure 2C). This severe thrombocytopenia was not detected in Adamts13^−/− mice that received AAV8-hAAT-mdtcs at doses of 2.6 × 10¹¹ vg/kg (Figure 2D) or 1.3 × 10¹² vg/kg (Figure 2E) 2 weeks before Stx2 challenge. All mice (5/5) in the Adamts13^−/− cohort without vector treatment died within 3 days, but only 1 mouse (1/5) within each of the two vector-treated cohorts died (mortality rate, 20%) (P < .001) (Figure 2G).

Blood smears and immunohistochemistry revealed the presence of red blood cell fragmentation and VWF-enriched microthrombi in the heart, kidneys, brain, and pancreas in Stx2-challenged Adamts13^−/− mice pretreated with PBS or AAV8-LacZ control vector, but these pathological changes were not observed in mice pretreated with AAV8-hAAT-mdtcs or wild-type mice (data not shown). These findings are consistent with the induction of a “TTP-like” syndrome in Adamts13^−/− mice not receiving AAV8-hAAT-mdtcs that was prevented in mice that received therapeutic doses of the vector. Neither elevation in plasma alanine aminotransferase (supplemental Figure 2) nor antibodies against the murine ADAMTS13 variant (supplemental Figure 3) were detected, even at 10-fold higher vector doses, supporting that expression in liver and secretion of the truncated metalloprotease is safe.

Together, these results demonstrate that AAV8-mediated liver expression of an ADAMTS13 variant is safe and efficacious in correcting prothrombotic and TTP phenotypes in mice. Our findings provide proof-of-concept supporting the development of an AAV-based gene transfer approach for treatment of hereditary TTP in humans.

The authors thank Dr David Ginsburg at the University of Michigan, Ann Arbor, for providing us with the Adamts13^−/− mice.

This study was supported by grants from the American Heart Association–National Established Investigator Award (AHA-EIA 0940100N) and the National Institutes of Health (HL-074124, project 3) (X.L.Z.).

Contribution: S.-Y.J., J.X., J.B., J.F.W., and X.L.Z. designed the research, performed the experiments, analyzed the data, and wrote the manuscript; and S.Z. performed experiments and revised manuscript. All authors reviewed and approved the final version of the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

The current affiliation for S.-Y.J. is the Department of Hematology, Affiliated Hospital of Yanbian University, Yanji, China. The current affiliation for J.X. is the Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Correspondence: X. Long Zheng, Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, 34th Street and Civic Center Blvd, 816G Abramson Research Center, Philadelphia, PA 19104; e-mail: zheng@email.chop.edu.