Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques

Abstract

The feasibility, safety, and efficacy of liverdirected gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models. A dose of 4 ؋ 10 12 vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. Persistence of the rAAV vgs as circular monomers and dimers and high-molecularweight concatamers was documented in liver tissue by Southern blot analysis for periods of up to 1 year. Vector particles were present in plasma, urine, or saliva for several days after infusion (as shown by polymerase chain reaction analysis), and the vgs were detected in spleen tissue at low copy numbers. An enzyme-linked immunosorption assay capable of detecting between 1% and 25% of normal levels of hFIX in rhesus plasma was developed by using hyperimmune serum from a rhesus monkey that had received an adenoviral vector encoding hFIX. Two macaques having 3 and 40 rAAV genome equivalents/cell, respectively, in liver tissue had 4% and 8% of normal physiologic plasma levels of hFIX, respectively. A level of hFIX that was 3% of normal levels was transiently detected in one other macaque, which had a genome copy number of 25 before abrogation by a neutralizing antibody (inhibitor) to hFIX. This nonhuman-primate model will be useful in further evaluation and development of rAAV vectors for gene therapy of hemophilia B. IntroductionRecombinant adeno-associated viral vectors (rAAVs) hold great promise for the treatment of hemophilia B. Preclinical studies in murine and canine models of hemophilia B have demonstrated persistent therapeutic expression of factor IX (FIX) leading to correction of the bleeding phenotype after intramuscular or portal vein administration of rAAV. 1-7 Liver-targeted delivery of rAAV resulted in significantly higher levels of FIX than observed after intramuscular administration of an equivalent dose of vector in immunodeficient mice. 8 More important, the development of neutralizing antibodies (NAbs) to human FIX (hFIX) is more common after intramuscular injection of rAAV than after intraportal administration of the same dose of vector. [8][9][10][11] Mice with persistent hFIX expression after liver-targeted delivery of rAAV could not mount a substantial antibody (Ab) response when challenged repeatedly with hFIX antigen in Freund adjuvant, a result suggesting that the liver has a unique ability to induce immune tolerance to neoantigens. 12 Endothelial cells of the liver, which can efficiently process and cross-present exogenous soluble antigens to CD8 ϩ T cells, are intimately involved in antigenspecific immune tolerance after liver-targeted gene transfer with rAAV vectors. 13 The findings of sustained expression of hFIX at therapeutic levels after a single bolus injection of rAAV vector into the portal vein of mice [3][4][5][6]8 and the absence of toxicity aft...

Immunity to adeno-associated virus vectors in animals and humans: a continued challenge

“…13,47,79,80 Consequently, with these promising preclinical data in mind, the first liver-directed gene therapy phase I/II trial in humans was initiated involving delivery of human F.IX for the treatment of hemophilia. 64 An AAV2 vector expressing human F.IX under the control of a liver-specific promoter was infused through the hepatic artery.…”

Intravenous administration of an AAV-2 vector for the expression of factor IX in mice and a dog model of hemophilia B

“…Previous studies have also shown the induction of a strong anti-AAV response following AAV administration to mice, dogs and monkeys, suggesting that readministration of AAV serotype 2 vectors may not be possible 3,8,30 by any route of administration. The use of alternative serotypes that are immunologically distinct to AAV-2, such as AAV-8, may allow repeat administration of a vector for increased therapeutic effect.…”

“…7 Hepatic portal vein (HPV) delivery of rAAV FIX vectors in mice, non-human primates and hemophilic B dogs is well tolerated without significant increases in markers of toxicity, no induction of anti-FIX inhibitors or nonneutralizing antibodies. 2,5,6,8 Although vector could be detected in the circulation immediately following administration, there has been no evidence of germline transmission. In murine models of hemophilia B, rAAV gene transfer of human FIX corrected the clotting deficiency as determined by functional correction of the bleeding disorder.…”

Long-term correction of hyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice

“…4,5 On the other hand, adeno-associated virus (AAV) vectors comprise another class of gene delivery vehicles, which have been shown to stably transduce nondividing cells such as hepatocytes, muscle fibers and neurons. [6][7][8] In this study, we evaluated a recombinant AAV vector carrying the PAH gene in a mouse model of PKU (PAH enu2 strain). [9][10][11] A missense mutation (F263S) in the PAH gene was introduced into BTBR mouse strain by chemical mutagenesis, resulting in a loss of enzyme activity.…”