Abstract

Adeno-associated viral vectors (AAVs) achieve stable therapeutic expression without long-term toxicity in adults with hemophilia. To avert irreversible complications in congenital disorders producing early pathogenesis, safety and efficacy of AAV-intrauterine gene transfer (IUGT) requires assessment. We therefore performed IUGT of AAV5 or -8 with liver-specific promoter-1 encoding either human coagulation factors IX (hFIX) or X (hFX) into Macaca fascicularis fetuses at ∼0.4 gestation. The initial cohort received 1 × 1012 vector genomes (vgs) of AAV5-hFIX (n = 5; 0.45 × 1013 vg/kg birth weight), resulting in ∼3.0% hFIX at birth and 0.6–6.8% over 19–51 mo. The next cohort received 0.2–1 × 1013 vg boluses. AAV5-hFX animals (n = 3; 3.57 × 1013 vg/kg) expressed <1% at birth and 9.4–27.9% up to 42 mo. AAV8-hFIX recipients (n = 3; 2.56 × 1013 vg/kg) established 4.2–41.3% expression perinatally and 9.8–25.3% over 46 mo. Expression with AAV8-hFX (n = 6, 3.12 × 1013 vg/kg) increased from <1% perinatally to 9.8–13.4% >35 mo. Low expressers (<1%, n = 3) were postnatally challenged with 2 × 1011 vg/kg AAV5 resulting in 2.4–13.2% expression and demonstrating acquired tolerance. Linear amplification–mediated-PCR analysis demonstrated random integration of 57–88% of AAV sequences retrieved from hepatocytes with no events occurring in or near oncogenesis-associated genes. Thus, early-IUGT in macaques produces sustained curative expression related significantly to integrated AAV in the absence of clinical toxicity, supporting its therapeutic potential for early-onset monogenic disorders.—Chan, J. K. Y., Gil-Farina I., Johana, N., Rosales, C., Tan, Y. W., Ceiler, J., Mcintosh, J., Ogden, B., Waddington, S. N., Schmidt, M., Biswas, A., Choolani, M., Nathwani, A. C., Mattar, C. N. Z. Therapeutic expression of human clotting factors IX and X following adeno-associated viral vector–mediated intrauterine gene transfer in early-gestation fetal macaques.

Highlights

  • Clinical gene therapy (GT) trials for severe mono- adeno-associated viral vectors (AAVs)-GT achieved dosegenic diseases have demonstrated survival benefit over dependent hematologic correction, with reassuring existing therapies

  • Plasmid expression of hFX expression was assessed by ELISA after transient transfection of HepG2 cells with Fugene (Roche, Basel, Switzerland), and clones were amplified with Megaprep (Qiagen, Limberg, The Netherlands). self-complementary AAV (scAAV)-LP1-hFIXco and scAAV-LP1-hFXco plasmids were used in adenovirus-free transient transfection of 293T cells to generate scAAV5 and scAAV8 pseudotypes, as described by Nakai et al [45]

  • fetal blood sampling (FBS) was successfully performed in the e5001, which was stillborn at 144 gestational day (GD) (0.9 G; Table 1)

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Summary

Introduction

Clinical gene therapy (GT) trials for severe mono- adeno-associated viral vectors (AAVs)-GT achieved dosegenic diseases have demonstrated survival benefit over dependent hematologic correction, with reassuring existing therapies. ABBREVIATIONS: AAV, adeno-associated viral vector; ALT, alanine transaminase; ELISPOT, enzyme-linked immunospot; FBS, fetal blood sampling; FIXco, codon-optimized factor; FXco, codon-optimized human FX transgene; GD, gestational day; GT, gene therapy; ICS, intracellular staining; IS, integration site; IUGT, intrauterine gene transfer; LAM, linear amplification–mediated; LP, liver-specific promoter; MNC, mononuclear cell; NAb, neutralized antibody; NHP, nonhuman primate; scAAV, self-complementary AAV; US, ultrasound; VC, vector challenge; VCN, vector copy number; vg, vector genome. We report unexpectedly prolonged and stable postnatal transgenic protein production with only transient transaminitis, a high level of vector integration with no hotspots, and evidence of immune tolerance

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