Stable Human FIX Expression After 0.9G Intrauterine Gene Transfer of Self-complementary Adeno-associated Viral Vector 5 and 8 in Macaques

Abstract

Intrauterine gene transfer (IUGT) offers ontological advantages including immune naiveté mediating tolerance to the vector and transgenic products, and effecting a cure before development of irreversible pathology. Despite proof-of-principle in rodent models, expression efficacy with a therapeutic transgene has yet to be demonstrated in a preclinical nonhuman primate (NHP) model. We aimed to determine the efficacy of human Factor IX (hFIX) expression after adeno-associated-viral (AAV)-mediated IUGT in NHP. We injected 1.0-1.95 × 10(13) vector genomes (vg)/kg of self-complementary (sc) AAV5 and 8 with a LP1-driven hFIX transgene intravenously in 0.9G late gestation NHP fetuses, leading to widespread transduction with liver tropism. Liver-specific hFIX expression was stably maintained between 8 and 112% of normal activity in injected offspring followed up for 2-22 months. AAV8 induced higher hFIX expression (P = 0.005) and milder immune response than AAV5. Random hepatocellular integration was found with no hotspots. Transplacental spread led to low-level maternal tissue transduction, without evidence of immunotoxicity or germline transduction in maternal oocytes. A single intravenous injection of scAAV-LP1-hFIXco to NHP fetuses in late-gestation produced sustained clinically-relevant levels of hFIX with liver-specific expression and a non-neutralizing immune response. These data are encouraging for conditions where gene transfer has the potential to avert perinatal death and long-term irreversible sequelae.

Obstacles and future of gene therapy for hemophilia

“…There is also an ongoing concern about the potential for episomal loss due to hepatocellular proliferation. Both of these mechanisms have been suggested to account for the observed decline of FIX plasma levels over time after AAV gene therapy in utero or perinatally in both sheep and NHPs [48,49]. Integration of the vector genome into the recipient’s DNA-utilizing vectors such as lentivirus, which allows for transgene replication during cell division, may provide for sustained transgene expression during normal growth and development.…”

In vitro and In vivo Model Systems for Hemophilia A Gene Therapy

“…First, vector potency and toxicity can be determined. Similar studies have been carried out with factor IX gene delivery [84–89]. Second, transgene expression, i.e., factor VIII levels produced by the vector can be measured.…”

Liver Gene Therapy Approaches for Acute Intermittent Porphyria: Metabolic Correction and Immunological Hurdles

“…87,91 In the liver, scAAV vector result in a 10-to 20-fold higher level of transgene expression compared with a similar dose of a classical AAV vector. 87 In 2011, Mattar et al 80 reported the first unequivocal evidence of a successful gene therapy trial for hemophilia B using a self-complementary rAAV serotype 8 vector carrying the coagulation factor IX coding sequence driven by a liver-specific promoter. The liver produced factor IX, which was secreted to the blood at levels sufficient to improve the bleeding phenotype.…”

“…[76][77][78][79][80]99 Random integration of vector sequences has been demonstrated in established cell lines 100 but only in some cases and at low frequency in primary cultures and in vivo. 78,101,102 In mouse liver, integration of rAAV vector has been quantified at a frequency of less than 2%.…”

“…76 However, rAAV integration site analysis in small animal models [77][78][79] and in large animal models, such as non-human primates (NHP) or even humans, indicated a random profile of vector integration. 80 Vectors based on rAAV have attracted much attention as potent gene-delivery vehicles, 38 because of the lack of pathogenicity of the wild-type virus and the ability to promote sustained and long-term therapeutic gene expression in mice and large animals 81,82 and transduce both dividing and non-dividing cells over a broad spectrum of host species. 83 AAV elicits a low intensity innate immunity response compared to adenoviral capsids.…”