Preclinical Development of an AAV8-hUGT1A1 Vector for the Treatment of Crigler-Najjar Syndrome

Abstract

Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed an (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc)AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo. Conversely, (ss)AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their (sc) counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss)AAV8-hUGT1A1 based on the triple transfection of HEK293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology-biodistribution study were also conducted using large-scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss)AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome.

Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases

“…The CNSI mice closely mimic the main features of the human syndrome, with neonatal severe unconjugated hyperbilirubinemia and early neonatal lethality (29,30,38). Although nonintegrative AAV-mediated gene therapy is very effective in rescuing the phenotype in adult CNSI animals (39,40), it requires high doses of liver-specific rAAV8 episomal vectors expressing the UGT1A1 transgene when administered to newborn mutant mice (13,41,42). However, therapeutic efficacy decreases over time and a second administration is necessary to achieve full correction in the long term (41,42).…”

“…or i.v. injected as described previously (30,39,40), with the indicated vectors, at the indicated dose, as described in Supplemental Table 5. Briefly, for EGFP editing experiments, WT mice were i.v.…”

Liver-directed gene therapy results in long-term correction of progressive familial intrahepatic cholestasis type 3 in mice

“…Liver-directed gene therapy using AAV vectors has proven to be safe and effective for the treatment of hemophilia B 14 and has great potential for the treatment of other inherited disorders as long as the liver tissue itself is not hyperproliferative. [15][16][17] It is well established that AAV-mediated gene transfer results in stable correction of quiescent tissue and that cell proliferation results in loss of the episomal transgene. [19][20][21] This fuels the paradigm that AAVmediated gene therapy in disorders that result in liver damage and subsequent hyperproliferation, such as PFIC3, can only lead to transient correction.…”

“…AAV vectors were produced using an adenovirus-free transient transfection method as previously described. 15,29 In brief, adherent human embryonic kidney cells (HEK293T) were transfected with AAV2 Rep and AAV8 Cap (pDP8.ape, Plasmid Factory, Bielefeld, Germany) and the ITRflanked transgene expression cassette. After 72 h of transfection cells were harvested, lysed by 2 freeze-thaw cycles and treated with DNAse, RNAse (Roche, Basel, Switzerland) and benzonase (Merck, Darmstadt, Germany).…”

“…AAV-mediated transfer of a functional copy of the clotting factor IX gene into hepatocytes has shown to be safe and provided a long-term reduction of bleeding episodes in patients with hemophilia B. 14 This approach is currently under clinical investigation for other indications such as Crigler-Najjar syndrome (NCT03466463), 15 Mucopolysaccharidosis type VI (NCT03173521) 16 and Pompe disease (NCT03533673). 17 The AAV system is established as the vector of choice because of its superior safety profile and nonintegrative nature.…”