In Utero Transfer of Adeno-Associated Viral Vectors Produces Long-Term Factor IX Levels in a Cynomolgus Macaque Model

Abstract

The safe correction of an inherited bleeding disorder in utero prior to the onset of organ damage is highly desirable. Here, we report long-term transgene expression over more than 6 years without toxicity following a single intrauterine gene transfer (IUGT) at 0.9G using recombinant adeno-associated vector (AAV)-human factor IX (hFIX) in the non-human primate model we have previously described. Four of six treated animals monitored for around 74 months expressed hFIX at therapeutic levels (3.9%–120.0%). Long-term expression was 6-fold higher in males and with AAV8 compared to AAV5, mediated almost completely at this stage by random genome-wide hepatic proviral integrations, with no evidence of hotspots. Post-natal AAV challenge without immunosuppression was evaluated in two animals exhibiting chronic low transgene expression. The brief neutralizing immune reaction elicited had no adverse effect and, although expression was not improved at the dose administered, no clinical toxicity was observed. This long-term surveillance thus confirms the safety of late-gestation AAV-hFIX transfer and demonstrates that postnatal re-administration can be performed without immunosuppression, although it requires dose optimization for the desired expression. Nevertheless, eventual vector genotoxicity and the possibility of germline transmission will require lifelong monitoring and further evaluation of the reproductive function of treated animals.

Fetal gene therapy for neurodegenerative lysosomal storage diseases

“…Liver-directed fetal gene therapy has previously demonstrated long-term expression of transgenic protein up to 6 years after a single injection in non-human primates with adeno-associated viral (AAV) vectors. 5 Theoretical advantages include target of progenitor or stem cells allowing similar effect at reduced dose for integrating vectors 6 and immunological immaturity limiting the risk of immune response against the vector or transgenic protein, which could allow vector re-injection if required. 5 Despite critical unmet needs, there is scant literature describing fetal administration of advanced therapies in animal models of neurodegenerative lysosomal storage diseases.…”

Therapeutic expression of human clotting factors IX and × following adeno‐associated viral vector‐mediated intrauterine gene transfer in early‐gestation fetal macaques

“…It is, however, critical to interrogate organ- and genotoxicity in addition to protein deficiency correction in GT applications—issues that have arisen in the successful gene transfer of young children with severe congenital immunodeficiency and spinal muscular atrophy (7, 32). Most intrauterine gene transfer (IUGT) animal models do not demonstrate the degree of transgenic protein production the we have described in nonhuman primates (NHPs), here and previously (29, 33). Murine IUGT with AAV1-human factor IV (hFIX) or AAV2-hFIX at 10 12 –10 13 vector genomes (vgs)/kg showed persistent low transgene levels, requiring AAV readministration for therapeutic expression (31).…”

“…These animal studies demonstrate the progressive loss of expression expected after fetal or neonatal treatment as the recipient grows (43). Introduction of GT vectors during fetal development to induce immune tolerance and facilitate repeat AAV administration after birth (33, 44–46) can be a useful strategy for stabilizing waning protein expression in the hemophilias and neurometabolic disorders (11, 47). Despite the encouraging outcomes of clinical trials, high-dose AAV has resulted in complications associated with integration, direct cellular toxicity, and systemic inflammation, resulting in hepatocellular carcinoma in rodents, neurodegeneration in piglets and liver failure in juvenile NHPs (48, 49).…”

“…Data regarding AAV-IUGT safety and efficacy can thus originate only from a high-fidelity NHP model in which longitudinal surveillance is clinically relevant (50). We have described long-term outcomes of liver-directed late-gestation IUGT in NHP by using the clinical vector scAAV-LP1-human codon-optimized human FIX transgene (FIXco) (5) in which a single injection at 0.9 gestation demonstrated therapeutic hFIX expression in most animals (29, 33). Low-expressing animals were safely boosted with only transient immune responses (33).…”

Rare Opportunities: CRISPR/Cas-Based Therapy Development for Rare Genetic Diseases

“…The risk of inadvertent germline transmission and the inability to predict possible success in human application from experimental models are concerns en route to clinical translation, but the landmark drop in required vector amounts and treatment cost, and, more fundamentally, the applicability to early-onset and prenatally lethal disorders, would be another game changer for gene therapy. Ongoing research into possible complications of treatment [ 101 ], improvements to efficiency [ 102 ], and successful tests in non-human primates, such as those for treatments of hemophilia B [ 103 ] and Gaucher disease [ 104 ], may help overcome bioethical and regulatory hurdles in implementing the technique for rare diseases.…”