Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep

Abstract

A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens.

Fig 1. Experimental scheme for assessment of induction of postnatal tolerance by IUGT.

IM, intramuscular; IV, intravenous; Luc, luciferase.

Fig 2. Assessment of GFP transgene specific antibodies at 1 month following IUGT.

Serum from fetal recipients of IUGT was assessed at 1 month post injection for the presence of GFP specific antibodies by ELISA. Positive control = serum from a neonatal lamb three weeks after receiving an intramuscular injection of AAV1.GFP; Non-injected control = serum from an aged matched fetus that did not undergo IUGT; AAV9 pre-IUGT = serum from the fetal recipient of AAV9 prior to IUGT; AAV8 (A) and AAV8 (B) refer to samples from 2 different fetuses injected with AAV8.GFP.

Fig 3. Lack of a postnatal humoral immune response to the GFP transgene product following IUGT.

Fetal recipients of AAV8.GFP or AAV6.2.GFP underwent postnatal IM challenge with AAV1.GFP. Serum anti-GFP antibodies prior to challenge and weekly for 4 weeks post challenge were assessed by ELISA. Control animals received the postnatal challenge without IUGT. The control animal generated a robust humoral (IgG) immune response to GFP (I). In contrast, recipients of IUGT with AAV8.GFP (A) or AAV6.2.GFP (E) did not generate anti-GFP antibodies. Results from 3 weeks post challenge are shown. Similar results were seen on analysis at 1, 2 and 4 weeks (data not shown). At 6 months of age, GFP expression was assessed at the site of postnatal challenge in the hindlimb muscle; AAV8 (B,C,D), AAV6.2 (F,G,H) and control (J,K,L). All animals demonstrated GFP expression which was associated with a significant inflammatory cell influx in the control but not the recipients of IUGT. IHC, immunohistochemistry; H&E, hematoxylin and eosin.

Fig 4. IUGT does not induce humoral immune tolerance to the viral vector capsid proteins.

Fetal recipients of AAV8.GFP (A) or AAV6.2.GFP (B) underwent postnatal challenge with AAV8.Luc or AAV6.2.Luc respectively at 3 months of age. The presence of IgG anti-AAV capsid protein antibodies in the serum was assessed weekly for 4 weeks post challenge. An anti-AAV capsid protein antibody response elicited at 3 weeks post challenge is demonstrated. Similar results were seen at 1, 2 and 4 weeks post challenge (data not shown).

Fig 5. Long-term hepatic GFP expression in lambs undergoing IUGT.

Hepatic expression of the GFP transgene was assessed serially in lambs undergoing IUGT with either AAV8.GFP or AAV6.2.GFP. Persistent, but decreased, expression at 6 months of age was seen in the recipient of AAV8 (1 month A,B,C; 6 months D,E). The recipient of AAV6.2 lost hepatic expression from 1 to 6 months of age (1 month F,G,H; 6 months I,J). IHC; immunohistochemistry.