Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy

Abstract

In a clinical study of recombinant adeno-associated virus-2 expressing human factor IX (AAV2-FIX), we detected 2 impediments to long-term gene transfer. First, preexisting anti-AAV neutralizing antibodies (NABs) prevent vector from reaching the target tissue, and second, CD8(+) T-cell responses to hepatocyte-cell surface displayed AAV-capsid-terminated FIX expression after several weeks. Because the vector is incapable of synthesizing viral proteins, a short course of immunosuppression, until AAV capsid is cleared from the transduced cells, may mitigate the host T-cell response, allowing long-term expression of FIX. To evaluate coad-ministration of immunosuppression, we studied AAV8 vector infusion in rhesus macaques, natural hosts for AAV8. We administered AAV8-FIX in 16 macaques via the hepatic artery and assessed the effects of (1) preexisting anti-AAV8 NABs, (2) a standard T-cell immunosuppressive regimen, and (3) efficacy and safety of AAV8-FIX. We found that low titers (1:5) of preexisting NABs abrogate transduction, whereas animals with undetectable NABs are safely and effectively transduced by AAV8-FIX. Coadministration of mycophenolate mofetil and tacrolimus with vector does not induce toxicity and does not impair AAV transduction or FIX synthesis. These findings enable a clinical study to assess the effects of immunomodulation on long-term FIX expression in patients with hemophilia B.

Figure 1.

Therapeutic levels of circulating hFIX achieved in AAV8-naive mice and rhesus monkeys. Levels of hFIX were determined by ELISA and presented on the left y-axis, and the corresponding percentage of normal FIX levels (5000 ng/mL) is presented on the right y-axis. (A) AAV8-naive monkeys received 5 × 10¹² vg/kg AAV8-hFIX vectors via direct intrahepatic artery injection. (B) C57Bl/6 mice received 1 × 10¹¹ vg/mouse (ie, 4 × 10¹² vg/kg) AAV8-or AAV2-hFIX by direct intraportal vein injection. Results presented here are means ± SEM from all data collected from 3 independent experiments comparing 3 lots of AAV8 and 2 lots of AAV2 vectors. Each time point consists of a minimum of 3 mice or a maximum of 21 mice.

Figure 2.

Determination of hFIX gene transfer efficiency and distribution in liver. Monkeys that received 5 × 10¹² vg/kg vector were biopsied at week 4 and humanely killed at week 25. (A) DNA FISH for hFIX (i and ii), and immunofluorescent staining for AAV-8 capsid protein (iii and iv) were performed on liver biopsies from an AAV8-injected animal (R3, i and iii) and from a noninjected control animal (R21, ii and iv). The detecting probe in DNA FISH and the detecting antibody in IF were labeled with Alexa fluor 488, so that positive signals appear as green punctuated dots (some identified by arrows in i and iii). Blue signal represents nuclei counterstained with DAPI (iii and iv). (B) Southern blotting on gDNAs isolated from individual lobes of rhesus liver collected at necropsy. LL indicates left lateral lobe; LM, left medial lobe; Q, quadrate lobe; RL, right lateral lobe; and RM, right medial lobe. Standards were generated with BamHI/BglII-digested plasmid DNA pAAV-hFIX spiked in pooled liver gDNAs from naive macaques at specified quantities. (C) Quantification of the copy numbers of transgene DNA per diploid genome in individual lobes of liver in each monkey as determined by Southern blotting analysis. Individual subjects, their preexisting AAV8 neutralizing titers, and the doses of AAV8-hFIX vector administered are as labeled.

Figure 3.

Monitoring levels of serum ALT in monkeys that received 5 × 10¹² vg/kg of vector. Normal high and low are means from a normal reference range established at the study facility. Values from noninjected control monkeys (R20-R24) from the same colony are also shown.

Figure 4.

Coadministration of AAV8-hFIX and immunosuppressants was well tolerated in rhesus macaques. Six AAV8-naive macaques were assigned to 2 groups. The No IS group consisted of R11, R12, and R13 and received 2 × 10¹³ vg/kg AAV8-hFIX16. The IS group consisted of R14, R15, and R16 and received the same dose of the vector in combination with MMF and tacrolimus. (A) Serum ALT levels. Dotted lines represent the normal reference range at the study facility. (B) Levels of hFIX detected in macaque plasma by ELISA (left y-axis) and their corresponding percentage of normal FIX levels (5000 ng/mL; right y-axis). Dotted line indicates when immunomodulatory therapy was stopped. (C) The relative levels of anti-AAV8 IgG detected in rhesus macaque plasma by ELISA before and after AAV8-hFIX infusion. Dotted line indicates when immunomodulatory therapy was stopped.