Corticosteroids (CCS; methylprednisolone, prednisolone and prodrug prednisone) bind to glucocorticoid receptors modifying diverse downstream transcriptional signaling. This includes annex I, MAPK phosphatase 1, and NF-κB resulting in anti-inflammatory and immunosuppressive properties (63). They have broad inhibitory effects on both innate and adaptive immune cells by reducing pro-inflammatory cytokine and chemokines, T- and to a lesser extent, B-cells production (64). CCS are used short-term in conjunction with systemically delivered gene therapies to negate transaminitis and associated CTL-induced injury transgene loss (30, 65), and reduce T-cell infiltrates in muscular fibers in non-human primates (NHP) (33). They are also adopted in approved gene therapies for inherited retinal dystrophy (25) and spinal muscle atrophy (SMA) (26).

Subsequently increasing doses of systemic rAAV have been delivered in preclinical and clinical studies with significant hepatic sequelae. High dose intravenous AAV9 (2×10¹⁴ vector genomes (vg)/kg) in NHP resulted in marked transaminitis and acute liver failure (66), posing concerns over dosage related hepatotoxicity (67). Furthermore, clinical phase II trial for X-linked myotubular myopathy delivered intravenous rAAV8.AT132 (NCT03199469) 3×10¹⁴vg/kg in high dosage group, with 16-weeks of prednisolone commencing 1 day prior to dosing. Three patients with pre-existing intrahepatic cholestasis (68) experienced severe hepatobiliary complications culminating in death. The exact mechanisms of the hepatotoxicity remain to be elucidated. These studies however build evidence that short-course CCS alone is likely to be insufficient to inhibit formation of capsid-reactive T cells (13) and rAAV-mediated immune response with systemic high dosages. Therefore, the addition of other immunosuppressive agents maybe beneficial. In a AAVrh10-microRNA study delivering 4.2×10¹⁴ vg intrathecally into two adult patients, the first developed meningoradiculitis after intrathecal infusion despite corticosteroids (IV methylprednisolone on day 0 and oral prednisone tapered over 4 weeks). In the second patient, the addition of rituximab and rapamycin to the regimen resulted in a lower increase of NAb and T-cell response (29) and these drugs are further discussed.

Rapamycin is a macrolide immunosuppressant that binds to the same intracellular target (immunophilin) as tacrolimus; however, rapamycin/FKPB12 complex inhibits a crucial cell-cycle kinase known as mammalian target of rapamycin (mTOR). Beneficial downstream effects include Treg generation, suppressing CTL and T helper (T_H) activation and at higher doses, B-cell proliferation and differentiation (69–71).

Rapamycin has beneficial effects on circumventing existing antibodies and studied in current hemophilia gene therapy trials. Hemophilia patients develop inhibitors (antibodies) to clotting factor replacement and another cause for exclusion in gene therapy trials. In a murine hemophilia A model, rapamycin (4mg/kg three times a week) was given in addition to B-cell depleting anti-CD20 antibodies to suppress T_H and Treg response suppressing inhibitor development (37). Intraperitoneal prednisolone with rapamycin was shown to inhibit B-cell activation in murine spleen and bone marrow, reducing pre-existing anti-capsid immunoglobulin G (IgG) by up to 93% after 8 weeks (72). Additionally, co-administrating AAV vectors with rapamycin encapsulated in synthetic vaccine particles (SVP[Rapa]) enabled re-dosing of AAV8 at 4 × 10¹²vg/kg in mice and NHP (39). SVP [Rapa] provided sufficient reduction of B and T cell activation in an antigen-selective manner, inhibited CTL liver infiltration, and efficiently blocked memory T cell response. Potential of intramuscular rAAV9 re-administration is currently investigated for Pompe disease (NCT02240407) (73), by attenuating T and B cell response with rapamycin and rituximab respectively. Preliminary results were successful in preventing formation of anti-capsid and anti-transgene antibodies (38), with aims to enable rAAV re-administration and maintain effectiveness in different underlying mutations.

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme for guanosine nucleotide synthesis, and type II IMPDH is upregulated in activated lymphocytes. Mycophenolate mofetil (MMF), prodrug of mycophenolic acid, preferentially inhibits type II IMPDH, suppressing T and B cells proliferation (74). In mice MMF reduced rAAV transduction efficiency by depleting guanosine triphosphate required for vector genome second strand synthesis (75), but this was not observed in higher animals. No difference in AAV8-hFIX transgene expression was observed when administered with tacrolimus in NHP (6), highlighting the difficulties of recapitulating human immune system in mouse models.

Ciclosporin and tacrolimus are immunosuppressants that inhibit calcineurin, a key signaling phosphatase, by binding to their respective immunophilins - cyclophilin and FKBP12 (76). A major downstream effect is suppression of interleukin (IL)-2 transcription, thereby inhibiting T cells differentiation, survival, and subsequent antibody production and CTL activities via effector T_H cells. Daily systemic administration of tacrolimus (0.06mg/kg/day) has been shown to prolong rAAV8 and rAAV9 expression in NHP skeletal muscle, up to 42 weeks from 8 and 16 weeks respectively (47). No generalized toxicity was reported but T-cell and macrophages infiltrations were observed.

The first approved gene therapy in Europe, alipogene tiparvovec (Glybera), incorporated 12-week immunosuppression regimen with ciclosporin (3mg/kg/day) and MMF (2g/day) (32). In the initial regimen, 9/14 subjects showed humoral and cellular response against rAAV1 (77). Subsequent study (AMT-011-02) modified the regimen to commence ciclosporin and MMF from day -3 with additional methylprednisolone on day 0 resulting in transient cellular responses without clinical sequalae (78).

Ciclosporin and tacrolimus were found to inhibit Treg proliferation and activity in vitro (79), and similar effects were observed in tacrolimus-treated allograft patients ex vivo (80); this could be detrimental in inhibiting the development of peripheral tolerance following rAAV administration. However, preclinical delivery of ciclosporin and non-depleting CD4 receptor antibody (NDCD4) have been shown to induce antigen-specific Treg, enabling AAV intravenous re-administration after 3 months (48).

Rituximab (RTX) is a chimeric mouse/human monoclonal antibody targeting CD20 present in pre‐B and mature B cells except plasma cells. It depletes B cells by inducing apoptosis, antibody dependent cell-mediated cytotoxicity and complement dependent cytotoxicity, thereby limiting antibody production and epitope presentation via MHC class II to T_H cells (81).

A preclinical model for hemophilia B showed RTX with ciclosporin dampened NAb response to human FIX and capsid without affecting Treg (51). As ciclosporin inhibits T_H cell, this further improves B-cell inhibition profile. Variable responses have been observed in RTX’s effect on reducing pre-existing AAV NAb. A small group of patients with rheumatoid arthritis were treated with combination of methotrexate and RTX, lowering anti-AAV2 and anti-AAV5 NAb in a subset of patients with variable magnitudes (50). For AAV2, 9/28 patients showed at least a half-log reduction, and inferred individuals with NAb titer ≤1:1000 were more likely to respond to RTX but the contribution of methotrexate is unknown. Considering the supportive evidence from previous AAVrh10-microRNA with RTX (29), further study in RTX application is warranted.

Imlifidase (Idefirix, Hansa Biopharma) is a IgG-degrading cysteine protease derived from Streptococcus pyogenes (IdeS), which specifically cleaves opsonizing IgG at the lower hinge region of the heavy chains, resulting in a F(ab’)₂ and a non-functioning dimeric Fc fragment (82). It could potentially overcome a limitation of RTX and cleave existing capsid-specific IgG. Using a laboratory version of IdeS with rAAV8, significant reductions in anti-AAV8 IgG and NAb levels, with enhanced liver transduction and transgene expression and observed in passively immunized murine models and naturally immunized NHP (53). Notably, the study also explored rAAV re-administration with IdeS pre-treatment in NHPs. In the first study (n=1), no induction of anti-capsid IgG and NAb, along with lower IgM and increased transgene level was observed for 21 days after second rAAV8-hFIX administration. However, this was not replicated in a larger cohort (n=5) immunized with rAAV-LK03, that developed anti-capsid IgM and IgG. Further studies are required as the IdeS dosing regimen differed between studies, and two rAAV-LK03 vectors (expressing GAA and hFVIII) were used in the latter study.

Proteasome inhibitors (PIs) are licensed for multiple myeloma. Second-generation carfilzomib is irreversible and more specifically inhibits chymotrypsin-like activity than bortezomib, the reversible first-generation inhibitor, which also inhibits lysosomal and calcium-activated cellular proteases (54, 83). After endosomal escape, rAAV particles either enter the nucleus for transgene expression, or become ubiquitylated then degraded by proteasome (84). The latter pathway results in unsuccessful transduction, and capsid-derived peptides are presented to CTL by MHC class I molecules, provoking elimination of transduced cells and loss of transgene expression (85). In addition, these inhibitors may have immunomodulatory role in suppressing dendritic cells function and downstream T-cell stimulation (86).

PIs have been investigated in preclinical models for their ability to increase rAAV availability and reduce CTL responses. Bortezomib has been shown to dose-dependently decrease cell surface MHC class I antigen presentation and inhibit CTL-mediated lysis after rAAV administration in vitro (87). Moreover, a single bortezomib dose given with rAAV8 dosing enhanced transgene expression by >50% for one year (compared to ~10%) in hemophilia A mice, and longer in-range clotting time for at least 10 months in hemophilia A dogs (57). Both bortezomib and carfilzomib enhance rAAV2 transduction in vitro, but bortezomib is more efficacious than carfilzomib in vivo when administered by retro-orbital injection with rAAV2 (56). Although no toxicity was found in the animal models, peripheral neuropathy and myelosuppression are adverse effects observed in humans (54). Emerging evidence showing variations in PI effectiveness across cell types and AAV serotypes (88), which warrants further study.

Second strand synthesis after capsid uncoating in nucleus is long-recognized as the rate-limiting step of rAAV transduction (89); an improvement in such efficacy could allow rAAV administration at lower dose. As traditional chemotherapeutics directly or indirectly induce DNA damage, thereby initiating DNA damage response (DDR) to repair lesions (90), it has been postulated that these repair mechanisms could increase conversion of rAAV genome into dsDNA (91), or divert DDR proteins that would otherwise impede dsDNA production (92). Several chemotherapy agents were evaluated previously (91, 93) and a high throughput screening study identified teniposide, a type II topoisomerase inhibitor pharmacologically similar to etoposide, as a potent transduction enhancer (94). Tail vein injection of rAAV2-Luc with teniposide (at doses of 1×10¹¹vg and 20mg/kg respectively) resulted in bioluminescence 2-log higher 48 hours post-administration without hepatotoxicity. This difference reduced to ~1 log at 8 days post-administration (study endpoint). Further study is required to determine whether the effect is sustained, and evaluate potential long-term effects of non-tissue-selective chemotherapy.

Oxidizing agents, such as arsenic trioxide (ATO) (59), and antioxidants, such as manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) (95), have been evaluated. Intraperitoneal ATO 5μg/g/day from day -2 to 2 showed 3.9-fold increase in luciferase assay 12 days after rAAV8 retro-orbital injection, with dose-dependent increase of intracellular reactive oxygen species that inhibit vector degradation pathways (59). Intraperitoneal MnTBAP 80mg/kg/day from day 0-4 reversibly downregulated CD4 on T cells, inhibiting T cell priming and humoral responses to initial rAAV1 dosing, and allowing re-administration of rAAV1 via a different route 28 days later (95).

Hydroxychloroquine is an anti-malarial that interferes with TLRs and cyclic GMP-AMP synthase (cGAS), dampening downstream pro-inflammatory cytokine and type I IFN production (60). A study injected hydroxychloroquine subretinally (18.75μM) with rAAV2, resulting in 5.9-fold improvement in photoreceptor transgene expression (61). However, endosomal acidification is essential for rAAV escape (84), and hydroxychloroquine increases endosomal and lysosomal pH (60), this effect may not be replicated or consistent with systemic application.

This study highlights importance of pharmacotherapy choice. rAAV2-hFIX (8×10¹²vg/kg) was delivered intrahepatically to NHP alongside 2-drug regimen of MMF and rapamycin compared to 3-drug adding Daclizumab (40). The addition of daclizumab resulted in decreased CD4⁺CD25⁺FoxP3⁺ Treg and consistent formation of inhibitory antibodies to hFIX; this was not observed in the 2-drug group. Daclizumab is a humanized monoclonal antibody targeting CD25 present on interleukin-2 receptor commonly found in activated T cells and CD4⁺CD25⁺FoxP3⁺ (96). This indicates careful selection of immunosuppressive agents is necessary as Treg play a critical role in regulating immune response to rAAV products, particularly observed in liver and muscle gene transfer (97).

Timing of T cell immunosuppressant regimen was evaluated with liver-directed rAAV2-hFIX, at 7.5×10¹²vg/kg via hepatic artery in NHP (41). Rabbit anti-thymocyte globulin (rATG), a rabbit polyclonal IgG, causes T-cell and plasma cell depletion and modulation of other immune effectors (98). Used with MMF (25 mg/kg) and rapamycin (4mg/kg, then 2mg/kg), a 35-day delay in rATG administration prevented formation of anti-transgene humoral response compared to commencing immunosuppression on day 0 (41). Neither group had cellular response to capsid or transgene, and 2 of 3 NHP in the delayed rATG group did not develop anti-capsid antibodies. It is possible by postponing rATG lowers the Th17/Treg ratio, allowing peripheral tolerance to the transgene product (41).

This intensive immunosuppressive therapy included T-cell-targeting ATG and tacrolimus, B-cell targeting rituximab, with MMF and methylprednisolone to deliver global immunosuppression (99). This 5-drug regimen with rAAV5-PBGD 1×10¹³vg/kg infusion resulted in reduced T-cell response in NHP, but did not prevent NAb emergence following regimen removal. This suggests that drug selection, initiation and duration of suppression, and role of global immunosuppression are important considerations.