AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer (2024)

Table 1

Receptors and Preferential Tissue Tropism of Natural AAV Vectors

Pre-existing Immunity to WT AAV in Humans

Induction of Immune Responses against AAV Vectors

Innate Capsid Immunogenicity

Several complex processes contribute to generating an immune response against an offending pathogen. Because AAV vectors lack any coding viral sequence, the main sources of foreign antigens brought in during gene transfer, aside from contaminant carryovers from the production and purification process, are derived from initial input of viral capsid and from the transgene product. The DNA component of AAV vectors, and possibly double-stranded RNA (dsRNA) produced by the vector itself,49 can also act as an adjuvant, concurring in the activation of innate immunity along with other host-specific factors (Figure2).

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Figure2

Factors that Influence AAV Vector Immunogenicity

The capsid, its genome, and the transgene product are the main potential immunogenic components of AAV vectors. Production of dsRNA driven by the promoter activity of ITRs can also act as a trigger for innate immunity. Additional host-dependent and vector-dependent factors can modulate the overall vector immunogenicity. These factors are mostly poorly understood, although the presence of innate immunity activators such as CpG and vector dose seem to correlate with vector-related immunotoxicities in some trials.

In recent years, innate immunity to AAV has gained increasing attention as one of the possible triggers of immune-mediated toxicities observed in clinical trials, although the overall lack of clinical evidence (e.g., detection of proinflammatory cytokines in the circulation) has been a challenge to clearly establish a direct causative relationship.

Innate immunity mounts rapidly, is non-specific, and does not result in immunological memory. Innate immune responses are initiated through the recognition of pathogen-associated molecular patterns (PAMPs), exhibited on pathogens, by PRRs (pattern recognition receptors) expressed at the surface or within immune cells. These PRRs recognize viral nucleic acids, as well as membrane glycoproteins, or even chemical messengers. Through a variety of signaling pathways, the engagement of PRRs mainly leads to the activation ofNF-κB (nuclear factor κB) and IRF (IFN-regulatory factor) transcription factors, both of which play a central role in inducing the expression of pro-inflammatory cytokines or type I IFNs, respectively.50 Type I IFNs have been described as being important forthe induction of anti-capsid CD8⁺ Tcell responses.51 In the preclinical setting, blocking the pathways of activation of innate immune responses has been shown to prevent anti-capsid cytotoxic51^,52 and humoral responses42invivo. In non-parenchymal liver cells, including Kupffer cells and liver sinusoidal endothelial cells (LSECs), the viral capsid has been seen to activate innate immunity mainly through binding to Toll-like receptor (TLR)2 expressed on the cell surface.53 Moreover, the double-stranded DNA vector genome, and in particular unmethylated CpG motifs, are recognized by the endosomal TLR9 in Kupffer cells,54 peripheral plasmacytoid dendritic cells(pDCs),52^,55 and monocyte-derived DCs.56 TLR9 engagement has been associated with enhanced capsid antigen presentation into major histocompatibility complex (MHC) class I and subsequent capsid-specific CD8⁺ Tcell activation.44^,52^,57 Also, the MyD88 (myeloid differentiating factor 88)-TLR9 pathway has been shown to mediate the induction of immune responses to liver- and muscle-targeted transgenes.56^,58

In addition to the vector DNA, a recent study suggested a possible contribution of dsRNA to the induction of innate immunity to AAV.49 This would explain why cellular responses are sometimes initiated weeks after vector administration in clinical trials.5^,41 According to this study, the promoter activity of the inverted terminalrepeats (ITRs) flanking the transgene expression cassette could potentially drive the production of dsRNA, which in turn wouldstimulate the MDA5 sensor in human hepatocytes transduced with AAV, leading to the expression of type I IFN. Interestingly, blockade of MDA5 decreased the IFN response and improved transgene expression in transduced cells invitro.49

Immune Responses against the Transgene Product

Several factors contribute to shape the immunogenicity of the transgene product in AAV gene transfer. These can be divided into host-specific factors, associated with the underlying disease or the genetic background of the vector recipient, and vector-specific factors, to group factors related to gene transfer (Table 2). Similar to anti-capsid immune responses, presentation of transgene-derived epitopes to CD8⁺ and CD4⁺ T lymphocytes can induce cytotoxic and humoral responses that have a negative impact on transgene stability.56^,58^,67^,68 One key factor determining the level of anti-transgene immune responses is the target organ for gene transfer, which is determined by the combination of the AAV capsid, the vector delivery route, and the tissue specificity of the promoter driving gene expression. In particular, systemic and intramuscular vector administration, with either ubiquitous or muscle-specific promoters, have been shown to be more immunogenic than gene transfer to immune privileged organs, as well as systemic administration with liver-specific promoters69^,70 (Table 2).

In the context of AAV trials, anti-transgene immune responses have been documented in only a few instances, mostly in the context of intramuscular delivery of AAV vectors. The first evidence of transgene-specific cytolytic Tcell activation came from a phase I/II trial of intramuscular gene transfer of a microdystrophin transgene.73 In this trial, lack of transgene expression was associated with the development of a CD8⁺ Tcell responses directed against epitopes mapping within the transgene amino acid sequence. Similarly, in a trial for α-1 antitrypsin (AAT) deficiency, following intramuscular delivery of an AAV vector, cytolytic Tcells were detected in association with decreased transgene expression in one of the participants,86 although the trial showed overall long-term expression of the transgene in most participants.4 More recently, in the context of systemic delivery of an AAV vector for the treatment of X-linked myotubular myopathy, anti-transgene antibodies were detected,87 although with no direct correlation with clinical endpoints (vide infra). Finally, transgene-reactive Tcells were also detected in a phase I/II trial of intracranial delivery of an AAV5 vector to the brain of children affected by mucopolysaccharidosis type IIIB.88

The significance of these early findings and their impact on endpointsof safety and efficacy are not entirely clear. What appears to be consistent is that targeting tissues that are not immune privileged, such as the muscle,89 with gene transfer may pose additional challenges related to transgene immunogenicity. To this end, concomitant expression of a transgene in a protolerogenic tissue such as the liver, at the same time as muscle, may help transgene engraftment.69^,79

The Tolerogenic Potential of Liver-Directed Gene Transfer

In the context of liver-directed gene transfer, transgene immunogenicity appears to be less of a potential concern compared to other tissues. Starting with the initial observation that mice expressing human factor IX (F.IX) in liver were immunologically tolerant tothe transgene product,80 several studies with AAV vectors in small and large animal models of genetic diseases show that expression ofan antigen in hepatocytes can promote robust antigen-specific immune tolerance (Figure3).80, 90, 91

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Figure3

Liver Gene Transfer Can Drive Transgene Immune Tolerance

Tolerance to a variety of transgenes expressed in the liver is mediated by a variety of mechanisms. Tregs are a common denominator of liver tolerance, as they mediate the suppression of both humoral and Tcell-mediated transgene immune responses. Additional mechanisms include anergy, exhaustion, and deletion of reactive Tcells. Key to tolerance induction appears to be a robust transgene expression in hepatocytes. Adapted from Sherman etal.92

Although several laboratories have investigated the mechanisms driving liver tolerance, results are not fully overlapping and dependon the experimental setting and model antigen used. The tolerogenic effect of liver gene transfer is likely to be mediated by the different cell types that can act as APCs in the liver. Reports indicate that liver-resident macrophages (Kupffer cells) and DCs have a less mature phenotype compared to professional APCs foundin the periphery.93^,94 This property seems to make them poor Tcell activators95 as antigens are presented in the absenceof sufficient co-stimulator ligands. Furthermore, it has beenshown that Kupffer cells can secrete IL-10, an anti-inflammatory cytokine, upon TLR stimulation.96, 97, 98 Another particularity is that LSECs areable to act as professional APCs presenting antigens through MHC class II, which seems to play a role in the induction of regulatory Tcells (Tregs).99^,100 Several studies have demonstrated the essential role of Tregs in the induction of liver-mediated toleranceto liver-targeted transgenes (Figure3).69^,90^,91^,101 In these studies, disruption of Treg homeostasis around the time of vector administration led to an immune response against the transgene. Conversely, administration of rapamycin, a drug known to favor Treg expansion,102 enhanced efficiency of induction of tolerance in the context of established immunity.103^,104

Tregs are not the only player involved in livertolerance. CD4⁺ Tcell anergy,105 apoptosis of reactive Tcells,80^,106 degradation of Tcells in hepatocytes,107 induction of CD8⁺ Tregs,108 and the acquisition of an exhausted phenotype by cytotoxic CD8⁺T cells69^,85, 109, 110 have also been described in the contextof hepatocellular antigen presentation (Figure3). Inductionoflivertolerance has been shown to be dose-dependent,111 ashigh levels of antigen presentation by hepatocytes through MHCclass I have been associated with a more efficient induction of CD8⁺ Tcellexhaustion and apoptosis,85^,112 although in the contextof AAV gene transfer this has not been fully demonstrated.

While preclinical data on liver tolerance are convincing, with compelling data on immunity eradication in small and large animal modelsof hemophilia76^,77^,111 and other diseases,79^,113 the open question is whether this concept will translate to humans carrying pre-existing immunity against a given therapeutic transgene.

Immune Responses to AAV Vectors in Clinical Trials

Recombinant AAV vectors are relatively simple from an immunogenicity point of view, as they do not encode viral proteins and theycomprise a protein capsid and a DNA genome, which can be single or double stranded. Pre-existing immunity originating fromthe exposure to WT AAV, however, can generate both humoral and cell-mediated immunity to the virus, which can cross-react with AAV vectors. While pre-existing humoral immunity represents one of the most efficient barriers to prevent successful gene transfer through systemic administration of AAV vectors (Figure4),114 theimpact of pre-existing Tcell immunity to AAV is not fully understood.

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Figure4

Humoral Immune Responses to AAV

Pre-existing immunity to AAV can block target tissue transduction when the vector is administered systemically directly into the bloodstream. While eradication of humoral immunity with immunosuppressive drugs can be challenging, as pharmacological targeting of B cells has inherent risks, pre-clinical evaluation of physical removal of antibodies with plasmapheresis has shown promising results. Isolation of target organs at the time of vector administration has also been explored. Recent data linked the acute toxicities observed following systemic administration of AAV vectors with complement activations. These toxicities may be mediated by anti-AAV antibodies and can be modulated by drugs targeting the complement activation pathways. Finally, after gene transfer, antibodies to AAV are induced and persist for the long term. Several potential approaches to vector readministration have been explored preclinically, with variable degrees of success.

Ocular Gene Transfer

The eye is a highly compartmentalized organ whose local environment is anatomically isolated from the peripheral immunity by theblood-ocular vasculature, and by the absence of lymphatic vessels.120^,121 Therefore, in the context of gene transfer, the eye hasbeen long thought to be at lower risk for activating innate and adaptive immune responses upon vector administration. Moreover, a phenomenon known as anterior chamber-associated immune deviation (ACAID) has been described in the eye upon recognition of immunogenic antigens, which involves the induction of Tregs, anti-inflammatory M2 macrophages, and the generation of an anti-inflammatory cytokine environment that promotes immunological tolerance.122^,123 This pro-tolerogenic environment is a protective adaptation aimed at preventing inflammatory responses that could affect vision. An immune deviation phenomenon similar to ACAID has been reported in the context of AAV vector administration to the subretinal space.124

The well-characterized immune privilege, the fact that circulating antibodies directed against the AAV capsid are not normally found in the eye,125 and the relatively low vector doses required to achieve therapeutic efficacy have driven the early successes of ocular gene transfer in the clinic.126^,127 Based on these promising results, and banking on more than two decades of research in the field of oculargene transfer, in recent years the field has experienced a dramatic expansion, with several gene transfer trials planned and ongoing, mostly based on the AAV vector platform.128

To date, AAV vector administration has been mainly performed intravitreally or subretinally (Figure5). Based on preclinical animal models and clinical trials, immunogenicity outcomes are greatly influenced by the route of vector administration to the eye, with subretinal vector delivery being less immunogenic than intravitreal administration.129 However, depending on the total vector dose administered, inflammatory responses can be detected regardless of the route of vector administration (vide infra).

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Figure5

AAV Vector Administration to the Eye

Two main routes of vector administration to the eye have been explored. Subretinal administration has been tested in several trials and has a demonstrated safety and efficacy profile in humans. While the approach appears to be feasible, safe, and associated with a low vector immunogenicity profile, it required a surgical procedure that is relatively invasive. Conversely, intravitreal vector administration requires a simple procedure for vector delivery. This route of administration seems to result in higher vector immunogenicity, resulting in inflammation after vector delivery. The safety and efficacy profile of intravitreal delivery of AAV vectors is being evaluated in several trials.

Liver Gene Transfer

The liver has been a long-suited target for gene transfer, as research efforts have been directed to targeting the liver for the correction of a variety of genetic and metabolic diseases167, 168, 169, 170, 171 and for other applications in which, for example, hepatocytes are turned into biofactories to supply protein therapeutics directly into the bloodstream.5^,81, 172, 173, 174 Several important features make the liver an ideal organ for gene therapy, including (1) the fact that hepatocytes arecentral to several metabolic functions and secrete a variety of proteins into the circulation; (2) its high degree of vascularization, which allows for easy transduction with any gene therapy vector delivered thorough the bloodstream; and (3) its unique immune protolerogenic environment (vide supra).

The first evidence of immune responses in the context of liver genetransfer was reported in a trial conducted by the Children’s Hospital of Philadelphia and Avigen, in which seven subjects with severe hemophilia B received a single-stranded AAV2 vector carrying the F.IX transgene under the control of a liver-specific promoter, through the hepatic artery (Table 4).32 Patients were divided intothree dose cohorts of 8× 10¹⁰, 4× 10¹¹, and 2× 10¹² vg/kg, respectively. Transient elevation of F.IX activity at levels around 10% of normal was detected only in the first patient treated at the highest dose. Differently from animals models, in which long-term expression is observed after liver gene transfer with AAV vectors,41^,175 4weeks after gene transfer, F.IX expression started to decline and eventually returned to pre-treatment level. A self-limited transient and asymptomatic rise in liver transaminases was also detected in the same time frame, suggesting the induction of cytotoxic immunity against the AAV2 capsid.32 Similar series of events were observed in aparticipant enrolled in the mid-dose cohort (subject G). An IFNγ ELISPOT assay and flow cytometry-based detection of AAV-specific CD8⁺ Tcells confirmed a Tcell response to the AAV2 capsid, which had a kinetics of appearance that closely mirrored the rise in serumtransaminases and decrease in transgene expression.32^,40 Anadditional important finding from this trial was that a second subject dosed with 2× 10¹² vg/kg, a dose expected to result in detectable transgene expression, but carrying pre-existing anti-AAVNAbs at a titer of just 1:17, had no evidence of liver expression.32

Based on the learning from this first trial, and on preclinical datashowing the potential superiority of the AAV8 serotype in transducing the hepatocytes,182 a second clinical study was initiated a few years later (ClinicalTrials.gov: NCT00979238). The vector genome carried a self-complementary genome, a strategy to enhance hepatocyte transduction.183 In addition, the F.IX sequence was codon optimized to further improve transgene expression. Six seronegative participants with severe hemophilia B were infused an AAV8 vector encoding the self-complementary, codon-optimized, liver-targeted F.IX transgene in a peripheral vein.63 In the low- and intermediate-dose cohorts, F.IX activity remained between 1% and 4% of normal, respectively, in the absence of immune-associated events. In the highest dose cohort (2× 10¹² vg/kg), however, F.IX activity reached maximum levelsof 8%–10% of normal, and patients again experienced an elevation of liver enzymes at 8weeks, which coincided with partialdecline in transgene expression and a rise in circulating capsid-specific Tcells detected by an IFNγ ELISPOT assay. Theelevation of liver enzymes was resolved with tapered treatmentwith high-dose steroids, after which transgene expression remained stable.63 Four more patients were enrolled in the high-dose cohort, two of which were transiently treated with prednisolone after experiencing a decline in F.IX expression,5 afterwhich transgene expression remained stable with a documented duration of up to 10 years (U.M. Reiss, 2019, National Hemophilia Foundation, conference).

Although effective at the currently used vector doses, ongoing studies will address whether this corticosteroid regimen will be effective at higher vector doses and with different AAV serotypes. Of note, evidence of Tcell activation in response to AAV capsid epitopes was documented in PBMCs isolated from participants from all dose cohorts, although liver toxicity was documented only in the high-dose cohort. High levels of anti-AAV8 antibodies could be detected in all participants following vector administration.

A similar set of clinical results was also recently published in thecontext of another hemophilia B clinical trial in which a bioengineered AAV capsid was administered to seronegative hemophilia B subjects at a dose of 5× 10¹¹ vg/kg. At this dose, 2 out of 10 participants required the administration of corticosteroids to managevector immunogenicity, resulting in long-term transgene expression in all subjects enrolled.62

Corticosteroids given reactively have been broadly used across trialsto modulate capsid reactivity. However, this measure did not succeed in all studies in rescuing transgene expression. In a phase I/II study initially sponsored by Baxalta/Shire (ClinicalTrials.gov: NCT01687608), a self-complementary AAV8 vector176 was given to eight hemophilia B subjects at doses ranging from 2× 10¹¹ to 3× 10¹² vg/kg. Transgene expression was transient in most subjects in this study, with the exception of one subject who did not experience an instance of liver enzyme elevation and expressed the F.IX transgene over the long term. Exome sequencing analysis in this subject revealed the presence of a polymorphism potentially affecting the immune system, which could explain the lack of immune responses detected after gene transfer.184 Similarly, a phase I/II study sponsored by Dimension Therapeutics was halted due to transaminase elevation and loss oftransgene expression not controlled by the administration of high-dose corticosteroids.181 Additional emerging data from other liver trials (ClinicalTrials.gov: NCT02991144, NCT03636438, NCT03517085, NCT03970278, and NCT03223194), and also trials in which high doses of AAV vectors are given systemically for the treatment of diseases not affecting the liver (ClinicalTrials.gov: NCT03368742 and NCT03362502),185^,186 show instances of liver toxicities that are likely to be related to vector immunogenicity.

A somewhat different outcome was documented in the context of trials in which AAV5 vectors were used to target the liver. This capsid serotype was first used in subjects affected by acute intermittent porphyria.171 In this study, vector doses up to 1.8× 10¹³ vg/kg were administered, with no evidence of transaminase elevation and no detection of capsid-specific Tcells, although also no clear evidence of therapeutic efficacy. Two additional studies were published, in which AAV5 vectors produced in a baculovirus system were infused at doses up to 5× 10¹² vg/kg178 and 6× 10¹³ vg/kg187 for the treatment of hemophilia B and A, respectively. In these two studies, elevated transaminase levels were detected and treated with corticosteroids, although their direct impact on transgene expression was not clearly established in the time frame of the initial observation period. Longer follow-up of participants in the hemophilia A trial showed a slow decline of expression during a few years after gene transfer, for which the exact etiology remains unknown.

Muscle Gene Transfer

Muscle is an important target tissue for gene transfer to treat a variety of neuromuscular,191 cardiac,192 and metabolic diseases.193 Moreover, even for diseases not affecting the muscle, targeting the muscle withAAV vectors has been shown to yield promising results.194, 195, 196 In this section we focus on muscle gene transfer. Diseases mostly affecting motor neurons are discussed separately.

From an immunological perspective, the muscle has unique challenges (reviewed in detail by Boisgerault and Mingozzi89). For example, the inflammation associated with dystrophies can result in enhanced transgene immunogenicity.73 Conversely, intramuscular gene transfer has been shown to induce the recruitment of FoxP3⁺ Tregs, which may play a role in contributing to the stability of transgene expression.56^,197 Additional challenges related to targeting the muscle include the limited distribution of AAV vectors delivered intramuscularly198^,199 and the need for high vector doses to achieve widespread transduction of skeletal muscle across the entire body.

Gene Transfer to Motor Neurons and the Central Nervous System

Systemic gene transfer to target motor neurons has been validated inthe context of the clinical development of a gene therapy treatment for spinal muscular atrophy (SMA) that is marketed by AveXis-Novartis as Zolgensma. Efficacy of this approach was been initially shown in 15 infantile patients with mutations on both alleles of the SMN1 gene and two copies of the SMN2 gene. Patients were injected systemically with an AAV9 vector carrying the SMN gene under the control of a ubiquitous promoter.186 Young pediatric patients were divided in two cohorts of 6.7× 10¹³ and 2.0× 10¹⁴ vg/kg. As in other systemic delivery studies, because most of the vector delivered systemically ends up in the liver, elevated liver transaminases were observed. Concomitantly, capsid-specific Tcells were detected in peripheral blood and were attenuated by a course of prednisolone. Overall, four participants enrolled in this study showed transient elevation in transaminases. Based on the groundbreaking clinical results,186^,212^,213 confirmed in a phase 3 trial (ClinicalTrials.gov: NCT03306277), the investigational gene therapy drug was recently approved.214

While there is little doubt about the potential therapeutic benefit of AAV-mediated gene transfer in the context of systemic neuromuscular diseases and in particular diseases affecting motor neurons, also in this context recent preclinical findings possibly related to vector immunogenicity are worth mentioning as potentially relevant. In a recent report,215 toxicities associated with systemic delivery of high doses of AAV vectors encoding for SMN were observed in non-human primates (NHPs) and piglets. Liver enzyme elevation, including acute liver failure in one animal, was observed in NHPs. Dorsal root ganglia (DRG) sensory neuron lesions were also detected in both species, with a higher degree of severity in piglets. Toxicities described in this study appeared to be independent of capsid cellular immune responses. Similar symptomatic DRG degeneration was also described in a preclinical study conducted by AveXis-Novartis in NHPs following the intrathecal delivery of an AAV9-SMN vector,216 prompting a hold on an ongoing trial of intrathecal vector delivery in SMA type 2 patients (ClinicalTrials.gov: NCT03381729). The nature of these preclinical findings is yet to be fully elucidated. To date, no similar toxicities were encountered in humans receiving the same AAV9-SMN vector systemically.186 Several questions about these findings are still unanswered, including whether the observed toxicities are driven by the vector capsid, the transgene product, or both and whether they are in some way immune mediated. Of note, no symptomatic toxicities have been reported in an ongoing gene transfer trial for giant axonal neuropathy in which high doses of an AAV9 vector are delivered intrathecally together with a course of immunomodulatory drugs (ClinicalTrials.gov: NCT02362438).

In contrast to systemic vector delivery, because of the physical separation to the systemic circulation mediated by the blood-brain barrier (BBB), brain delivery of AAV vectors has several peculiarities, including (1) that the cerebrospinal fluid (CSF) generally contains lower levels of antibodies able to neutralize AAV vectors compared to what is found in the general circulation; and (2) that the brain has a distinct subset of resident immune cells composed mainly of specialized macrophages called microglia, which, because of the lack of access to brain samples, are difficult to study. Immune responses in the human central nervous system (CNS) following gene transfer seem to be less frequent than in other body sites.217 However, the tools we are using to track these responses are likely to be not sensitive enough to detect, for example, local asymptomatic, discrete responses that could lead to gradual clearance of transduced cells.

Pre-existing NAbs to AAV do not appear to be an issue in the context of direct AAV vectors into the CNS. For instance, it has been shown that the presence of circulating anti-AAV-NAbs up to a 1:128 titer had no inhibitory effect on efficacy of the AAV9-GFP gene transfer when delivered to the CSF in NHPs.218 Conversely, the concentration of AAV9-NAbs in circulation increases post-vector infusion, both systemically and in the CSF.219

As mentioned, the brain possesses specialized resident immune cells that are yet to be studied in terms of their response to the AAV vectors. Microglia are initial responders to pathogens or tissue damage and are responsible for initiating an inflammatory response in brain. Alternatively, while phagocytosing dead or dying cells, microglia actually prevent the release of pro-inflammatory signals from necrotic tissue and thus limit further brain damage.220 Additionally, microglia can also phagocytose viable cells that were infected by a virus, as a consequence, for example, of the intracellular calcium dysregulation and phosphatidylserine externalization observed in the context of adenovirus infection.221 However, it is not certain whether transduction of neurons by AAV vector could elicit levels of cellular stress that can lead to clearance of transduced cells by microglia.

The infiltration of microglia to inflammation sites can be followed in tissue sections by fluorescent microscopy detecting ionized calcium binding adaptor molecule 1 (Iba1)-positive cells. The activation statecan be inferred from the cellular morphology, where a resting microglial cell has elongated ramified processes that rapidly retract upon recognition of a pathogen or other inflammatory stimuli. Microglial cells will, in these circ*mstances, become a mobile effector cell.222 Obviously, this type of analysis is not feasible for the purpose of tracking immune responses during clinical trials, which are commonly limited to testing anti-capsid and anti-transgene antibodies and Tcells in peripheral blood or CSF.

Direct delivery of AAV vectors into the brain causes little or no response as measured by IFNγ ELISPOT in PBMCs from infused subjects (reviewed elsewhere223). One important feature to consider in this particular clinical setting is that the doses of AAV vectors administered thus far were relatively small. Nevertheless, a direct injection in brain can also be associated with a local inflammatory response. Similar to muscle, systemic infusion of AAV vectors into the CSF may decrease vector immunogenicity, although, because it requires higher vector doses, it does increase systemic vector exposure. For example, in a mouse model of Niemann-Pick disease type A (NPD-A), it was shown that the delivery of AAV9-ASM to the CSF through the cerebellomedullary (CM) cistern led to transgene expression within the central and peripheral nervous systems. When compared to the direct intracerebellar injection, this route of delivery did not trigger inflammation.224 Similarly, administration of AAV9 vectors into the CSF of dogs resulted in widespread transduction of the brain,219 with also leakage of the vector into the systemic circulation and transduction of the liver.

Across the ongoing trials, the approaches to modulation of potential immunogenicity associated with gene transfer in the CNS vary significantly, ranging from concomitant use of combination of immunosuppressive drugs (ClinicalTrials.gov: NCT02362438, NCT03612869) for an extended period to no immunosuppression (EudraCT: 2015-000359-26). A published example comes from a clinical trial in subjects with Canavan disease with mutations on the gene encoding for aspartoacylase (ASPA) who received an AAV2-ASPA vector infused intracranially.225 No immunosuppression was used during this trial, and only 3 out of 10 subjects developed low to moderately high levels of AAV2 NAbs in blood compared to baseline. In another trial, patients with metachromatic leukodystrophy (MLD) were administered AAVrh.10-arylsulfatase A (ARSA) by intracerebral injection (ClinicalTrials.gov: NCT01801709). A short course of steroids was used to avoid potential immune reactions starting 1day prior to the vector infusion and given for 10days. Anti-capsid antibodies were detected in blood and CSF of all patients. Only one out of four participants developed anti-ARSA antibodies detectable in blood but not in CSF (C.G. Bonnemann, 2019, National Center for Advancing Translational Sciences/NIH, conference). Another example of immune suppression combined with the CNS gene transfer may be illustrated by the AAV9-mediated delivery of gigaxonin for cross-reactive immunologic material (CRIM)-negative giant axonal neuropathy patients (ClinicalTrials.gov: NCT02362438). To mitigate the risk for CRIM-negative participants to mount an immune response against the transgene, an immunosuppressive regimen including overlapping dosing of methylprednisolone, prednisone, tacrolimus, and rapamycin was used. The approach was successful in preventing inflammation, as in immunosuppressed participants no pleocytosis was detected. After vector infusion, anti-AAV9 NAbs appeared in blood and CSF. Of note, an IFNγ ELISPOT assay demonstrated the presence of AAV9-specific, but not gigaxonin-specific, Tcells circulating in blood in subjects dosed with the vector and corticosteroids alone (C.G. Bonnemann, 2019, National Center for Advancing Translational Sciences/NIH, conference).

Conclusions

The gene therapy field is experiencing one of its most exciting periods. Long-term efficacy has been achieved in several clinical trials, and gene therapy drug candidates are reaching late-stage clinical development and market approval. Diseases previously untreatable or for which suboptimal treatments were available are now being cured, delivering previously unimaginable outcomes to patients. Since the first reports of immune-related toxicities associated with AAV vector administration, much progress has been made in understanding the interactions between viral vectors and the host immune system. Yet, new unforeseen complexities are still emerging from trials in which high vector doses are administered systemically. This is clear evidence that many questions about AAV vector immunogenicity are still unanswered.

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