Over the past decade, vectors derived from adeno-associated virus (AAV) have established themselves as a powerful tool for in vivo gene transfer, allowing long-lasting and safe transgene expression in a variety of human tissues. Nevertheless, clinical trials demonstrated how B and T cell immune responses directed against the AAV capsid, likely arising after natural infection with wild-type AAV, might potentially impact gene transfer safety and efficacy in patients. Seroprevalence studies have evidenced that most individuals carry anti-AAV neutralizing antibodies that can inhibit recombinant AAV transduction of target cells following in vivo administration of vector particles. Likewise, liver- and muscle-directed clinical trials have shown that capsid-reactive memory CD8+ T cells could be reactivated and expanded upon presentation of capsid-derived antigens on transduced cells, potentially leading to loss of transgene expression and immune-mediated toxicities. In celebration of the 25th anniversary of the European Society of Gene and Cell Therapy, this review article summarizes progress made during the past decade in understanding and modulating AAV vector immunogenicity. While the knowledge generated has contributed to yield impressive clinical results, several important questions remain unanswered, making the study of immune responses to AAV a priority for the field of in vivo transfer.

Anti-transgene immune responses elicited after intramuscular (i.m.) delivery of recombinant adenoassociated virus (rAAV) have been shown to hamper long-term transgene expression in large-animal models of rAAV-mediated gene transfer. To overcome this hurdle, an alternative mode of delivery of rAAV vectors in nonhuman primate muscles has been described: the locoregional (LR) intravenous route of administration. Using this injection mode, persistent inducible transgene expression for at least 1 year under the control of the tetracycline-inducible Tet-On system was previously reported in cynomolgus monkeys, with no immunity against the rtTA transgene product. The present study shows the long-term follow-up of these animals. It is reported that LR delivery of a rAAV2/1 vector allows long-term inducible expression up to at least 5 years post gene transfer, with no any detectable host immune response against the transactivator rtTA, despite its immunogenicity following i.m. gene transfer. This study shows for the first time a long-term regulation of muscle gene expression using a Tet-On-inducible system in a largeanimal model. Moreover, these findings further confirm that the rAAV LR delivery route is efficient and immunologically safe, allowing long-term skeletal muscle gene transfer.

Pre-existing immunity to AAV capsid may compromise the safety and efficiency of rAAV-mediated gene transfer in patients. Anti-capsid cytotoxic immune responses have proven to be a challenge to characterize because of the scarcity of circulating AAV-specific CD8 + T lymphocytes which can seldom be detected with conventional flow cytometry or ELISpot assays. Here, we used fluorescent MHC class I tetramers combined with magnetic enrichment to detect and phenotype AAV8-specific CD8 + T cells in human PBMCs without prior amplification. We showed that all healthy individuals tested carried a pool of AAV8-specific CD8 + T cells with a CD45RA + CCR7 − terminally-differentiated effector memory cell (T EMRA) fraction. Ex vivo frequencies of total AAV-specific CD8 + T cells were not predictive of IFNγ ELISpot responses but interestingly we evidenced a correlation between the proportion of T EMRA cells and IFNγ ELISpot positive responses. T EMRA cells may then play a role in recombinant AAV-mediated cytotoxicity in patients with preexisting immunity. Overall, our results encourage the development of new methods combining increased detection sensitivity of AAV-specific T cells and their poly-functional assessment to better characterize and monitor AAV capsid-specific cellular immune responses in the perspective of rAAV-mediated clinical trials.

Preventing untoward immune responses against a specific antigen is a major challenge in different clinical settings such as gene therapy, transplantation, or autoimmunity. Following intramuscular delivery of recombinant adeno-associated virus (rAAV)-derived vectors, transgene rejection can be a roadblock to successful clinical translation. Specific immunomodulation strategies potentially leading to sustained transgene expression while minimizing pharmacological immunosuppression are desirable. Tolerogenic dendritic cells (TolDC) are potential candidates but have not yet been evaluated in the context of gene therapy, to our knowledge. Following intramuscular delivery of rAAV-derived vectors expressing an immunogenic protein in the nonhuman primate model, we assessed the immunomodulating potential of autologous bone marrow-derived TolDC generated in the presence of IL10 and pulsed with the transgene product. TolDC administered either intradermally or intravenously were safe and well tolerated. While the intravenous route showed a modest ability to modulate host immunity against the transgene product, intradermally delivery resulted in a robust vaccination of the macaques when associated to intramuscular rAAV-derived vectors-based gene transfer. These findings demonstrate the critical role of TolDC mode of injection in modulating host immunity. This study also provides the first evidence of the potential of TolDC-based immunomodulation in gene therapy.

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Background: Allergen-specific type 2 CD4 1 T H 2 cells are critically involved in the pathogenesis of IgE-mediated allergic diseases. However, the heterogeneity of the T H 2 response has only recently been appreciated. Objective: We sought to characterize at the single-cell level the ex vivo phenotype, transcriptomic profile, and T-cell receptor (TCR) repertoire of circulating CD4 1 T cells specific to the major dog allergens Can f 1, Can f 4, and Can f 5 in subjects with and without dog allergy. Methods: Dog allergen-specific memory CD4 1 T cells were detected ex vivo by flow cytometry using a CD154-based enrichment assay and single-cell sorted for targeted gene expression analysis and TCR sequencing. Results: Dog allergen-specific T-cell responses in allergic subjects were dominantly of T H 2 type. T H 2 cells could be phenotypically further divided into 3 subsets, which consisted ofcells. All these subsets were nonexistent within the allergen-specific T-cell repertoire of healthy subjects. Single-cell transcriptomic profiling confirmed the T H 2-biased signature in allergen-specific T cells from allergic subjects and revealed a T H 1/T H 17 signature in nonallergic subjects. TCR repertoire analyses showed that dog allergen-specific T cells were diverse and allergic subjects demonstrated less clonality compared to nonallergic donors.Finally, TCR and transcriptomic analyses revealed a close relationship between T H 2-like, T H 2, and T H 2A cells, with the last ones representing the most terminally differentiated and highly polarized subtype. Conclusions: Our study demonstrates heterogeneity within allergen-specific T H 2 cells at the single-cell level. The results may be utilized for improving immune monitoring after allergen immunotherapy and for designing targeted immunomodulatory approaches. (J Allergy Clin Immunol 2021;nnn:nnn-nnn.)

Recombinant Adeno-Associated Virus (rAAV) is considered as one of the most successful and widely used viral vectors for in vivo gene therapy. However, host immune responses to the vector and/or the transgene product remain a major hurdle to successful AAV gene transfer. In contrast to antivector adaptive immunity, the initiation of the innate immunity towards rAAV is still poorly understood but is directly dependent on the interaction between the viral vector and innate immune cells. Here, we used a quantitative transcriptomic-based approach to determine the activation of inflammatory and anti-viral pathways after rAAV8-based infection of monocyte-derived dendritic cells (moDCs) obtained from 12 healthy human donors. We have shown that rAAV8 particles are efficiently internalized, but that this uptake does not induce any detectable transcriptomic change in moDCs in contrast to an adenoviral infection, which upregulates anti-viral pathways. These findings suggest an immunologically favorable profile for rAAV8 serotype with regard to in vitro activation of moDC model. Transcriptomic analysis of rAAV-infected innate immune cells is a powerful method to determine the ability of the viral vector to be seen by these sensor cells, which remains of great importance to better understand the immunogenicity of rAAV vectors and to design immune-stealth products.

PBS-formulated rAAV2/8 PGK GFP-HY were injected subretinally in C57Bl/6 female mice. At day 7, mice were challenged subcutaneously with the UTY and DBY peptides adjuvanted in CFA, and the immune response was analyzed at day 14 by IFNγ ELISpot, cytokine titration and proliferation assays. Our results revealed that: (i) The subretinal injection of 10E8 to 2.10E9 vg/mouse of rAAV2/8 PGK GFP-HY did not induce a significant HY-specific peripheral immune modulation in contrast to the ACAID obtained after subretinal injection of UTY and DBY peptides (50µg each); (ii) Higher doses of rAAV2/8 PGK GFP-HY (5.10E10 vg/mouse) triggered increased Th1 and Tc1 cellular immune responses against the transgene product in peripheral lymphoid organs. Lower doses of vector did not increase peripheral immune responses toward the transgene product following the challenge. We have thus far failed to demonstrate the induction of ACAID by rAAV2/8 subretinal gene transfer. We show that rAAV2/8 vector-mediated subretinal gene transfer is not necessarily ignored at the immunological level. High doses of vector can effectively trigger anti-transgene T-cell responses with the potential for elimination of transgene-expressing cells. Clearly, anti-transgene-specific immune monitoring should be refined at least in preclinical models, to improve the biosafety and the long-term efficacy of rAAV-mediated ocular gene transfer.