Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency.

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

An empowered, clinically viable hematopoietic stem cell gene therapy for the treatment of multisystemic mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a rare X-linked recessive lysosomal storage disorder due to a mutation in the lysosomal enzyme iduronate-2-sulfatase (IDS) gene. IDS deficiency leads to a progressive, multisystem accumulation of glycosaminoglycans (GAGs) and results in central nervous system (CNS) manifestations in the severe form. We developed up to clinical readiness a new hematopoietic stem cell (HSC) gene therapy approach for MPS II that benefits from a novel highly effective transduction protocol. We first provided proof of concept of efficacy of our approach aimed at enhanced IDS enzyme delivery to the CNS in a murine study of immediate translational value, employing a lentiviral vector (LV) encoding a codon-optimized human IDS cDNA. Then the therapeutic LV was tested for its ability to efficiently and safely transduce bona fide human HSCs in clinically relevant conditions according to a standard vs. a novel protocol that demonstrated superior ability to transduce bona fide long-term repopulating HSCs. Overall, these results provide strong proof of concept for the clinical translation of this approach for the treatment of Hunter syndrome.

Recent advances in hematopoietic gene therapy for genetic disorders.

Hematopoietic gene therapy is based on the transplantation of gene-modified autologous hematopoietic stem cells and since the inception of this approach, many technological and medical improvements have been achieved. This review focuses on the clinical studies that have used hematopoietic gene therapy to successfully treat several rare and severe genetic disorders of the blood or immune system as well as some non-hematological diseases. Today, in some cases hematopoietic gene therapy has progressed to the point of being equal to, or better than, allogeneic bone marrow transplant. In others, further improvements are needed to obtain more consistent efficacy or to reduce the risks posed by vectors or protocols. Several hematopoietic gene therapy products showing both long-term efficacy and safety have reached the market, but economic considerations challenge the possibility of patient access to novel disease-modifying therapies. © 2023 Published by Elsevier Masson SAS on behalf of French Society of Pediatrics.

Recent Advances Using Genetic Therapies Against Infectious Diseases and for Vaccination.

The development of prophylatic or therapeutic medicines for infectious diseases is one of the priorities for health organizations worldwide. Innovative solutions are required to achieve effective, safe, and accessible treatments for most if not all infectious diseases, particularly those that are chronic in nature or that emerge unexpectedly over time. Genetic technologies offer versatile possibilities to design therapies against pathogens. Recent developments such as mRNA vaccines, CRISPR gene editing, and immunotherapies provide unprecedented hope to achieve significant results in the field of infectious diseases. This review will focus on advances in this domain, showcasing the cross-fertilization with other fields (e.g., oncology), and addressing some of the logistical and economic concerns important to consider when making these advances accessible to diverse populations around the world.

Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

Evaluation of diversity indices to estimate clonal dominance in gene therapy studies.

In cell and gene therapy, achieving the stable engraftment of an abundant and highly polyclonal population of gene-corrected cells is one of the key factors to ensure the successful and safe treatment of patients. Because integrative vectors have been associated with possible risks of insertional mutagenesis leading to clonal dominance, monitoring the relative abundance of individual vector insertion sites in patients' blood cells has become an important safety assessment, particularly in hematopoietic stem cell-based therapies. Clinical studies often express clonal diversity using various metrics. One of the most commonly used is the Shannon index of entropy. However, this index aggregates two distinct aspects of diversity, the number of unique species and their relative abundance. This property hampers the comparison of samples with different richness. This prompted us to reanalyze published datasets and to model the properties of various indices as applied to the evaluation of clonal diversity in gene therapy. A normalized version of the Shannon index, such as Pielou's index, or Simpson's probability index is robust and useful to compare sample evenness between patients and trials. Clinically meaningful standard values for clonal diversity are herein proposed to facilitate the use of vector insertion site analyses in genomic medicine practice.

Intravitreal air tamponade after AAV2 subretinal injection modifies retinal EGFP distribution.

The subretinal injection protocol for the only approved retinal gene therapy (voretigene neparvovec-rzyl) includes air tamponade at the end of the procedure, but its effects on the subretinal bleb have not been described. In the present study, we evaluated the distribution of enhanced green fluorescent protein (EGFP) after subretinal injection of AAV2 in non-human primates (NHP) without (group A = 3 eyes) or with (group B = 3 eyes) air tamponade. The retinal expression of EGFP was assessed 1 month after subretinal injection with in vivo fundus photographs and fundus autofluorescence. In group A (without air), EGFP expression was limited to the area of the initial subretinal bleb. In group B (with air), EGFP was expressed in a much wider area. These data show that the buoyant force of air on the retina causes a wide subretinal diffusion of vector, away from the injection site. In the present paper, we discuss the beneficial and deleterious clinical effects of this finding. Whereas subretinal injection is likely to become more common with the coming of new gene therapies, the effects of air tamponade should be explored further to improve efficacy, reproducibility, and safety of the protocol.

Severe hematopoietic stem cell inflammation compromises chronic granulomatous disease gene therapy.

X-linked chronic granulomatous disease (CGD) is associated with defective phagocytosis, life-threatening infections, and inflammatory complications. We performed a clinical trial of lentivirus-based gene therapy in four patients (NCT02757911). Two patients show stable engraftment and clinical benefits, whereas the other two have progressively lost gene-corrected cells. Single-cell transcriptomic analysis reveals a significantly lower frequency of hematopoietic stem cells (HSCs) in CGD patients, especially in the two patients with defective engraftment. These two present a profound change in HSC status, a high interferon score, and elevated myeloid progenitor frequency. We use elastic-net logistic regression to identify a set of 51 interferon genes and transcription factors that predict the failure of HSC engraftment. In one patient, an aberrant HSC state with elevated CEBPß expression drives HSC exhaustion, as demonstrated by low repopulation in a xenotransplantation model. Targeted treatments to protect HSCs, coupled to targeted gene expression screening, might improve clinical outcomes in CGD.

Time to evolve: predicting engineered T cell-associated toxicity with next-generation models.

Despite promising clinical results in a small subset of malignancies, therapies based on engineered chimeric antigen receptor and T-cell receptor T cells are associated with serious adverse events, including cytokine release syndrome and neurotoxicity. These toxicities are sometimes so severe that they significantly hinder the implementation of this therapeutic strategy. For a long time, existing preclinical models failed to predict severe toxicities seen in human clinical trials after engineered T-cell infusion. However, in recent years, there has been a concerted effort to develop models, including humanized mouse models, which can better recapitulate toxicities observed in patients. The Accelerating Development and Improving Access to CAR and TCR-engineered T cell therapy (T2EVOLVE) consortium is a public-private partnership directed at accelerating the preclinical development and increasing access to engineered T-cell therapy for patients with cancer. A key ambition in T2EVOLVE is to design new models and tools with higher predictive value for clinical safety and efficacy, in order to improve and accelerate the selection of lead T-cell products for clinical translation. Herein, we review existing preclinical models that are used to test the safety of engineered T cells. We will also highlight limitations of these models and propose potential measures to improve them.

Lentiviral standards to determine the sensitivity of assays that quantify lentiviral vector copy numbers and genomic insertion sites in cells.

With an increasing number of gene therapy clinical trials and drugs reaching the market, it becomes important to standardize the methods that evaluate the efficacy and safety of gene therapy. We herein report the generation of lentiviral standards which are stable, cloned human cells prepared from the diploid HCT116 cell line and which carry a known number of lentiviral vector copies in their genome. These clones can be used as reference cellular materials for the calibration or qualification of analytical methods that quantify vector copy numbers in cells (VCN) or lentiviral vector genomic integration sites (IS). Cellular standards were used to show the superior precision of digital droplet PCR (ddPCR) over quantitative PCR (qPCR) for VCN determination. This enabled us to develop a new sensitive and specific VCN ddPCR method specific for the integrated provirus and not recognizing the transfer plasmid. The cellular standards, were also useful to assess the sensitivity and limits of a ligation-mediated PCR (LM-PCR) method to measure IS showing that at least 1% abundance of a single IS can be detected in a polyclonal population but that not all IS can be amplified with similar efficiency. Thus, lentiviral standards should be systematically used in all assays that assess lentiviral gene therapy efficacy and safety.

Hematopoietic stem and progenitors cells gene editing: Beyond blood disorders.

Lessons learned from decades-long practice in the transplantation of hematopoietic stem and progenitor cells (HSPCs) to treat severe inherited disorders or cancer, have set the stage for the current ex vivo gene therapies using autologous gene-modified hematopoietic stem and progenitor cells that have treated so far, hundreds of patients with monogenic disorders. With increased knowledge of hematopoietic stem and progenitor cell biology, improved modalities for patient conditioning and with the emergence of new gene editing technologies, a new era of hematopoietic stem and progenitor cell-based gene therapies is poised to emerge. Gene editing has the potential to restore physiological expression of a mutated gene, or to insert a functional gene in a precise locus with reduced off-target activity and toxicity. Advances in patient conditioning has reduced treatment toxicities and may improve the engraftment of gene-modified cells and specific progeny. Thanks to these improvements, new potential treatments of various blood- or immune disorders as well as other inherited diseases will continue to emerge. In the present review, the most recent advances in hematopoietic stem and progenitor cell gene editing will be reported, with a focus on how this approach could be a promising solution to treat non-blood-related inherited disorders and the mechanisms behind the therapeutic actions discussed.

WAS Promoter-Driven Lentiviral Vectors Mimic Closely the Lopsided WASP Expression during Megakaryocytic Differentiation.

Transplant of gene-modified autologous hematopoietic progenitors cells has emerged as a new therapeutic approach for Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency with microthrombocytopenia and abnormal lymphoid and myeloid functions. Despite the clinical benefits obtained in ongoing clinical trials, platelet restoration is suboptimal. The incomplete restoration of platelets in these patients can be explained either by a low number of corrected cells or by insufficient or inadequate WASP expression during megakaryocyte differentiation and/or in platelets. We therefore used in vitro models to study the endogenous WASP expression pattern during megakaryocytic differentiation and compared it with the expression profiles achieved by different therapeutic lentiviral vectors (LVs) driving WAS cDNA through different regions of the WAS promoter. Our data showed that all WAS promoter-driven LVs mimic very closely the endogenous WAS expression kinetic during megakaryocytic differentiation. However, LVs harboring the full-length (1.6-kb) WAS-proximal promoter (WW1.6) or a combination of the WAS alternative and proximal promoters (named AW) had the best behavior. Finally, all WAS-driven LVs restored the WAS knockout (WASKO) mice phenotype and functional defects of hematopoietic stem and progenitor cells (HSPCs) from a WAS patient with similar efficiency. In summary, our data back up the use of WW1.6 and AW LVs as physiological gene transfer tools for WAS therapy.

Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs.

In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or ß hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.

Lentiviral gene therapy for X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells1,2. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34+ hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4-1.8 copies per neutrophil) and the persistence of 16-46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.

Biosafety Studies of a Clinically Applicable Lentiviral Vector for the Gene Therapy of Artemis-SCID.

Genetic deficiency of the nuclease DCLRE1C/Artemis causes radiosensitive severe combined immunodeficiency (RS-SCID) with lack of peripheral T and B cells and increased sensitivity to ionizing radiations. Gene therapy based on transplanting autologous gene-modified hematopoietic stem cells could significantly improve the health of patients with RS-SCID by correcting their immune system. A lentiviral vector expressing physiological levels of human ARTEMIS mRNA from an EF1a promoter without post-transcriptional regulation was developed as a safe clinically applicable candidate for RS-SCID gene therapy. The vector was purified in GMP-comparable conditions and was not toxic in vitro or in vivo. Long-term engraftment of vector-transduced hematopoietic cells was achieved in irradiated Artemis-deficient mice following primary and secondary transplantation (6 months each). Vector-treated mice displayed T and B lymphopoiesis and polyclonal T cells, had structured lymphoid tissues, and produced immunoglobulins. Benign signs of inflammation were noted following secondary transplants, likely a feature of the model. There was no evidence of transgene toxicity and no induction of hematopoietic malignancy. In vitro, the vector had low genotoxic potential on murine hematopoietic progenitor cells using an immortalization assay. Altogether, these preclinical data show safety and efficacy, and support further development of the vector for the gene therapy of RS-SCID.

Vectofusin-1 Improves Transduction of Primary Human Cells with Diverse Retroviral and Lentiviral Pseudotypes, Enabling Robust, Automated Closed-System Manufacturing.

Cell and gene therapies are finally becoming viable patient treatment options, with both T cell- and hematopoietic stem cell (HSC)-based therapies being approved to market in Europe. However, these therapies, which involve the use of viral vector to modify the target cells, are expensive and there is an urgent need to reduce manufacturing costs. One major cost factor is the viral vector production itself, therefore improving the gene modification efficiency could significantly reduce the amount of vector required per patient. This study describes the use of a transduction enhancing peptide, Vectofusin-1®, to improve the transduction efficiency of primary target cells using lentiviral and gammaretroviral vectors (LV and RV) pseudotyped with a variety of envelope proteins. Using Vectofusin-1 in combination with LV pseudotyped with viral glycoproteins derived from baboon endogenous retrovirus, feline endogenous virus (RD114), and measles virus (MV), a strongly improved transduction of HSCs, B cells and T cells, even when cultivated under low stimulation conditions, could be observed. The formation of Vectofusin-1 complexes with MV-LV retargeted to CD20 did not alter the selectivity in mixed cell culture populations, emphasizing the precision of this targeting technology. Functional, ErbB2-specific chimeric antigen receptor-expressing T cells could be generated using a gibbon ape leukemia virus (GALV)-pseudotyped RV. Using a variety of viral vectors and target cells, Vectofusin-1 performed in a comparable manner to the traditionally used surface-bound recombinant fibronectin. As Vectofusin-1 is a soluble peptide, it was possible to easily transfer the T cell transduction method to an automated closed manufacturing platform, where proof of concept studies demonstrated efficient genetic modification of T cells with GALV-RV and RD114-RV and the subsequent expansion of mainly central memory T cells to a clinically relevant dose.

Temporary Reduction of Membrane CD4 with the Antioxidant MnTBAP Is Sufficient to Prevent Immune Responses Induced by Gene Transfer.

Unexpectedly, the synthetic antioxidant MnTBAP was found to cause a rapid and reversible downregulation of CD4 on T cells in vitro and in vivo. This effect resulted from the internalization of membrane CD4 T cell molecules into clathrin-coated pits and involved disruption of the CD4/p56Lck complex. The CD4 deprivation induced by MnTBAP had functional consequences on CD4-dependent infectious processes or immunological responses as shown in various models, including gene therapy. In cultured human T cells, MnTBAP-induced downregulation of CD4 functionally suppressed gp120- mediated lentiviral transduction in a model relevant for HIV infection. The injection of MnTBAP in mice reduced membrane CD4 on lymphocytes in vivo within 5 days of treatment, preventing OVA peptide T cell immunization while allowing subsequent immunization once treatment was stopped. In a mouse gene therapy model, MnTBAP treatment at the time of adenovirus-associated virus (AAV) vector administration, successfully controlled the induction of anti-transgene and anti-capsid immune responses mediated by CD4+ T cells, enabling the redosing mice with the same vector. These functional data provide new avenues to develop alternative therapeutic immunomodulatory strategies based on temporary regulation of CD4. These could be particularly useful for AAV gene therapy in which novel strategies for redosing are needed.

Toward a Scalable Purification Protocol of GaLV-TR-Pseudotyped Lentiviral Vectors.

Lentiviral vectors (LV) that are used in research and development as well as in clinical trials are in majority vesicular stomatitis virus G glycoprotein (VSVg) pseudotyped. The predominance of this pseudotype choice for clinical gene therapy studies is largely due to a lack of purification schemes for pseudotypes other than VSVg. In this study, we report for the first time the development of a new downstream process protocol allowing high-yield production of stable and infectious gibbon ape leukemia virus (GaLV)-TR-LV particles. We identified critical conditions in tangential flow filtration (TFF) and chromatographic steps for preserving the infectivity/functionality of LV during purification. This was carried out by identifying for each step, the critical parameters affecting LV infectivity, including pH, salinity, presence of stabilizers, temperature, and by defining the optimal order of these steps. A three-step process was developed for GaLV-TR-LV purification consisting of one TFF and two chromatographic steps (ion-exchange chromatography and size exclusion chromatography) permitting recoveries of >27% of infectious particles. With this process, purified GaLV-pseudotyped LV enabled the transduction of 70% human CD34+ cells in the presence of the Vectofusin-1 peptide, whereas in the same conditions nonpurified vector transduced only 9% of the cells (multiplicity of infection 20). Our protocol will allow for the first time the purification of GaLV-TR-LV that are biologically active, stable, and with sufficient recovery in the perspective of preclinical studies and clinical applications. Obviously, further optimizations are required to improve final vector yields.

Successful engraftment of gene-corrected hematopoietic stem cells in non-conditioned patients with Fanconi anemia.

Fanconi anemia (FA) is a DNA repair syndrome generated by mutations in any of the 22 FA genes discovered to date1,2. Mutations in FANCA account for more than 60% of FA cases worldwide3,4. Clinically, FA is associated with congenital abnormalities and cancer predisposition. However, bone marrow failure is the primary pathological feature of FA that becomes evident in 70-80% of patients with FA during the first decade of life5,6. In this clinical study (ClinicalTrials.gov, NCT03157804 ; European Clinical Trials Database, 2011-006100-12), we demonstrate that lentiviral-mediated hematopoietic gene therapy reproducibly confers engraftment and proliferation advantages of gene-corrected hematopoietic stem cells (HSCs) in non-conditioned patients with FA subtype A. Insertion-site analyses revealed the multipotent nature of corrected HSCs and showed that the repopulation advantage of these cells was not due to genotoxic integrations of the therapeutic provirus. Phenotypic correction of blood and bone marrow cells was shown by the acquired resistance of hematopoietic progenitors and T lymphocytes to DNA cross-linking agents. Additionally, an arrest of bone marrow failure progression was observed in patients with the highest levels of gene marking. The progressive engraftment of corrected HSCs in non-conditioned patients with FA supports that gene therapy should constitute an innovative low-toxicity therapeutic option for this life-threatening disorder.

Immune Response and Intraocular Inflammation in Patients With Leber Hereditary Optic Neuropathy Treated With Intravitreal Injection of Recombinant Adeno-Associated Virus 2 Carrying the ND4 Gene: A Secondary Analysis of a Phase 1/2 Clinical Trial.

Importance: Intravitreal gene therapy is regarded as generally safe with limited mild adverse events, but its systemic effects remain to be investigated. Objective: To examine the association between immune response and intraocular inflammation after ocular gene therapy with recombinant adeno-associated virus 2 carrying the ND4 gene (rAAV2/2-ND4). Design, Setting, and Participants: This secondary analysis of an open-label, dose-escalation phase 1/2 randomized clinical trial of rAAV2/2-ND4 included data from February 13, 2014 (first patient visit), to March 30, 2017 (last patient visit at week 96), the first 2 years after injection. Patients older than 15 years with diagnosed ND4 Leber hereditary optic neuropathy (LHON) and visual acuity of at least counting fingers were enrolled in 1 of 5 cohorts. Four dose cohorts of 3 patients each were treated sequentially. An extension cohort of 3 patients received the dose of 9 × 1010 viral genomes per eye. Interventions: Patients received increasing doses of rAAV2/2-ND4 (9 × 109, 3 × 1010, 9 × 1010, and 1.8 × 1011 viral genomes per eye) as a single unilateral intravitreal injection. Patients were monitored for 96 weeks after injection; ocular examinations were performed regularly, and blood samples were collected for immunologic testing. Main Outcomes and Measures: A composite ocular inflammation score (OIS) was calculated based on grades of anterior chamber cells and flare, vitreous cells, and haze according to the Standardization of Uveitis Nomenclature. The systemic immune response was quantified by enzyme-linked immunospot (cellular immune response), enzyme-linked immunosorbent assay (IgG titers), and luciferase assay (neutralizing antibody [NAb] titers). Results: The present analysis included 15 patients (mean [SD] age, 47.9 [17.2] years; 13 men and 2 women) enrolled in the 5 cohorts of the clinical trial. Thirteen patients experienced intraocular inflammation after rAAV2/2-ND4 administration. Mild anterior chamber inflammation and vitritis were reported at all doses, and all cases were responsive to treatment. A maximum OIS of 9.5 was observed in a patient with history of idiopathic uveitis. Overall, OIS was not associated with the viral dose administered. No NAbs against AAV2 were detected in aqueous humor before treatment. Two patients tested positive for cellular immune response against AAV2 at baseline and after treatment. Humoral immune response was not apparently associated with the dose administered or with the immune status of patients at baseline. No association was found between OISs and serum NAb titers. Conclusions and Relevance: In this study, intravitreal administration of rAAV2/2-ND4 in patients with LHON was safe and well tolerated. Further investigations may shed light into the local immune response to rAAV2/2-ND4 as a potential explanation for the observed intraocular inflammation.