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Review

Clinical and Ocular Inflammatory Inhibitors of Viral-Based Gene Therapy of the Retina

by
Marc Ohlhausen
1 and
Christopher D. Conrady
1,2,*
1
Department of Ophthalmology and Visual Sciences, Truhlsen Eye Center, University of Nebraska Medical Center, Omaha, NE 68105, USA
2
Department of Pathology, Microbiology, and Immunology, University of Nebraska Medical Center, Omaha, NE 68198, USA
*
Author to whom correspondence should be addressed.
Acta Microbiol. Hell. 2024, 69(3), 187-203; https://doi.org/10.3390/amh69030018
Submission received: 15 August 2024 / Revised: 1 September 2024 / Accepted: 5 September 2024 / Published: 11 September 2024
(This article belongs to the Special Issue Feature Papers in Medical Microbiology in 2024)
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Abstract

:
Gene therapy is an emerging field of medicine that can target and treat previously untreatable blinding or lethal diseases. Within the field of ophthalmology, gene therapy has emerged to treat retinal degenerative disorders, but its exact role is in its infancy. While this exciting frontier is rapidly expanding, these typically viral-based gene therapy vectors trigger a host immune response. Thus, a better understanding of the host immune response to gene therapies is critical, in that harnessing immunity to these vectors may improve treatment efficacy and reduce the risk of vision loss from inflammation. As such, we will discuss innate and adaptive immunity to gene therapy vectors, and avenues through which this response may be harnessed to improve visual outcomes.

1. Introduction

Gene therapy is an exciting therapeutic modality with rapidly expanding conditions treated. There is a particular focus on inherited retinal conditions, given that many are monogenic in etiology and lend themselves to treatment via gene therapy. Many of the affected cells in these degenerative disorders are photoreceptors and the retinal pigment epithelium (RPE), an important detail when comparing different routes of administration.
The underlying mechanism of gene therapy involves altering target cells through the introduction of transgene genetic material so that these cells are able to produce a certain therapeutic substance in vivo. Gene augmentation, correction, and expression modification are all examples of strategies used for this purpose [1]. The most commonly employed approaches in current gene therapies are gene augmentation and editing [2].
Target cells ideally need only a single treatment to produce the transgene product for an extended period, possibly indefinitely. The eye has been at the forefront of gene therapy development as it is an ideal environment given its small size, compartmentalization, ease of access and evaluation with sophisticated imaging, confined space, immune-privileged status, and a blood–ocular barrier that reduces risk of systemic dispersion of the drug [3,4].

1.1. Vector Types

There are viral and non-viral methods of gene therapy, with viral-based being the most common. The different types of viral vectors include adenovirus (AV), adeno-associated virus (AAV), and lentiviruses. All three of these viruses can infect dividing and non-dividing cells of their target organs [5]. AAV-based gene therapy is generally considered to have the superior safety profile compared to other gene vectors and is the most utilized in current clinical trials. AV is a DNA virus that was the first virus to be utilized in clinical trials, but has fallen out of favor due to the significant inflammatory response and destruction of transduced cells in primates [6,7]. An advantage of AV is its ability to carry large genes [5]. Lentiviruses are RNA retroviruses derived from primate lentiviruses, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), and simian immunodeficiency virus [8].
AAV is now the most used vector for ocular gene therapy trials. It is a small, single-stranded DNA virus in the Parvoviridae family. There have been 13 serotypes of AAV identified so far [9]. Various hybrid AAV vectors have also been created that combine components of different serotypes to enhance transduction, modulate immunogenicity, or restrict tropism to specific cells [9,10,11]. Dual vector strategies have allowed for the delivery of larger therapeutic transgenes, as a single AAV vector cannot hold more than about 5 kb of DNA [12]. Recombinant AAV (rAAV) lacks protein-coding viral DNA and is therefore used to generate gene therapeutics as either a single stranded or self-complementary rAAV [13,14].
Because natural serotypes of AAV are commonly encountered, up to 70% of people have antibodies (Abs) against one or multiple AAV subtypes prior to gene therapy treatment [1]. The presence of antiviral Abs against a certain strain of AAV has been associated with lower or no gene function following gene therapy in non-human primates (NHPs) [15]. Approximately 70% of the population has Abs against AAV2, while only 38% have Abs against AAV8, a variant typically isolated in NHPs [5,16]. However, there is cross-reactivity of Abs across different serotypes in NHPs [15].
Non-viral gene therapy strategies involve the delivery of DNA-like plasmids, oligodeoxynucleotides, and RNA molecules using non-viral chemical and physical vectors. The use of non-viral vectors is particularly useful for introducing genetic material that is too large to be carried using viral vectors such as AAV [17]. Eight antisense oligonucleotides were approved by the United States Food and Drug Administration (FDA) between 2016 and 2020 [17]. These non-viral methods carry a superior safety profile compared to viral vectors due to the lower risk of immune stimulation and insertional oncogenesis. The major disadvantage is a shorter expression duration of the transduced genetic material [18].
Insertional oncogenesis is a possible adverse event of all insertional forms of gene therapy. Genetic material insertion may result in a change in chromatin or gene structure and subsequent tumor suppressor gene inactivation or oncogene activation, contributing to the development of malignancy [19]. The delivery of genetic material through an extragenomic circular episome that does not integrate into the human genome decreases the risk of insertional oncogenesis when using AAV vectors [5].

1.2. Routes of Delivery

The primary routes of drug delivery are intravitreal, subretinal, and suprachoroidal (Figure 1). Intravitreal and suprachoroidal injections can be completed in an outpatient clinical setting, but in its current form, subretinal delivery requires surgical intervention including a pars plana vitrectomy (PPV) at the time of administration in a hospital or ambulatory surgical center. Each of these methods have their own advantages and disadvantages and are associated with varying levels of efficacy and inflammatory responses (Table 1).
Intravitreal injections have the advantage of being relatively convenient and are administered similarly to other medications used by retinal specialists. A major limitation is the difficulty these therapies encounter in reaching cells of the outer retina. Greater outer retinal transduction has been seen following internal limiting membrane (ILM) removal, suggesting a role of this retinal layer in preventing vector penetration in NHPs [20]. There have been some interesting developments involving engineered AAV capsids created through directed evolution, which were better able to transduce cells of the outer retina [21,22,23,24]. These findings were first demonstrated in a mouse model, before more recently being demonstrated in NHPs [24]. Further testing must be completed, but this could have important implications for the efficacy of intravitreal gene therapy in the future.
A limitation of intravitreal gene therapy is the increased risk of systemic exposure. A potential route of systemic exposure for ocular AAV gene therapies is through the optic nerve lymphatics. Yin et al. examined whether ablation of the deep cervical lymph nodes could prolong the therapeutic effect of repeat AAV intravitreal injections and found that it was successful in mitigating the immune response created by a second AAV injection and improved transduction efficiency in mice [25]. As an alternative to lymphatic ligation, intravitreal injection of soluble vascular endothelial growth factor receptor (sVEGFR3) was administered to inhibit lymphatic drainage and allowed the second dose of AAV to be as effective as the first [25].
Subretinal delivery requires a higher level of skill and time in the operating room but has many advantages. In order to deliver the drug, a macular retinotomy is completed using a small gauge cannula and the gene therapy vector is injected into the subretinal space [26]. An advantage of this technique is that the viral vectors are delivered directly to the outer retinal cells, the primary targets for transduction [8,27,28,29]. Intravitreal AAV2 and AAV8 have been shown to mainly transduce retinal ganglion cells, particularly M-type [30,31]. Subretinal delivery of AAV8 compared to AAV2 demonstrated that AAV8 was superior in targeting photoreceptors, an important cell type in many retinal degenerative disorders [30].
There are risks associated with a vitrectomy when associated with administering subretinal therapies including cataract progression, macular hole formation, retinal tears or detachments, endophthalmitis and reflux of viral vector into the vitreous. There have also been reports of retinal thinning and hyper-reflective spots following subretinal delivery of AAV [32]. Another disadvantage of subretinal therapy is the focal treatment of typically the macula that could limit its utility when treating conditions that affect all retinal cells or those predominantly in the periphery.
Similar to intravitreal injections, suprachoroidal injections can be performed in the clinic and do not require surgery. Delivery can be accomplished via microcatheter, hypodermic needle, or specially designed microneedles. The microneedle approach is like that used for triamcinolone acetonide injections in non-infectious uveitis [33,34]. Qualities that make this approach particularly well-suited to gene therapy delivery include direct delivery of the vector to the outer retina, but in a more widespread manner than subretinal injection. There is also limited anterior segment exposure to the drug possibly reducing inflammatory reactions and unwanted intraocular side effects.
In a trial of suprachoroidal AAV8 injection, there was a lower humoral immune response and neutralizing antibody production compared to intravitreal injections in NHPs [35]. This is hypothesized to be due to greater systemic exposure of intravitreal therapies through trabecular outflow, compared to the less efficient outflow of subretinal and suprachoroidal therapies through the uveoscleral pathway [35]. There was also diffuse, peripheral transduction of RPE cells compared to more focal delivery of subretinal injections [35]. One possible drawback is the loss of in vivo gene expression at 3 months, thought to be related to exposure of the vectors to macrophages along with the extremely high choroidal blood flow causing greater immune exposure and responses [35]. Another potential drawback is inadvertent subretinal injection, although this was an infrequent complication in previous studies utilizing this delivery method in humans [34,35].

1.3. Mechanism of Inflammation

The immunogenic components of AAV vectors are primarily the capsid and DNA vector [4]. Specific regions of the viral DNA, including the promoter and inverted terminal repeat regions, have been shown to elicit varying immune responses [36]. Following host cell transduction, an immune response to the transgene RNA and protein products may develop. Both innate and humoral immune responses to gene therapy products in the ocular environment are comparable to those seen with invading pathogens [37]. Nonspecific inflammation and acquired immunity can block the effects of vectors and reduce transduced cell viability.
The innate immune response involves a rapid and nonspecific reaction with limited long-term immunological memory. Host pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns such as viral nucleic acids and membrane glycoproteins, leading to the upregulation of downstream inflammatory markers and cytokine production [38]. Damage-associated molecular patterns are endogenous molecules that are released from damaged or dying cells and can also be recognized by and activate PRRs [32,39]. PRRs are expressed by innate immune cells such as macrophages, monocytes, granulocytes, natural killer cells, dendritic cells, and also many retinal cells [32,37]. PRRs include toll-like receptors (TLRs), retinoic acid-inducible gene-I-like (RIG-I-like) receptors (RLRs), nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), cyclic GMP-AMP synthase (cGAS), and absent in melanoma 2 (AIM2)-like receptors (ALRs) [32]. Once a PRR is activated, downstream effects lead to increased production of pro-inflammatory cytokines, type I interferons, and other chemical mediators, including within the retina itself [32,37,40,41]. This produces an immediate, nonspecific host immune response while promoting the development of adaptive immunity [37,40]. Most, if not all, of these innate sensors have been implicated in inducing the host inflammatory response [42].
The innate immune response to AAV vectors has been extensively studied, though mostly in extraocular settings such as hepatocytes. The findings from these studies may not be universally applicable to the ocular compartment, but they do provide insight to the systemic response to ocular gene therapy if it should escape into systemic circulation. Responses can be classified as either anti-capsid or anti-nucleic acid. Anti-capsid responses have been shown to derive from TLR-2 recognition and signaling, whereas TLR-9 recognizes viral DNA with unmethylated CpG-motifs [40,43,44,45,46]. TLR-2 activation in human Kupffer cells and liver sinusoidal endothelial cells resulted in increased inflammatory cytokine production via the transcription factor nuclear factor-kB (NF-kB). Identification of viral DNA by TLR-9 activates myeloid differentiating factor 88 (MyD88) and the type I interferon cascade, shown to result in silencing of transgenes over time, which has been of special interest with AAV vectors [47,48,49]. Retinal expression of TLR-2, TLR-9, RLR, AIM2/NLR, and cGAS was confirmed in previous analyses [32]. Reichel et al. found that there was significant upregulation of genes from the RLR and AIM2/NLR pathways in NHPs with clinically evident inflammation following subretinal gene therapy compared to sham injections [31]. The activation of PRRs by AAV also plays an important role in promotion of adaptive immune responses via downstream pro-inflammatory cytokines and type I interferons that increase vascular permeability, induce endothelial activation, promote leukocyte transmigration across post-capillary venules, and recruit and activate immune cells (Figure 2) [37,40,45,50]. Activation of these innate sensors also ultimately drives adaptive immunity [51].
While immunity to AAV vectors is becoming an area of significant scientific interest due to their common use in gene therapy, the innate immune response to lentiviral approaches under development is unclear within the eye. Fortunately, there is typically minimal preexisting immunity (i.e., adaptive responses) to these vectors when compared to AAV serotypes [52]. What is known is that subretinal or intravitreal injection of lentiviral vectors expressing GFP in rats resulted in transgene expression within the RPE, but also induced F4/80 expression, a marker found on macrophages; and antibody production, especially with higher doses [53,54]. This is especially significant, as macrophages and subpopulations of microglia contribute to pathological changes of retinal degenerative disorders such as AMD [55,56,57,58,59].
Finally, non-viral delivery methods such as plasmids are in development and have shown promise in transducing the retina [60]. While the study utilizing these plasmids did not note significant inflammation, the mice were on an immunosuppressive regimen prior to and following injection [60]. However, there was less gene expression in the plasmid with more CpG motifs, which the authors suggested may be due to innate immune mechanisms that would be activated by foreign DNA such as TLR-9 [37,60]. While very little is known in regard to the inflammatory responses to lentiviral and non-viral delivery methods, the activation of innate immune pathways such as TLR-dependent pathways and macrophage recruitment is clear. Activation of these responses will pose its own set of challenges to therapy efficacy and safety, especially with the known roles of these responses in pathological changes of retinal degenerative conditions [56,61].
The adaptive immune response develops over days and is initiated by the presentation of antigens to B and T cells by antigen-presenting cells (APCs). The activation and clonal expansion of B and CD4+ and CD8+ T cells creates a specific, targeted response to the antigen, antibody production, and lasting immunological memory. APCs can be found throughout the eye, and consist of resident macrophage-like microglial cells, perivascular macrophages, and dendritic cells [62,63,64]. APCs may take up AAV antigens and present them in major histocompatibility complex class I and II to activate CD8+ and CD4+ T cells. Cellular responses mediated by CD4+ and CD8+ T cells are directed against either the capsid or the transgene products [1,32]. An important consideration when using gene therapy to treat patients with null point mutations is the possibility that the resulting transgene product will be recognized as foreign and elicit an immune response [32]. Transgene-specific T cells have been identified in the systemic circulation of patients treated with retinal gene therapy and have been correlated with increased retinal toxicity in some studies [30,65,66]. There is also variation in the T-cell response between the different AAV serotypes, with a more robust response to AAVrh32.33 and AAV2 compared to AAV8, for example [67,68].
The humoral response to AAV involves neutralizing Abs, Abs that interact with Fc receptors, and complement-activating Abs and complexes [69,70]. Neutralizing Abs prevent the binding of the virus to its target cell, keeping the genetic material from being released intracellularly. By binding to Fc receptors, Abs induce apoptosis of infected cells via antibody-dependent cellular cytotoxicity (ADCC) [69]. And lastly, by activating complement factors, infected cells or viral particles can be lysed or marked for phagocytosis.
Exposure to wild-type AAV, which occurs commonly in the general population, can lead to pre-existing cellular and humoral immunological memory, which subsequent AAV therapies can potentially reactivate [38,71]. In humans, antibody formation against capsid proteins of AAV encountered in the environment can start as early as 2 years of age [72]. These pre-existing systemic anti-AAV antibodies have been shown to decrease efficacy of gene therapies. The presence of pre-existing neutralizing antibody titers in the serum of NHPs strongly correlated with weak, decaying or lack of transgene expression following intravitreal AAV2 gene therapy administration [15]. Following AAV gene therapy, capsule-specific Abs have been found in the systemic circulation of patients and NHPs, but it is unclear to what extent they enter the eye [73,74,75].

1.4. Inflammatory Response Based on Delivery Method

The inflammatory response elicited from various ocular gene therapy delivery modalities are associated with differing severity levels and characteristics. Intravitreal injections are thought to induce a predominantly humoral immune response. The Abs produced in response to intravitreal therapies can target and eliminate the transduced cells of the retina and significantly reduce the efficacy of the treatment, as has been seen in mice, rabbits, NHPs, and human clinical trials [31,76,77,78,79]. Some studies have also correlated antibody titers to the degree of intraocular inflammation observed [74]. Though, others have found a disconnect between the intraocular and humoral inflammatory responses, and the inflammatory response did not always manifest as a visible intravitreal reaction [35,80]. The neutralizing Abs produced in response to intravitreal therapy in one eye can result in a loss of efficacy in the fellow eye if the same vector is introduced [79].
The inflammatory response to viral DNA plus capsid versus only viral capsid following intravitreal injection of AAV vectors into NHP eyes was compared [81]. Aqueous inflammation was seen only in the presence of the viral genome within the viral capsids, along with a more sustained inflammatory reaction in the vitreous [81]. Injection of only the capsid induced low levels of vitreous inflammation but no anterior chamber reaction [81]. Reducing the total viral capsid dose by removing empty capsids reduced inflammation and improved viral transduction. Systemic neutralizing Abs to AAV were formed regardless of the presence of the viral genome [81].
The subretinal space is known to have a unique immune response to antigens, mirroring that seen in anterior-chamber-associated immune deviation (ACAID) [82,83]. In ACAID, the introduction of antigens in the anterior chamber results in suppression of the systemic antigen-specific T-helper (Th) cells and the downregulation of innate and adaptive immune responses [5,32,82]. With subretinal gene therapy, it is thought that RPE cells that have undergone transduction may act as APCs of the retina and subsequently produce a population of immunosuppressive Th2 cells [82]. There are even reports of a suppressed pre-existing immune response against antigens after their injection in the subretinal space [82]. Combined, these findings have raised the question of whether steroids after subretinal injection are helpful to reduce immune reaction or if this practice is counterproductive [31,82]. Unfortunately, immune deviation within the posterior segment of the eye is poorly defined.
Clinically evident ocular inflammation is frequently encountered following subretinal gene therapy like intravitreal routes. Some of the inflammation can be attributed to the invasiveness of the procedure, although in a study by Reichel et al., when AAV8-injected NHP eyes were compared to those which were sham-injected, there was significantly more inflammatory gene overexpression in the AAV8 group [31]. In this study, the anterior and posterior chamber inflammation was compared between NHPs who underwent subretinal or intravitreal injection with or without an AAV8 vector [31]. It was found that cells in the anterior chamber peaked at 3 days post-injection, and posterior chamber cell peaked at 1 week. The highest overall scores for inflammation were found in the eyes that underwent subretinal surgery combined with AAV8 exposure. Fluorescein angiography showed that perfusion characteristics remained unchanged and the blood–retina barrier remained intact without leakage in all cases [31]. In the subretinal group, temporary loss of retinal outer segments due to retinal detachment and then re-attachment was found to be reversible by day 90 post-injection.
Multiple studies have demonstrated that there appears to be minimal humoral response—and subsequently, fewer neutralizing antibodies produced in response—to subretinal versus intravitreal gene therapy [31,79,84]. Even when neutralizing antibodies were generated following a subretinal injection, fellow eyes could be retreated successfully in NHPs during the development of Luxturna [1]. However, there is always the possibility that leakage of the subretinal therapy into the vitreous cavity may occur following injection, with subsequent outflow through the conventional pathway and systemic inflammatory response comparable to intravitreal injection [29]. The cellular immune response to subretinal AAV8 gene therapy in a murine model was characterized by Chandler et al. [85]. A significant increase in CD45+ retinal leukocytes was seen at day 14 post-subretinal injection of an AAV8 vector encoding green fluorescent protein (GFP), with predominant cells being macrophages, natural killer cells, CD4+ and CD8+ T cells, and natural killer T cells. The cellular response persisted at 28 days post-injection and suggested a Th1 cell-mediated effector immunity [36,85].
For AAV vectors administered through either intravitreal or subretinal routes, there has been a demonstrated dose-dependent relationship with the immune response. A dose-dependent humoral response has been demonstrated between the amount of AAV2 or AAV8 vector used, manifesting as higher levels of systemic neutralizing antibody levels to the vector capsid [30,75]. A similar dose-dependent relationship was found between clinically apparent ocular inflammation and the strength of intravitreal AAV8-RS1 [74]. Systemic antibodies against AAV8 also increased in a dose-dependent fashion in this study, but no antibodies against the RS1 genetic material were identified [74].
In the limited research currently available on suprachoroidal gene therapy, a less robust systemic humoral immune response has been demonstrated compared to intravitreal injections. A more localized chorioretinitis was seen in cases of suprachoroidal injection compared to intravitreal injections [35]. It has been proposed that localized steroids could be beneficial in these scenarios, but our understanding of innate and adaptive immune responses to suprachoroidal gene therapy is currently lacking.

2. Current Applications

2.1. Clinical Trials

There are many different ocular applications of gene therapy currently under investigation to treat posterior segment disorders such as retinal degeneration, intraocular inflammation, and aberrant angiogenesis, among others [33]. Most of these are directed towards the treatment of retinal diseases, though their use for some corneal and glaucoma indications continues to be explored. The conditions with the greatest number of gene therapy clinical trials are Leber’s congenital amaurosis (LCA), Leber’s hereditary optic neuropathy, neovascular age-related macular degeneration, achromatopsia, retinitis pigmentosa, and choroideremia, though there are many others (Table 2).
In 2017, the United States Food and Drug Administration approved the use of gene therapy in pediatric patients affected by Leber Congenital Amaurosis with the RPE65 mutation using the drug voretigene neparvovec-rzyl (Luxturna) [27,86]. This treatment is delivered via a subretinal approach and utilizes an AAV2 viral vector. To date, this remains the only FDA-approved ocular gene therapy. While multiple gene therapy trials have not met their primary endpoints, we hope that with a better understanding of one of the major inhibitors to efficacy, ocular inflammation, that more agents will be approved to reduce the global burden of blindness [87].

2.2. Ocular Inflammation Management

The generation of intraocular inflammation remains a significant obstacle to widespread adoption of gene therapies. Varying strategies have been used to limit ocular inflammation. Almost all human trials have involved some sort of pretreatment with corticosteroids, along with an extended post-treatment steroid taper [1]. A sample of these regimens was examined in a workshop convened by the Foundation Fighting Blindness in September 2020 to discuss intraocular inflammation during viral vector-mediated gene therapy for inherited retinal disease [1]. The most common strategies employed a course of oral prednisone at either 60 mg/day or 1 mg/kg/day for 1 to 3 days prior to therapy, then an oral prednisone taper lasting between 18 days and 2 months following the injection [1,74,88,89,90]. The wide variety of anti-inflammatory protocols prevented the workshop from making a recommendation on optimal strategies at that time. The current recommendations for prophylactic anti-inflammatory therapy for Luxturna, the only ocular gene therapy product currently available on the market, is for systemic oral corticosteroids equivalent to prednisone at 1 mg/kg/day (maximum of 40 mg/day) for a total of 7 days (starting 3 days before therapeutic administration), followed by tapering the dose over the following 10 days.
Overall, inflammatory reactions have predominantly been mild and well-controlled with oral and topical steroids, though there are some exceptions. One patient in a phase 1 trial of subretinal AAV2-REP1 for choroideremia experienced a presumed localized intraretinal immune response after stopping oral steroids. This resulted in significantly decreased best corrected visual acuity and long-term disruption of outer retinal structures on spectral-domain optical coherence tomography following resolution of the event [90]. In the INFINITY trial utilizing intravitreal ADVM-022 (AAV vector carrying aflibercept) to treat diabetic macular edema, three patients in the high-dose intravitreal therapy group developed panuveitis and subsequent hypotony, requiring PPV and silicone oil placement [5]. Minimizing off-target exposure by vitreous fluid lavage may limit inflammatory responses following administration of subretinal gene therapy as well [91]. Ultimately, while these aforementioned strategies utilizing steroids and vitreous lavages temporarily reduce ocular inflammation, the use of longer-acting nonsteroidal anti-inflammatory medications that are routinely used in uveitis may be required to reduce the risk of inflammatory insult to the eye following gene therapy and also allow larger vector delivery [33,92]. This has been especially apparent with AAV vectors targeting hemophilia due to loss of transduced hepatocytes over time due to cell-mediated immunity [93]. However, strategies employing these nonsteroidal medications are untested. Lastly, there is emerging interest in other AAV or even hybrid AAV serotypes that have shown altered tropism (Table 3), lower rates of pre-existing neutralizing antibodies, and reduced immune activation to diminish or negate the host innate and adaptive immune response to the therapy altogether [16,48,72,94,95]. Thus, identification of safe and less toxic AAVs through directed evolution or viral engineering utilized in lower concentrations may negate most of the immune response altogether by reducing TLR-9-activating CpG motifs or adding TLR-9 inhibitory sequences [1,48,96,97,98].

3. Conclusions

Ocular gene therapy remains an extremely promising future treatment modality for various conditions, but the irreversible nature and unknown long-term adverse events warrant some caution. Therapies have been associated with varying levels of intraocular inflammation that can differ based on the vector type and mode of delivery. Ongoing efforts have been made to prophylactically treat this inflammation or successfully address it when it occurs, with mostly success. Previous studies have allowed for better characterization of the innate and adaptive immune responses to ocular gene therapy, allowing us to better understand future directions vector delivery systems should take and what the most effective anti-inflammatory strategies may be going forward. However, our relatively limited understanding of the host response has hampered our ability to produce larger, more efficacious gene therapy vectors and should remain an area of heightened interest within the ophthalmic community.

Author Contributions

Study conception and design: M.O. and C.D.C.; manuscript preparation: M.O. and C.D.C.; editing: M.O. and C.D.C. All authors have read and agreed to the published version of the manuscript.

Funding

C.D.C. was supported by the National Institutes of Health K08 award EY034892, the Knights Templar Eye Foundation career starter grant and competitive renewal, the National Institute of Medical Sciences U54 GM115458 IDeA Clinical and Translational Research Early Career Investigator Program, and startup funds provided by the University of Nebraska Medical Center.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Amh 69 00018 g001
Figure 1. Routes of gene therapy delivery for posterior segment diseases. Created utilizing BioRender, (Toronto, ON, Canada).
Figure 1. Routes of gene therapy delivery for posterior segment diseases. Created utilizing BioRender, (Toronto, ON, Canada).
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Figure 2. Innate to adaptive immunity of the retina.
Figure 2. Innate to adaptive immunity of the retina.
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Table 1. Advantages and disadvantages of intravitreal, subretinal, and suprachoroidal methods of viral gene therapy delivery. ACAID, anterior chamber associated immune deviation; RPE, retinal pigment epithelium.
Table 1. Advantages and disadvantages of intravitreal, subretinal, and suprachoroidal methods of viral gene therapy delivery. ACAID, anterior chamber associated immune deviation; RPE, retinal pigment epithelium.
IntravitrealSubretinalSuprachoroidal
Advantages•Clinic administered•Minimal humoral response•Clinic administration
•Diffuse treatment of entire retina•ACAID promoting immune tolerance•Better exposure to outer retina/RPE
•Able to reach outer retina/RPE•Limits intraocular exposure and outflow through trabecular meshwork
•Diffuse treatment of entire retina
Disadvantages•Greater systemic exposure and humoral response•Requires surgical instillation•Few trials
•The highest potential for intraocular inflammation•Surgical complications•Possibly shorter duration of effect
•Retinal thinning and hyperreflective foci•Newer, less familiar technique
•Focal treatment•Risk of subretinal injection
Bold: This is to highlight that it is associated with the highest rates of intraocular inflammation.
Table 2. Ophthalmic gene therapy clinical trials.
Table 2. Ophthalmic gene therapy clinical trials.
NCT NumberPhaseConditionGene/Genetic Material DeliveredDrugVector/MoleculeRouteStatus
NCT044184272Diabetic Macular Edema, Diabetic RetinopathyafliberceptAAV.7m8-afliberceptAAV2IntravitrealComplete
NCT055369732nAMDafliberceptAAV.7m8-afliberceptAAV2IntravitrealActive
NCT051582962/3Retinitis Pigmentosa, Usher SyndromeUSH2AUltevursenantisense RNA oligonucleotideIntravitrealActive
NCT037802571/2Retinitis Pigmentosa, Usher SyndromeUSH2AQR-421aantisense RNA oligonucleotideIntravitrealComplete
NCT032935243LHONND4GS010 (rAAV2/2-ND4)AAV2IntravitrealActive
NCT031409691/2LCACEP290 QR-110antisense RNA oligonucleotideIntravitrealComplete
NCT026527803LHONND4GS010 (rAAV2/2-ND4)AAV2IntravitrealComplete
NCT034061043LHONND4GS010 (rAAV2/2-ND4)AAV2IntravitrealComplete
NCT049457722Retinitis PigmentosaMCO-010MC0-010AAV2IntravitrealComplete
(optogenetic)
NCT021613801LHONND4scAAV2-P1ND4v2AAV2IntravitrealActive
NCT049194731/2Retinitis PigmentosavMCO-IvMCO-IAAV2IntravitrealComplete
NCT054171262Stargardt DiseasevMCO-010vMCO-010AAV2IntravitrealComplete
(optogenetic)
NCT052936261/2LHONND4NR082 (rAAV2-ND4)AAV2IntravitrealActive
NCT041236261/2Autosomal Dominant Retinitis Pigmentosa (P23H)RHOQR-1123antisense RNA oligonucleotideIntravitrealActive
NCT037487841nAMDafliberceptAAV.7m8-afliberceptAAV2IntravitrealComplete
NCT039131432/3LCACEP290QR-110antisense RNA oligonucleotideIntravitrealActive
NCT026527673LHONND4GS010 (rAAV2/2-ND4)AAV2IntravitrealComplete
NCT044834401ChoroideremiaCHM4D-110AAV2IntravitrealActive
NCT023178871/2X-Linked RetinoschisisRS1AAV8-scRS/IRBPhRSAAV8IntravitrealActive
NCT025567361/2Retinitis PigmentosaChR2RST-001AAV2IntravitrealActive
NCT030662581/2nAMDAnti-VEGF monoclonal antibody fragmentRGX-314AAV8IntravitrealComplete
NCT031449991Dry AMDsCD59AAVCAGsCD59AAV2IntravitrealComplete
NCT031532932/3LHONG11778A ND4rAAV2-ND4AAV2IntravitrealActive
NCT035855561nAMDsCD59AAVCAGsCD59AAV2IntravitrealComplete
NCT010249981nAMDsFLT01AAV2-sFLT01AAV2IntravitrealComplete
NCT061968271/2LCARPE65LX101 (rAAV2-RPE65)AAV2SubretinalActive
NCT063004761/2Stargardt DiseaseABCA4JWK006AAV hybridSubretinalActive
NCT032788731/2AchromatopsiaCNGB3AAV2/8-hCARp.hCNGB3AAV2SubretinalComplete
CNGA3AAV2/8-hG1.7p.coCNGA3
NCT007499571/2LCARPE65rAAV2-CB-hRPE65AAV2SubretinalComplete
NCT033746571/2Retinitis PigmentosaRLBP1CPK850 (scAAV8-RLBP1)AAV8SubretinalActive
NCT025999221/2AchromatopsiaCNGB3AGTC-401 (rAAV2tYF-PR1.7-hCNGB3)AAV2SubretinalActive
NCT026715392ChoroideremiaREP1rAAV2.REP1AAV2SubretinalComplete
NCT025531352ChoroideremiaREP1AAV2-REP1AAV2SubretinalComplete
NCT027814801/2LCARPE65AAV2/5-OPTIRPE65AAV2/5SubretinalComplete
NCT026105821/2AchromatopsiaCNGA3rAAV.hCNGA3AAV8SubretinalActive
NCT045163693LCARPE65voretigene neparvovec-rzyl (AAV2-hRPE65v2)AAV2SubretinalActive
NCT004815461LCARPE65rAAV2-CBSB-hRPE65AAV2SubretinalActive
NCT030013101/2AchromatopsiaCNGB3AAV2/8-hCARp.hCNGB3AAV2/8SubretinalComplete
NCT014960401/2LCARPE65rAAV-2/4.hRPE65AAV2/4SubretinalComplete
NCT008213401LCARPE65rAAV2-hRPE65AAV2SubretinalComplete
NCT057918641/2CLN2 (Batten disease)TPP1RGX-381 (AAV9.CB7.hCLN2)AAV9SubretinalActive
NCT037487841nAMDAfliberceptADVM-022 (AAV.7m8-aflibercept)AAV2SubretinalComplete
NCT038724791/2LCACEP290EDIT-101CRISPR/Cas9 therapySubretinalActive
NCT014821951Retinitis PigmentosaMERTKrAAV2-VMD2-hMERTKAAV2SubretinalComplete
NCT046115031/2Retinitis PigmentosaPDE6ArAAV.hPDE6AAAV2/8SubretinalActive
NCT013014431nAMDEndostatin and angiostatinRetinostatlentiviralSubretinalComplete
NCT047941013X-Linked Retinitis PigmentosaRPGRAAV5-hRKp.RPGRAAV5SubretinalActive
NCT037584041/2AchromatopsiaCNGA3AAV2/8-hG1.7p.coCNGA3AAV2/8SubretinalComplete
NCT009996093LCARPE65voretigene neparvovec-rzyl (AAV2-hRPE65v2)AAV2SubretinalActive
NCT048327242nAMDAnti-VEGF-A antigen binding fragmentRGX-314AAV8SubretinalComplete
NCT014612131/2ChoroideremiaREP1rAAV.REP1AAV2SubretinalComplete
NCT035855561nAMDsCD59AAVCAGsCD59AAV2SubretinalComplete
NCT023418071/2ChoroideremiaCHMAAV2-hCHMAAV2SubretinalComplete
NCT024076782ChoroideremiaREP1AAV2.REP1AAV2SubretinalComplete
NCT029355171/2AchromatopsiaCNGA3AGTC-402 (rAAV2tYF-PR1.7-hCNGA3)AAV2SubretinalActive
NCT020773611/2ChoroideremiaREP1rAAV2.REP1AAV2SubretinalComplete
NCT014948051/2nAMDsFlt-1rAAV.sFlt-1AAV2SubretinalComplete
NCT005164771LCARPE65voretigene neparvovec-rzyl (AAV2-hRPE65v2)AAV2SubretinalComplete
NCT010249981nAMDsFLT01AAV2-sFLT01AAV2SubretinalComplete
NCT006437471/2LCARPE65rAAV2/2.hRPE65p.hRPE65AAV2SubretinalComplete
NCT032528471/2X-Linked Retinitis PigmentosaRPGRAAV2/5-RPGRAAV2/5SubretinalComplete
Table 3. AAV tropism and rates of neutralizing antibodies from preclinical and clinical trials.
Table 3. AAV tropism and rates of neutralizing antibodies from preclinical and clinical trials.
RPEPhotoreceptorsGanglion CellsnAbsAdditional Notes
AAV1YesNoNo+++
AAV2YesYesYes++++Brain transduction
AAV4YesNoNoUnk
AAV5YesNoNo+
AAV6YesNoNo++
AAV8YesYesNo+
AAV9YesNoNo+Systemic transduction
HybridYesYesYesLow
Based on prior work [10,16,72,95,99]. Hybrid, altered AAV strains; nAbs, neutralizing antibodies; RPE, retinal pigment epithelium; Unk, unknown. Lowest (+) to highest (++++) likelihood to induce neutralizing antibodies.
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Ohlhausen, M.; Conrady, C.D. Clinical and Ocular Inflammatory Inhibitors of Viral-Based Gene Therapy of the Retina. Acta Microbiol. Hell. 2024, 69, 187-203. https://doi.org/10.3390/amh69030018

AMA Style

Ohlhausen M, Conrady CD. Clinical and Ocular Inflammatory Inhibitors of Viral-Based Gene Therapy of the Retina. Acta Microbiologica Hellenica. 2024; 69(3):187-203. https://doi.org/10.3390/amh69030018

Chicago/Turabian Style

Ohlhausen, Marc, and Christopher D. Conrady. 2024. "Clinical and Ocular Inflammatory Inhibitors of Viral-Based Gene Therapy of the Retina" Acta Microbiologica Hellenica 69, no. 3: 187-203. https://doi.org/10.3390/amh69030018

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