Human iPSC-derived photoreceptor transplantation in the cone dominant 13-lined ground squirrel.

Several retinal degenerations affect the human central retina, which is primarily comprised of cones and is essential for high acuity and color vision. Transplanting cone photoreceptors is a promising strategy to replace degenerated cones in this region. Although this approach has been investigated in a handful of animal models, commonly used rodent models lack a cone-rich region and larger models can be expensive and inaccessible, impeding the translation of therapies. Here, we transplanted dissociated GFP-expressing photoreceptors from retinal organoids differentiated from human induced pluripotent stem cells into the subretinal space of damaged and undamaged cone-dominant 13-lined ground squirrel eyes. Transplanted cell survival was documented via noninvasive high-resolution imaging and immunohistochemistry to confirm the presence of human donor photoreceptors for up to 4 months posttransplantation. These results demonstrate the utility of a cone-dominant rodent model for advancing the clinical translation of cell replacement therapies.

Downregulation of lysosomal trafficking in ARPE19 cells leads to decreased transfection efficiency at high passage.

PURPOSE: ARPE19 cells are a commonly used cell culture model for the study of retinal pigment epithelial cell biology and pathologies. However, numerous studies have demonstrated that ARPE19 undergo morphologic, transcriptomic and genomic alterations over time and with increasing passage number. Herein, we explore the mechanisms underlying increased resistance to the delivery of exogenous genetic material via transfection in ARPE19 cells using mass spectrometry. METHODS: ARPE19 cells (N=5 wells/reagent) were seeded in 6-well plates at passages 24 through 30. At 70% confluency an mCherry reporter construct was delivered via transfection using Lipofectamine 3000, Lipofectamine LTX, Lipofectamine Stem, or PEI (polyethylenimine) reagents. After 72 hours, transfection efficiency was quantified by fluorescence microscopy and flow cytometry. Mass spectrometry and immunofluorescence of ARPE19 cells were performed at passages 24 and 30 to evaluate altered protein synthesis and localization between passage numbers. RESULTS: ARPE19 transfection showed a maximum transfection efficiency of 32.4% at P26 using Lipofectamine 3000 reagent. All lipofectamine based reagents demonstrated statistically significant decreases in transfection efficiency between passages 24 and 30. Mass spectrometry analysis revealed 18 differentially expressed proteins, including down-regulation of clathrin light chain B (CLTB) and legumain (LGMN) that was confirmed via immunofluorescence imaging, which indicated altered intracellular localization. CONCLUSIONS: ARPE19 cells demonstrate passage number dependent changes in lipofectamine-based transfection efficiency. Mass spectrometry and immunofluorescence indicates the observed decrease in transfection efficiency involves the dysregulation of endocytosis and intracellular endolysosomal trafficking at later passages. TRANSLATIONAL RELEVANCE: This study contributes to mounting evidence for changes in ARPE19 cell physiology with increasing passage number. This information is of value for the continued use of ARPE19 cells as a model system for RPE biology and the development of therapeutics.

Tolerability and tropism of recombinant adeno-associated virus vectors in the African green monkey (Chlorocebus sabaeus) anterior chamber.

While many studies have investigated the use of recombinant adeno-associated vectors (rAAV) in the posterior chamber for treatment of inherited retinal diseases, fewer studies have looked at rAAV's ability to transduce cells within the anterior chamber. This study focuses on evaluating the tropism and tolerability of three rAAV serotypes-rAAV2/6, rAAV2/9, and rAAV2/2[MAX]-expressing a green fluorescent protein (GFP) reporter following intracameral injection in the non-human primate (NHP) African green monkey (Chlorocebus sabaeus) model. Injection of high dose (1 × 1012 vg/eye) rAAV vector resulted in transient inflammation characterized by aqueous flare and cellular infiltrate that resolved without intervention in all serotypes. Post-mortem histology revealed widespread expression of GFP in cells of the trabecular meshwork and iris in high dose rAAV2/6, rAAV2/9, and particularly rAAV2/2[MAX] eyes, indicating that rAAV vectors of these serotypes have broad tropism for cells of the anterior chamber and may facilitate the treatment of blinding disorders, such as glaucoma.

Improving retinal vascular endothelial cell tropism through rational rAAV capsid design.

Vascular endothelial cells (VEC) are essential for retinal homeostasis and their dysfunction underlies pathogenesis in diabetic retinopathy (DR) and exudative age-related macular degeneration (AMD). Studies have shown that recombinant adeno-associated virus (rAAV) vectors are effective at delivering new genetic material to neural and glial cells within the retina, but targeting VECs remains challenging. To overcome this limitation, herein we developed rAAV capsid mutant vectors with improved tropism towards retinal VEC. rAAV2/2, 2/2[QuadYF-TV], and rAAV2/9 serotype vectors (n = 9, capsid mutants per serotype) expressing GFP were generated by inserting heptameric peptides (7AA) designed to increase endothelial targeting at positions 588 (2/2 and 2/2[QuadYF-TV] or 589 (2/9) of the virus protein (VP 1-3). The packaging and transduction efficiency of the vectors were assessed in HEK293T and bovine VECs using Fluorescence microscopy and flow cytometry, leading to the identification of one mutant, termed EC5, that showed improved endothelial tropism when inserted into all three capsid serotypes. Intra-ocular and intravenous administration of EC5 mutants in C57Bl/6j mice demonstrated moderately improved transduction of the retinal vasculature, particularly surrounding the optic nerve head, and evidence of sinusoidal endothelial cell transduction in the liver. Most notably, intravenous administration of the rAAV2/2[QuadYF-TV] EC5 mutant led to a dramatic and unexpected increase in cardiac muscle transduction.

rAAV-mediated over-expression of acid ceramidase prevents retinopathy in a mouse model of Farber lipogranulomatosis.

Farber disease (FD) is a rare monogenic lysosomal storage disorder caused by mutations in ASAH1 that results in a deficiency of acid ceramidase (ACDase) activity and the abnormal systemic accumulation of ceramide species, leading to multi-system organ failure involving neurological decline and retinopathy. Here we describe the effects of rAAV-mediated ASAH1 over-expression on the progression of retinopathy in a mouse model of FD (Asah1P361R/P361R) and its littermate controls (Asah1+/+ and Asah1+/P361R). Using a combination of non-invasive multimodal imaging, electrophysiology, post-mortem histology and mass spectrometry we demonstrate that ASAH1 over-expression significantly reduces central retinal thickening, ceramide accumulation, macrophage activation and limits fundus hyper-reflectivity and auto-fluorescence in FD mice, indicating rAAV-mediated over-expression of biologically active ACDase protein is able to rescue the anatomical retinal phenotype of Farber disease. Unexpectedly, ACDase over-expression in Asah1+/+ and Asah1+/P361R control eyes was observed to induce abnormal fundus hyper-reflectivity, auto-fluorescence and retinal thickening that closely resembles a FD phenotype. This study represents the first evidence of a gene therapy for Farber disease-related retinopathy. Importantly, the described gene therapy approach could be used to preserve vision in FD patients synergistically with broader enzyme replacement strategies aimed at preserving life.

Prostaglandin-based rAAV-mediated glaucoma gene therapy in Brown Norway rats.

Prostaglandin analogs are first-line treatments for open angle glaucoma and while effective at lowering intraocular pressure, they are undermined by patient non-compliance, causing atrophy of the optic nerve and severe visual impairment. Herein, we evaluate the safety and efficacy of a recombinant adeno-associated viral vector-mediated gene therapy aimed at permanently lowering intraocular pressure through de novo biosynthesis of prostaglandin F2α within the anterior chamber. This study demonstrated a dose dependent reduction in intraocular pressure in normotensive Brown Norway rats maintained over 12-months. Crucially, therapy could be temporarily halted through off-type riboswitch activation, reverting intraocular pressure to normal. Longitudinal multimodal imaging, electrophysiology, and post-mortem histology revealed the therapy was well tolerated at low and medium doses, with no major adverse effects to anterior chamber health, offering a promising alternative to current treatment strategies leading to clinically relevant reductions in intraocular pressure without the need for adherence to a daily treatment regimen.

Identification of Novel Retinal Pericyte-Targeting rAAV Vectors Through Directed Evolution.

Purpose: Retinal pericytes play a vital role in maintaining retinal homeostasis, and their dysfunction underlies pathogenesis in such vascular eye diseases as diabetic retinopathy and wet age-related macular degeneration. Consequently, retinal pericytes are attractive therapeutic targets for gene therapy, but effectively targeting pericytes with recombinant adeno-associated virus (rAAV) vectors remains a challenge. Methods: We introduced genetic modifications into the surface-exposed variable regions of the rAAV2/2 capsid to generate a complex library (>1 × 107) of capsid mutants that were then screened for preferential tropism toward retinal pericytes. Using the Tg(Cspg4-DsRed.T1)1Akik/J reporter mouse model, which has red fluorescent pericytes that can be isolated via flow cytometry in order to recover vector genomes, we performed three rounds of screening and identified seven putative mutants capable of transducing retinal pericytes. Results: Following intravitreal administration of mutant vectors packaging ubiquitously expressing green fluorescent protein reporters and postmortem flow cytometry of enzymatically digested retinae, two mutants in particular, Peri-E and Peri-G, demonstrated significantly greater transduction of retinal pericytes than unmodified rAAV2/2 (1.4-fold and 2.8-fold, respectively). Conclusions: Although difficult to characterize the effect of each point mutation in the context of multiple amino acid variations from the wild-type AAV2 sequence, we identified several point mutations that may play critical roles in limiting HSPG binding, evading neutralization by murine A20 monoclonal antibodies, modulating antigenicity, and evading ubiquitination to ultimately improve transduction efficiency of retinal pericytes. Translational Relevance: Identification of novel retinal pericyte targeting rAAV vectors enables the development of new, long-lasting gene therapies for retinal diseases such as diabetic retinopathy and wet age-related macular degeneration.

Pre-retinal delivery of recombinant adeno-associated virus vector significantly improves retinal transduction efficiency.

Intravitreal injection is the most widely used injection technique for ocular gene delivery. However, vector diffusion is attenuated by physical barriers and neutralizing antibodies in the vitreous. The 13-lined ground squirrel (13-LGS), as in humans, has a larger relative vitreous body volume than the more common rodent models such as rats and mice, which would further reduce transduction efficiency with the intravitreal injection route. We report here a "pre-retinal" injection approach that leads to detachment of the posterior hyaloid membrane and delivers vector into the space between vitreous and inner retina. Vectors carrying a ubiquitously expressing mCherry reporter were injected into the deep vitreous or pre-retinal space in adult wild-type 13-LGSs. Then, adeno-associated virus (AAV)-mediated mCherry expression was evaluated with non-invasive imaging, immunofluorescence, and flow cytometry. Compared to deep vitreous delivery, pre-retinal administration achieved pan-retinal gene expression with a lower vector dose volume and significantly increased the number of transduced cone photoreceptors. These results suggest that pre-retinal injection is a promising tool in the development of gene therapy strategies in animal models and is a potential approach for use in human research, particularly in younger individuals with an intact posterior hyaloid membrane and stable vitreous.

Development of a Preclinical Laser Speckle Contrast Imaging Instrument for Assessing Systemic and Retinal Vascular Function in Small Rodents.

Purpose: To develop and test a non-contact, contrast-free, retinal laser speckle contrast imaging (LSCI) instrument for use in small rodents to assess vascular anatomy, quantify hemodynamics, and measure physiological changes in response to retinal vascular dysfunction over a wide field of view (FOV). Methods: A custom LSCI instrument capable of wide-field and non-contact imaging in small rodents was constructed. The effect of camera gain, laser power, and exposure duration on speckle contrast variance was standardized before the repeatability of LSCI measurements was determined in vivo. Finally, the ability of LSCI to detect alterations in local and systemic vascular function was evaluated using a laser-induced branch retinal vein occlusion and isoflurane anesthesia model, respectively. Results: The LSCI system generates contrast-free maps of retinal blood flow with a 50° FOV at >376 frames per second (fps) and under a short exposure duration (>50 µs) with high reliability (intraclass correlation R = 0.946). LSCI was utilized to characterize retinal vascular anatomy affected by laser injury and longitudinally measure alterations in perfusion and blood flow profile. Under varied doses of isoflurane, LSCI could assess cardiac and systemic vascular function, including heart rate, peripheral resistance, contractility, and pulse propagation. Conclusions: We present a LSCI system for detecting anatomical and physiological changes in retinal and systemic vascular health and function in small rodents. Translational Relevance: Detecting and quantifying early anatomical and physiological changes in vascular function in animal models of retinal, systemic, and neurodegenerative diseases could strengthen our understanding of disease progression and enable the identification of new prognostic and diagnostic biomarkers for disease management and for assessing treatment efficacies.

Electroretinogram of the Cone-Dominant Thirteen-Lined Ground Squirrel during Euthermia and Hibernation in Comparison with the Rod-Dominant Brown Norway Rat.

Purpose: The majority of small animal species used in research are nocturnal, with retinae that are anatomically and functionally dissimilar from humans, complicating their use as disease models. Herein we characterize the retinal structure and electrophysiological function of the diurnal, cone-dominant 13-lined ground squirrel (13-LGS) retina during euthermia and in hibernation. Methods: Full-field electroretinography (ERG) was performed in 13-LGS and Brown Norway (BN) rat models to establish baseline values for retinal function in each species, including following intravitreal injection of pharmacologic agents to selectively block the contributions of ON- and OFF-bipolar cells. The effect of hibernation-associated retinal remodeling on electrophysiological function was assessed in 13-LGS during torpor and emergence, with correlative histology performed using transmission electron microscopy. Results: Under light-adapted conditions, the a-, b-, and d-wave amplitude of the 13-LGS was significantly greater than that of the BN rat. Retinal function was absent in the 13-LGS during hibernation and correlated to widespread disruption of photoreceptor and RPE structure. Remarkably, both retinal function and structure recovered rapidly on emergence from hibernation, with ERG responses reaching normal amplitude within 6 hours. Conclusions: ERG responses for both BN rats and 13-LGS reflect the relative proportions of cone photoreceptors present within the retinae, indicating that the cone-dominant 13-LGS may be a potentially useful model for studying human central retinal function and disease. That retinal remodeling and restoration of electrophysiological function occurs rapidly on emergence from hibernation implies the 13-LGS may also be a useful tool for studying aspects of retinal physiology and recovery from injury.

Validating a low-cost laser speckle contrast imaging system as a quantitative tool for assessing retinal vascular function.

The ability to monitor progression of retinal vascular diseases like diabetic retinopathy in small animal models is often complicated by their failure to develop the end-stage complications which characterize the human phenotypes in disease. Interestingly, as micro-vascular dysfunction typically precedes the onset of retinal vascular and even some neurodegenerative diseases, the ability to visualize and quantify hemodynamic changes (e.g. decreased flow or occlusion) in retinal vessels may serve as a useful diagnostic indicator of disease progression and as a therapeutic outcome measure in response to treatment. Nevertheless, the ability to precisely and accurately quantify retinal hemodynamics remains an unmet challenge in ophthalmic research. Herein we demonstrate the ability to modify a commercial fundus camera into a low-cost laser speckle contrast imaging (LSCI) system for contrast-free and non-invasive quantification of relative changes to retinal hemodynamics over a wide field-of-view in a rodent model.

A Comparison of Inducible Gene Expression Platforms: Implications for Recombinant Adeno-Associated Virus (rAAV) Vector-Mediated Ocular Gene Therapy.

The ability to temporally control levels of a therapeutic protein in vivo is vital for the development of safe and efficacious gene therapy treatments for autosomal dominant or complex retinal diseases, where uncontrolled transgene overexpression may lead to deleterious off-target effects and accelerated disease progression. While numerous platforms exist that allow for modulation of gene expression levels - ranging from inducible promoters to destabilizing domains - many have drawbacks that make them less than ideal for use in recombinant adeno-associated virus (rAAV) vectors, which over the past two decades have become the mainstay technology for mediating gene delivery to the retina. Herein, we discuss the advantages and disadvantages of three major gene expression platforms with regard to their suitability for ocular gene therapy applications.

Presynaptic Expression of LRIT3 Transsynaptically Organizes the Postsynaptic Glutamate Signaling Complex Containing TRPM1.

Throughout the CNS, interactions between pre- and postsynaptic adhesion molecules establish normal synaptic structure and function. Leucine-rich repeat (LRR) domain-containing proteins are a large family that has a diversity of ligands, and their absence can cause disease. At the first retinal synapse, the absence of LRIT3 expression leads to the disassembly of the postsynaptic glutamate signaling complex (signalplex) expressed on depolarizing bipolar cell (DBC) dendrites. The prevalent view is that assembly of the signalplex results from direct postsynaptic protein:protein interactions. In contrast, we demonstrate that LRIT3 is expressed presynaptically, in rod photoreceptors (rods), and when we restore LRIT3 expression in Lrit3-/- rods, we restore expression of the postsynaptic glutamate signalplex and rod-driven vision. Our results demonstrate that, in the retina, the LRR-containing protein LRIT3 acts as a transsynaptic organizer of the postsynaptic complex required for normal synaptic function.

Acid Ceramidase Deficiency in Mice Leads to Severe Ocular Pathology and Visual Impairment.

Farber disease (FD) is a debilitating lysosomal storage disorder characterized by severe inflammation and neurodegeneration. FD is caused by mutations in the ASAH1 gene, resulting in deficient acid ceramidase (ACDase) activity. Patients with ACDase deficiency exhibit a broad clinical spectrum. In classic cases, patients develop hepatosplenomegaly, nervous system involvement, and childhood mortality. Ocular manifestations include decreased vision, a grayish appearance to the retina with a cherry red spot, and nystagmus. That said, the full effect of ACDase deficiency on the visual system has not been studied in detail. We previously developed a mouse model that is orthologous for a known patient mutation in Asah1 that recapitulates human FD. Herein, we report evidence of a severe ocular pathology in Asah1P361R/P361R mice. Asah1P361R/P361R mice exhibit progressive retinal and optic nerve pathology. Through noninvasive ocular imaging and histopathological analyses of these Asah1P361R/P361R animals, we revealed progressive inflammation, the presence of retinal dysplasia, and significant storage pathology in various cell types in both the retina and optic nerves. Lipidomic analyses of retinal tissues revealed an abnormal accumulation of ceramides and other sphingolipids. Electroretinograms and behavioral tests showed decreased retinal and visual responses. Taken together, these data suggest that ACDase deficiency leads to sphingolipid imbalance, inflammation, dysmorphic retinal and optic nerve pathology, and severe visual impairment.

A Destabilizing Domain Allows for Fast, Noninvasive, Conditional Control of Protein Abundance in the Mouse Eye - Implications for Ocular Gene Therapy.

Purpose: Temporal and reversible control of protein expression in vivo is a central goal for many gene therapies, especially for strategies involving proteins that are detrimental to physiology if constitutively expressed. Accordingly, we explored whether protein abundance in the mouse retina could be effectively controlled using a destabilizing Escherichia coli dihydrofolate reductase (DHFR) domain whose stability is dependent on the small molecule, trimethoprim (TMP). Methods: We intravitreally injected wild-type C57BL6/J mice with an adeno-associated vector (rAAV2/2[MAX]) constitutively expressing separate fluorescent reporters: DHFR fused to yellow fluorescent protein (DHFR.YFP) and mCherry. TMP or vehicle was administered to mice via oral gavage, drinking water, or eye drops. Ocular TMP levels post treatment were quantified by LC-MS/MS. Protein abundance was measured by fundus fluorescence imaging and western blotting. Visual acuity, response to light stimulus, retinal structure, and gene expression were evaluated after long-term (3 months) TMP treatment. Results: Without TMP, DHFR.YFP was efficiently degraded in the retina. TMP achieved ocular concentrations of ∼13.6 µM (oral gavage), ∼331 nM (drinking water), and ∼636 nM (eye drops). Oral gavage and TMP eye drops stabilized DHFR.YFP as quickly as 6 hours, whereas continuous TMP drinking water could stabilize DHFR.YFP for ≥3 months. Stabilization was completely and repeatedly reversible following removal/addition of TMP in all regimens. Long-term TMP treatment had no impact on retina function/structure and had no effect on >99.9% of tested genes. Conclusions: This DHFR-based conditional system is a rapid, efficient, and reversible tool to effectively control protein expression in the retina.

Development of an inducible anti-VEGF rAAV gene therapy strategy for the treatment of wet AMD.

Vascular endothelial growth factor (VEGF) is a key mediator in the development and progression of choroidal neovascularization (CNV) in patients with wet age-related macular degeneration (AMD). As a consequence, current treatment strategies typically focus on the administration of anti-VEGF agents, such as Aflibercept (Eylea), that inhibit VEGF function. While this approach is largely successful at counteracting CNV progression, the treatment can require repetitive (i.e. monthly) intravitreal injections of the anti-VEGF agent throughout the patient's lifetime, imposing a substantial financial and medical burden on the patient. Moreover, repetitive injection of anti-VEGF agents over a period of years may encourage progression of retinal and choroidal atrophy in patients with AMD, leading to a decrease in visual acuity. Herein, we have developed a single-injection recombinant adeno-associated virus (rAAV)-based gene therapy treatment for wet AMD that prevents CNV formation through inducible over-expression of Eylea. First, we demonstrate that by incorporating riboswitch elements into the rAAV expression cassette allows protein expression levels to be modulated in vivo through oral supplementation on an activating ligand (e.g. tetracycline). We subsequently utilized this technology to modulate the intraocular concentration of Eylea following rAAV delivery, leading to nearly complete (p = 0.0008) inhibition of clinically significant CNV lesions in an established mouse model of wet AMD. The results shown in this study pave the way for the development of a personalized gene therapy strategy for the treatment of wet AMD that is substantially less invasive and more clinically adaptable than the current treatment paradigm of repetitive bolus injections of anti-VEGF agents.

Small and Micro-Scale Recombinant Adeno-Associated Virus Production and Purification for Ocular Gene Therapy Applications.

Over the past two decades recombinant adeno-associated virus (rAAV) vectors have emerged as the gold standard for transferring genetic material to cells of the retina. The ability to effectively produce small batches of rAAV vector at high enough purity for in vitro and in vivo applications in a cost-effective manner is paramount. This is particularly the case when conducting preclinical experiments to screen novel serotypes, promoters or transgenes, where production of numerous vector batches is required. Current vector production methods often produce large quantities of vector, limiting the cost-effectiveness and practicality of such screening experiments, which often require only small volumes of vector to carry out. Herein, we describe a method to produce high titer (1012-1013 vector genomes (vg)/mL) rAAV vector on small (~100 µL) or micro (~15 µL) scale for in vitro and in vivo applications.

Improvement of Photoreceptor Targeting via Intravitreal Delivery in Mouse and Human Retina Using Combinatory rAAV2 Capsid Mutant Vectors.

Purpose: Effective intravitreal gene delivery to cells of the central retina (i.e., photoreceptors) would be of substantial benefit for treating patients with retinal diseases, such as achromatopsia, where retinal detachment from a subretinal may be harmful. Previous studies demonstrated that mutation of the recombinant adeno-associated virus (rAAV) capsid through introduction of peptide insertions or amino acid substitutions dramatically alters vector tropism. Herein, we evaluate the photoreceptor transduction efficiency of three rAAV2/2-based capsid mutant vectors: rAAV2/2[7m8], rAAV2/2[QuadYF+TV], and a chimeric vector incorporating both mutations (termed rAAV2/2[MAX]) following intravitreal delivery in mice. Furthermore, we evaluate the transduction efficiency of rAAV2/2[MAX] using explanted human central retinal samples to address clinical translatability. Methods: Vectors containing a GFP or mCherry reporter gene were intravitreally injected into C57BL/6J or Nrl-EGFP mice, respectively. Transduction was assessed in vivo utilizing a custom multiline confocal scanning laser ophthalmoscope. Injected Nrl-EGFP mouse retinas were used to quantify transduced photoreceptors using flow cytometry. Postmortem human retinal tissue was cultured following administration of rAAV2/2[MAX]. C57BL/6J retinas and human explants were cryosectioned to determine vector tropism. Results: The chimeric vector rAAV2/2[MAX] transduced significantly higher proportions of the retina than did either single mutant serotypes following intravitreal delivery in murine retina, including inner retinal cells and photoreceptors. Vector rAAV2[MAX] demonstrated transduction of human photoreceptors and ganglion cells. Conclusions: Transduction observed via rAAV2/2[MAX] indicates that combining mutations with complementary mechanisms of action in a single vector results in enhanced transduction. rAAV2/2[MAX] also presented the ability to transduce human photoreceptors and ganglion cells, indicating potential for efficient intravitreal vector delivery.

Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4-/- mouse and bipolar cells in the rd1 mouse and human retina ex vivo.

Gene therapy using adeno-associated viral (AAV) vectors for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4-/- and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4-/- mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treat retinal degenerations in which high levels of transgene expression are required.

Systemic Vascular Transduction by Capsid Mutant Adeno-Associated Virus After Intravenous Injection.

The ability to effectively deliver genetic material to vascular endothelial cells remains one of the greatest unmet challenges facing the development of gene therapies to prevent diseases with underlying vascular etiology, such as diabetes, atherosclerosis, and age-related macular degeneration. Herein, we assess the effectiveness of an rAAV2-based capsid mutant vector (Y272F, Y444F, Y500F, Y730F, T491V; termed QuadYF+TV) with strong endothelial cell tropism at transducing the vasculature after systemic administration. Intravenous injection of QuadYF+TV resulted in widespread transduction throughout the vasculature of several major organ systems, as assessed by in vivo bioluminescence imaging and postmortem histology. Robust transduction of lung tissue was observed in QuadYF+TV-injected mice, indicating a role for intravenous gene delivery in the treatment of chronic diseases presenting with pulmonary complications, such as α1-antitrypsin deficiency. The QuadYF+TV vector cross-reacted strongly with AAV2 neutralizing antibodies, however, indicating that a targeted delivery strategy may be required to maximize clinical translatability.