Ocular Gene Therapy Laboratory

Validating Laser Speckle Contrast Imaging as a Quantitative Tool for Measuring Retinal Vascular Function in Rodents
Dwani Patel¹, Thomas B. Connor², Daniel M. Lipinski^2,3

1. Cell Biology, Neurobiology, Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
2. Department of Ophthalmology and Visual Science, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
3. Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom;

Abstract Number: 172 - A0581

Purpose: Rodent models of diabetes mellitus fail to develop the characteristic proliferative retinal vascular abnormalities of end-stage diabetic retinopathy – limiting their use for studying disease pathogenesis and the effectiveness of therapeutic interventions. Thus, developing methodologies to measure early changes in vascular function, which occur in small animal species like in humans, would significantly benefit the development of novel therapies. Using a rodent model of branch retinal vein occlusion (BRVO), here we evaluate whether laser speckle contrast imaging (LSCI), a non-invasive technique capable of visualizing hemodynamic changes with high spatiotemporal resolution, can be used to quantify changes in rodent retinal vascular function.

Methods: A commercial fundus camera (Phoenix Micron IV) was adapted to enable full field illumination of the mouse retina using a 640nm coherent laser light source and a speckle image analysis software was developed in Matlab. Using a syringe pump, blood was forced at various flow rates (0-2 mL/min) through polyethylene tubing (0.43mm ID) to evaluate our ability to quantify relative blood flow rate using ex vivo LSCI. Next, we performed LSCI in C57BL/6J mice (N=5) pre- and post-euthanasia to confirm that observed changes in speckle pattern derive from the movement of blood cells through the retinal vessels. Lastly, fluorescein angiography and LSCI were performed in age and gender-matched C57BL/6J mice (N=10) before and after (D1 and D7) BRVO induction using a 532nm diode laser.

Results: Speckle contrast intensity correlated with flow rate in our ex vivo capillary tubing and in vivo mouse models. Our imaging system and analysis software are capable of high spatial (~10μm) and temporal (≥10ms) resolution, enabling visualization and quantification of blood flow even in retinal microvessels. Pre- and post-euthanasia LSCI confirmed that change in contrast intensity derived solely from movement of blood through retinal vessels. LSCI of BRVO models showed a reduction in venous drainage and blood flow rate in occluded vessels relative to unaffected vessels.

Conclusions: LSCI can be applied to visualize and detect changes in rodent retinal vascular perfusion with high spatial and temporal resolution. LSCI may be useful for assessing early vascular changes in retinal diseases and as an outcome measure to determine therapeutic treatment efficacy.

Biodistribution and tolerability of rAAV vectors in the anterior chamber for the treatment of primary open angle glaucoma
Kristina Ertel¹, Daniel M. Lipinski^2,3

1. Cell Biology Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
2. Ophthalmology and Visual Science, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
3. Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom;

Abstract Number: 2922 - A0109

Purpose: Poor adherence to existing topical treatments for lowering intra-ocular pressure (IOP) is a major risk factor in developing vision compilations in primary open angle glaucoma (POAG). Consequently, developing a long-acting, single use gene therapy to permanently lower IOP would significantly improve visual outcomes and patient quality of life. One barrier to development of such a therapy is the ability to safely and efficiently transduce cells of the anterior chamber. Herein, we evaluate the tropism, safety and tolerability of four recombinant adeno-associated virus (rAAV) serotypes following intracameral injection using in vivoimaging and post-mortem histology.

Methods: C57BL/6J mice were injected with 2.6x109 viral genomes of rAAV2/1, 2/6, 2/9, or 2/2[MAX] packaging a GFP reporter gene driven by a small chicken beta actin promoter (N=20). GFP expression within the anterior chamber was assessed in vivofour weeks post injection using confocal scanning laser ophthalmoscopy (cSLO). Anterior chamber optical coherence tomography (OCT) imaging was used to evaluate corneal health and thickness following rAAV administration. Slit lamp examination and flare scoring was performed to assess inflammatory response to rAAV following intracameral injection. Following in vivoevaluations, eyes from all animals were enucleated and fixed for histological evaluation to confirm rAAV tropism.

Results: Corneal OCT and slit lamp evaluation showed no evidence of inflammation or increased corneal thickness compared to baseline levels. cSLO imaging of rAAV2/6 injected eyes revealed widespread corneal transduction. Injection of rAAV[MAX] and 2/9 also transduced the cornea, while rAAV 2/1 showed transduction of the iris and angle.

Conclusions: Herein, we evaluate the transduction tropism and tolerance of differing rAAV serotypes in the anterior chamber. Importantly, we observed that intracameral administration of rAAV is well tolerated and does not induce an observable immune response or corneal alterations. rAAV2/6 resulted in highly efficient and specific transduction of corneal endothelial cells and may have use in the development of gene therapy treatments for endothelial dystrophies. rAAV2/2[MAX] was additionally able to transduce trabecular meshwork cells, making it an attractive candidate for mediating a gene delivery in glaucoma.

A comparison of the electroretinogram in the cone-dominant thirteen-lined ground squirrel and the rod-dominant Brown Norway rat
Hanmeng Zhang^1,2, Benjamin S. Sajdak^1,2, Dana K. Merriman³, Joseph Carroll^1,2, Maureen A. McCall⁴, Daniel M. Lipinski^2,5

1. Department of Cell biology, Neurobiology and Anatomy, Medical college of wisconsin, Milwaukee, Wisconsin, United States
2. Department of Ophthalmology & Visual Science, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
3. Department of Biology, University of Wisconsin Oshkosh, Oshkosh, Wisconsin, United States
4. Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville, Kentucky, United States
5. Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom;

Abstract Number: 5971 - A0468

Purpose: Rodents have been used extensively as models of ocular disease owing to their small size, low cost of maintenance and ease of genetic manipulation. Unfortunately, the retinae of the most commonly utilized species – mice and rats – are rod-dominant and poorly recapitulate both the anatomy and function of the human central retina. By contrast, the retina of the thirteen-lined ground squirrel (13-LGS) is highly cone-rich, making it an attractive potential model for studying diseases that primarily affect cone structure and function in humans. Herein we use electroretinography (ERG) to assess the visual function of the diurnal 13-LGS in comparison to that of the nocturnal rod-dominant Brown Norway (BN) rat.

Methods: Five-month old 13-LGS (n=6) and BN rats (n=6) initially underwent dark- (-4 to 1 log cd.s/m2) and light-adapted (0 to 1 log cd.s/m2) single flash (1Hz, 4ms) and flicker (5, 15 and 30Hz) ERG recordings to assess baseline retinal function of each species. The relative contributions of ON- and OFF-bipolar cells to the 13-LGS and BN rat ERG were assessed using a light-adapted, long-duration (500ms) flash stimulus following intravitreal injection of glutamatergic (2mM APB) and kainate (10μM ACET) receptor agonists.

Results: Dark-adapted a- and b-wave amplitudes were higher in the BN rat model compared to the 13-LGS under low light conditions (-4 to -1 log cd.s/m2), reflecting the relatively greater proportion of rod photoreceptors. The a- and b-wave amplitudes were greater in the 13-LGS at higher intensity light stimuli under both dark-adapted (0.48 to 1 log cd.s/m2) and light-adapted (0 to 1 cd.s/m2, 25 cd/m2 background) conditions, reflecting the greater contribution of cones to the overall ERG response. Intravitreal injection of 2mM APB and 10μM ACET significantly decreased b-wave and d-wave response (by paired-t test), respectively, in both the BN rat and 13-LGS models, indicating post-synaptic signaling pathways are similar in both species.

Conclusions: ERG responses for both BN rats and 13-LGS reflected the relative proportions of cone photoreceptors present within the retinae. That the 13-LGS is highly cone-dominant may make it a useful model for studying human central retinal function.

Intravitreal Delivery of rAAV2 Vectors to the 13-Lined Ground Squirrel Retina
Benjamin S. Sajdak^1,2, Kristina J. Ertel¹, Hanmeng Zhang¹, Emily R. Nettesheim³, Dana K. Merriman⁴, Daniel M. Lipinski^1,3, Joseph Carroll^1,3

1. Cell Biology, Neurobiology, & Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
2. McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
3. Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
4. Biology, University of Wisconsin Oshkosh, Oshkosh, Wisconsin, United States;

Abstract Number: 2896 - A0083

Purpose: To deliver rAAV2 vectors to 13-lined ground squirrel (13-LGS) cone photoreceptors via intravitreal injection and assess artefacts arising from these injections that may impact subsequent longitudinal imaging studies.

Methods: Newborn (P1; n=43) and adolescent (P70-163; n=11) 13-LGS received intravitreal injections of a capsid mutant rAAV2/2 vector containing the [7m8] peptide insertion (R588_Q589insLALGETTRPA) and five point mutations (Y272F, Y444F, T491V, Y500F, Y730F) termed herein, rAAV2[MAX], packaging an mCherry reporter gene under control of either a ubiquitously expressing CBA promoter or a photoreceptor specific GRK1 promoter. Cornea/lens damage was examined using a Bioptigen Envisu R2200 OCT. Retinal transduction of mCherry was assessed in vivo using a Heidelberg Spectralis confocal SLO, and ex vivo with immunocytochemistry. Cone transduction of mCherry was quantified with flow cytometry using dissociated 13-LGS retina incubated with antibodies to M- and S-opsin.

Results: Iris synechia was found in 65% of newborn-injected eyes, but only 1 (4%) of the adolescent-injected eyes. The outcome of iris synechia in newborn-injected 13-LGS was independent of injection substance (p = 0.29; Chi Square test), suggesting it was due to the injection procedure. Conversely, 27% of adolescent-injected eyes had hyper-reflective puncta in anterior and/or posterior chambers, but this was not seen in any newborn-injected eyes. This presumed inflammatory response was specific to rAAV2[MAX] injected eyes with the CBA promoter. Successful but variable cone transduction was seen using flow cytometry regardless of injection age or promoter used. From 0.03% to 15.1% of opsin positive cells also expressed mCherry. Newborn injections of rAAV2[MAX]-CBA resulted in strong lens expression of mCherry, and cone transduction was minimal in these animals. Preliminary flow cytometry results suggest that 13-LGS cones are transduced most efficiently with rAAV2[MAX]-CBA administered during adolescence.

Conclusions: Intravitreal injections of rAAV2[MAX] with either CBA or GRK1 promoters can effectively target cone photoreceptors in the 13-LGS. Injecting these vectors in newborn animals often results in mild iris synechia and lens damage, which in most cases does not inhibit retinal imaging with OCT. Injecting these vectors in adolescent animals can result in inflammation, which in only one instance inhibited OCT retinal imaging.

Role of presynaptic LRIT3 expression in the restoration of dim light vision
Nazarul Hasan¹, Gobinda Pangeni², Catherine Cobb¹, Emily R. Nettesheim³, Daniel M. Lipinski^3,5, Maureen A. McCall2,⁴, Ronald G. Gregg^1,4

1. Biochemistry and Molecular Genetics, University of Louisville, Louisville, Kentucky, United States
2. Ophthalmology and Visual Sciences, University of Louisville, Louisville, Kentucky, United States
3. Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
4. Anatomical Sciences & Neurobiology, University of Louisville, Louisville, Kentucky, United States
5. Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom;

Abstract Number: 4788

Purpose: Complete congenital stationary night blindness (cCSNB) is a genetically heterogeneous disorder of the retina characterized by impairment of low light vision and loss of the b-wave of the electroretinogram (ERG). In mouse models of cCSNB, normal ON retinal ganglion cell responses are absent. Mutations in Grm6, Trpm1, Nyx, Gpr179 and Lrit3, which encode proteins critical to signal transmission in one class of retinal interneurons, the ON bipolar cells, cause cCSNB. LRIT3 is a leucine rich repeat protein that when absent causes loss of TRPM1 and Nyctalopin expression from the dendritic tips of rod bipolar cells, and in addition mGluR6, GPR179 and the RGS complex of proteins from dendritic tips of cone ON bipolar cells. Here we investigate the mechanism by which LRIT3 impacts the rod bipolar cells signalplex, and its cellular location.

Methods: Using a fluorescence based in situhybridization we examined the expression of Lrit3 mRNA in fixed retinal sections of wild type mice. To express LRIT3 in rod photoreceptors we used a human rhodopsin promoter to control expression in rAAVs. The expression construct was packaged in the rAAV2/2[MAX] capsid. rAAVs were intravitreally injected in mice at postnatal day 5 (P5). We characterized retinal function of rAAV-injected Lrit3-/-mice using ERG and multi-electrode array (MEA). Localization of LRIT3 and TRPM1 were examined by immunohistochemistry.

Results: We show that LRIT3 is a presynaptic protein and its expression in rod photoreceptors via rAAV restores function. LRIT3 restored its expression on the rod axon terminals where it co-localized with mGluR6. This also restored postsynaptic TRPM1 expression to the rod bipolar cells dendrites. Functional analyses showed that AAV mediated LRIT3 expression in rods partially restored the scotopic b-wave of the ERG in Lrit3-/- mice. MEA recordings of retinas from AAV injected Lrit3-/- mice, showed a significant restoration of normal retinal ganglion cell ON responses.

Conclusions: Our data show that LRIT3 acts in a trans-synaptic manner to localize the rod bipolar cell signalplex protein TRPM1. This finding is the first example of such a mechanism in the retina. These data also show the potential for gene therapy in LRIT3 mutant cCSNB patients by AAV mediated gene delivery.

Effect of inducible VEGF-trap expression on CNV formation in a murine model of wet AMD following intravitreal administration of a capsid mutant rAAV-riboswitch vector

Christopher A. Reid, Emily R. Nettesheim, Thomas B. Connor¹, Daniel M. Lipinski^1,2

Preclinical Approaches in Gene Therapy for Neurosensory Disorders Session: 4.55pm – 5.00pm. Salon A-1/2

Purpose: Age-related macular degeneration (AMD) is one of the leading causes of vision loss in the developed world. In 15% of patients, AMD progresses to an exudative stage characterized by chorodial neovascularization (CNV) and loss of central vision. Development of CNV is triggered largely through extracellular vascular endothelial growth factor A (VEGFA) signaling. consequently, current treatments for wet AMD are based around repetitive administration of VEGF traps (e.g. Eylea). Although anti-VEGF treatments have proven to be extremely effective at preventing CNV, continuous anti-VEGF exposure over a period of years has been shown to cause increased retinal pigment epithelium (RPE) and photoreceptor atrophy, leading to a decrease in visual acuity. Here, we evaluate the efficacy of a tetracycline responsive ‘OFF-type’ riboswitch (TC45) in regulating Eylea expression in the mouse retina following recombinant adeno-associated viral (rAAV) delivery.

Methods: Six-week-old C57BL/6J mice were intravitreally injected with PBS or 5.0x1010vg of rAAV encapsulating either a constitutively active (smCBA-Eylea) or a tetracycline-inducible (smCBA-Eylea-1x-TC45) Eylea construct. smCBA-Eylea-1x-TC45 injected mice were placed in two groups with mice receiving standard diet (ON-state; high levels of Eylea expression) or tetracycline diet (OFF-state; reduced Eylea expression). CNV was induced using an 810nm diode laser six weeks following injection. Seven days following laser injury, CNV formation was evaluated using fluorescein angiography with leakage independently graded by three blinded individuals. Finally, eyes were harvested and the concentration of Eylea was determined by ELISA.

Results: Constitutive expression of Eylea (smCBA-Eylea) significantly reduced the severity of CNV formation and leakage compared to PBS sham injected eyes (p<0.0001). Moreover, smCBA-Eylea-1x-TC45 injected mice receiving standard diet (Eylea expression ‘ON’) had a significantly reduced number of clinically significant lesions compared to smCBA-Eylea-1x-TC45 injected mice receiving tetracycline diet (Eylea expression ‘OFF’) (p=0.0008). Tetracycline mediated activation of the TC45 riboswitch resulted in a significant decrease in Eylea concentration in smCBA-Eylea-1x-TC45 injected mice (p<0.05).

Conclusions: Over-expression of Eylea in the mouse retina following rAAV delivery significantly reduced the severity of CNV formation. Furthermore, tetracycline mediated TC45-activation significantly reduced the expression of Eylea, resulting in an increase of clinically significant lesions. In summary, riboswitch mediated regulation of Eylea opens the door for the development of a personalized gene therapy strategy for the treatment of wet AMD. Ultimately, we propose that neovascularization in wet AMD can be controlled following a single administration of an rAAV.riboswitch.anti-VEGF vector that allows for periodic over-expression of a VEGF trap simply through dosing of the activating ligand (e.g. an oral drug). This strategy would effectively eliminate the need for invasive, repetitive (i.e. monthly) intra-ocular injections, greatly improving treatment safety and patient quality of life.

Protease Inhibitors Overexpression Eliminate the Toxicity Effect on ERG In Vivo by AAV-Mediated Gene Delivery and Protect ARPE19 Cells from Oxidative Stress and Cell Death In Vitro

Hanmeng Zhang¹, Christopher A. Reid¹, Emily R. Nettesheim¹, Daniel M. Lipinski^2,3

Neurologic Diseases (Including Ophthalmic and Auditory Diseases) II: Thursday May 17, 2018 5:15 PM - 7:15 PM, Room: Stevens Salon C, D

Purpose: Ciliary neurotrophic factor (CNTF) has been demonstrated to be effective at rescuing rod- and cone-photoreceptors in animal models of RP, but at the cost of suppressing electrophysiological responses. We previously conducted a transcriptome analysis of 23,365 mouse genes in order to determine the mechanisms underlying long-term neuroprotection of cone photoreceptors following recombinant adeno-associated virus (rAAV)-mediated over-expression of CNTF. Of the 460 genes found to be significantly differentially expressed in high dose rAAV.CNTF treated eyes comparedreated controls, peptidase inhibitor gene families were identified as being most highly over-expressed (up to 90-fold). As peptidase inhibitors are known to be anti-apoptotic, protective against oxidative stress, and to prevent degradation of the cell membrane and extracellular matrix, we sought to investigate whether they can directly protect against cone photoreceptor degeneration without ERG suppression.

Methods: Wild-type (C57Bl/6j) mice received a unilateral intravitreal injection of rAAV2/2 vector expressing either CNTF, Serpina5, Serpina3k, Wfdc6a or Timp3; the contralateral eye received a sham buffer injection of equal volume (2μl). All animals were followed up by repetitive electroretinography (ERG) to determine whether over-expression of proteolysis inhibitors has a similar suppressive effect as CNTF. A novel in vitro co-culture assay was used to determine the anti-oxidative and anti-apoptotic effects of CNTF (positive control) and each protease inhibitor. HEK293T cell monolayers were transfected with plasmid DNA expressing either CNTF, Serpina3K, Serpina5, Timp3 or Wfdc6a. ARPE19 cells were then plated in tissue culture inserts and suspended above the transfected HEK293T cell monolayer, allowing for diffusion of secreted factors (CNTF or inhibitors) from HEK293T cell monolayer to interact with the APRE19 cells. Oxidative stress and cell survival assays were performed on ARPE19 cells after incubation with 1mM H2O2 for one hour. A CellROX staining assay was used to detect oxidative stress levels while a calcein/ethidium bormide staining assay was used to measure cell viability. Fluorescence signals were quantified by plate reader and normalized to Hoechst staining, to control for cell number variability.

Results: 1) Four weeks post injection, ERG revealed that AAV2/2-meadiated over-expression of CNTF significantly reduced the mean amplitude of both a- wave and b- wave in dark- and light-adapted conditions. By contrast, AAV-mediated over-expression of protease inhibitors did not affect either dark- or light-adapted ERG responses in any group. Our in vitro co-culture assay revealed that CNTF and protease inhibitors were all protective against oxidative stress compared to untransfected H2O2 only controls. Encouragingly, the live/dead assay demonstrated significantly more living cells and fewer dead cells in all groups relative to control. Among those, CNTF and Serpina5 significantly increased cell survival, while CNTF, Serpina5 and Serpina3k significantly prevented cell death.

Conclusions: The preliminary data presented herein demonstrate that 1) over-expression of proteolysis inhibitors does not negatively affect ERG function in vivo compared to CNTF, and 2) that proteolysis inhibitors are protective against oxidative stress and cell death in vitro. Based on this work, we are currently evaluating the long-term rAAV-medaited protective effects of proteolysis inhibitor overexpression using a well establish mouse model of retinitis pigmentosa.

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

Dwani Patel¹, Damien Marsic³, Sergei Zolothukin³, & Daniel Lipinski1,^2,4

1. Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin
2. Department of Ophthalmology and Visual Sciences, Medical College of Wisconsin
3. Department of Pediatrics, University of Florida College of Medicine
4. Nuffield Laboratory of Ophthalmology, University of Oxford

Abstract 394: Thursday 17th May, 5.15pm – 7.15pm, Stevens Salon C,D

Purpose: The ability to deliver genetic material to cells of the retinal vasculature may facilitate the development of long-term gene therapy treatments for sight threatening diseases, such as diabetic retinopathy or age-related macular degeneration. While recombinant adeno-associated virus (rAAV) vectors have been used extensively to target retinal neurons and glia, their utility for vascular gene transfer remains limited due to extremely poor transduction efficiency - regardless of administration route. Herein, we employed a library screening approach to identify novel rAAV2-based capsid variants with improved ability to target retinal vascular pericytes following intravitreal delivery.

Methods: Directed evolution was used to generate a combinatorial library of rAAV2 based capsid variants where mutations existed only in the variable regions of the capsid. Three rounds of in vivo selection were carried out to identify mutants with the greatest tropism for retinal pericytes. Briefly, 15-20 Cspg4-DsRed reporter mice per round received bilateral intravitreal injections of 1x109-10 vector genomes. Retinal pericytes were harvested 6 days later by fluorescence-activated cell sorting (FACS) of enzymatically dissociated retinae followed by recovery of vector genomes (integrated and episomal) using column DNA purification. Following rounds one and two, the recovered capsid DNA sequences were amplified by PCR to prepare an enriched pericyte-targeting capsid library for the subsequent round of in vivo selection. After three rounds of selection, capsid DNA from retinal pericytes was amplified, inserted into pACG2-m56, and transformed, at which point 100-200 random clones were analyzed by next generation sequencing.

Results: Following three rounds of screening, library complexity had reduced significantly, leading to the identification of seven novel capsid mutant variants with greatly enhanced ability to target retinal pericytes. A ubiquitously expressing GFP reporter construct was packaged into each of these novel mutant capsids and injected intravitreally into to Cspg4-DsRed reporter mice. A combination of confocal scanning laser ophthalmoscopy, post-mortem histology, and flow cytometry revealed that the novel capsid mutant vectors identified had substantially improved ability to target retinal pericytes than the unmodified rAAV2 serotype on which the library was based.

Conclusions: Conducting multiple rounds of library screening under increasingly stringent conditions lead to a rapid decrease in library complexity and the isolation of several novel capsid mutant sequences with improved ability to target retinal pericytes. Despite the relative scarcity of retinal pericytes (>1,500 per eye) FACS followed by column-based DNA purification proved to be an effective technique for isolating target (i.e. red fluorescent) cells and recovering internalized vector genomes. The ability to target retinal pericytes using rAAV vector technology is highly encouraging for the development of a future gene therapy for diabetic retinopathy, a disease in which pericyte cell death precedes the development of sight threatening vascular complication, including proliferation and edema.

Riboswitch mediated modulation of transgene expression following rAAV delivery to the mouse retina
Christopher A. Reid & Daniel M. Lipinski

Poster B0088: 8.30am – 10.15am and 2.45pm – 3.45pm May 10th

Members-in-training outstanding poster award: 12pm – 3pm, May 9th

Purpose: Recombinant adeno-associated virus (rAAV) vectors have emerged as promising tools for mediating gene therapy for diseases of the retina. Effectively controlling gene expression levels following vector delivery is paramount to the success of potential gene therapies, where uncontrolled over-expression of the therapeutic transgenes can lead to toxicity. Traditionally, inducible promoter systems have been employed. Unfortunately, due to the limited coding capacity of AAV, and the large size of the regulatory elements required to make such systems work effectively, their inclusion is frequently not feasible. Here, we evaluate the use of small (~100bp) ligand responsive self-cleavable riboswitches to modulate gene expression in the mouse retina.

Methods: Six riboswitches (K19, Tc40x45, GuaM8HDV, L2Bulge18tc, L2Bulge9 and Theo6HDV) were evaluated in HEK293T cells to determine optimal copy number (largest dynamic range) and dose-responsiveness to its activating ligand using a dual luciferase assay. Next, the optimal copy number of each riboswitch was cloned into a rAAV GFP reporter cassette and packaged in an AAV2 capsid. Each GFP-riboswitch cassette was intravitreally injected into C57Bl/6j mice in combination with an AAV2 control vector harboring a non-inducible mCherry reporter gene. Four weeks post-injection, mCherry and GFP fluorescence levels were quantified in vivo using a custom ‘Multiline’ confocal scanning laser ophthalmoscope (cSLO). Mice subsequently received a dose of 1000mg/kg of its activating ligand, and fluorescence levels were quantified 2 and 24 hours post-gavage.

Results: Cell culture experiments revealed significant changes in firefly luminescence in response to dosing of the appropriate ligand (p<0.01, One-way ANOVA, N=4 all groups). In vivo results demonstrated dosing mice with the activating ligand of each riboswitch could achieve a highly significant change in GFP fluorescence at 2 hours post-gavage compared to pre-treatment levels (p<0.01, paired t test, N=6). Importantly, GFP fluorescence was recovered to pre-treatment levels 24 hours after receiving the activating ligand.

Conclusions: All riboswitch constructs were proficient at modulating transgene expression in vivo. Concurrent dual color cSLO imaging allowed riboswitch-mediated changes in GFP fluorescence to be normalized (to mCherry), effectively standardizing our in vivo measurements and greatly increasing detection sensitivity. Due to their small size and lack of immunogenicity, incorporation of riboswitches in rAAV constructs could have potential clinical applications where tightly controlled gene expression is paramount.

Intravitreal transduction profile of recombinant adeno-associated virus in murine and human retina
Kristina J. Ertel, Christpher A. Reid & Daniel M. Lipinski

Poster B0344: 8.30am – 10.15am and 2.45pm – 3.45pm May 7th

Purpose: The ability to target cells of the inner and outer retina via intravitreal injection is vital for treatment of diseases where detachment of the retina may be detrimental. It has previously been demonstrated that the tropism of recombinant adeno-associated virus (rAAV) vectors can be altered through mutation of the virion by site-directed mutagenesis or inclusion of short targeting peptides. Herein, we compare the transduction profiles of three rAAV2- based capsid mutant vectors –rAAV2.7m8, rAAV2.QuadYF-TV and a hybrid vector termed rAAV.MAX– following intravitreal injection in mice. Additionally, we use human retinal tissue to determine the ability of each vector to transduce macular cone photoreceptors – a critical consideration for clinical translation of intravitreal gene delivery.

Methods: rAAV vectors packaging a ubiquitously expressing mCherry reporter gene were injected intravitreally into adult NRL-EGFP and WT mice. Retinal fluorescence levels were quantified in vivo using a custom multiline confocal scanning laser ophthalmoscope (m-cSLO) allowing dual color fluorescence imaging. Post-mortem, double positive retinal cells were quantified by flow cytometry to determine the percent of photoreceptors transduced. Human retinal tissue from the Lion’s Eye Bank of Wisconsin was maintained in organotypic culture following administration of capsid mutant vectors packaging a GFP reporter gene. WT mouse eyes and human retinal explants were cryosectioned to histologically evaluate tropism.

Results: rAAV2.MAX exhibited greater retinal penetrance and photoreceptor transduction following intravitreal injection than either unmodified rAAV2 or the original capsid mutant vectors. Dual color m-cSLO imaging facilitated normalization of retinal fluorescence levels in vivo, allowing for quantification of rAAV-mediated transgene expression. rAAV2-MAX vector administration on human macular tissue resulted in substantial transduction of central photoreceptors.

Conclusions: The increased tissue penetrance and photoreceptor transduction properties of the rAAV2.MAX vector demonstrates the effects of capsid mutation and peptide insertion within the AAV capsid are cumulative. rAAV2.MAX was able to mediate gene transfer to both murine and human photoreceptors from the vitreous thus encouraging for the future treatment of retinal diseases where subretinal gene delivery may represent an unacceptable surgical risk.

Micro-RNA induced silencing of cytotoxic transgenes leads to increased recombinant adeno-associated virus (AAV) titers
Chris Reid , William Hauswirth & Daniel M. Lipinski

Paper presentation 2295: 4.15 – 4.30pm, May 2nd.

Purpose: The production of high titer recombinant adeno-associated virus (AAV) vector is essential for the successful treatment of genetic diseases affecting the retina and choroid, where anatomical constraints may substantially limit injectable volumes. Problematically, cytotoxicity arising from over-expression of the transgene during vector production frequently leads to a reduction in vector yield. This is particularly true of transgenes intended for ‘suicide’ gene therapy, such as herpes simplex virus thymidine kinase (HSVtk) or Bcl-2 associated X protein (BAX), which may be useful for the treatment of retinoblastoma. Herein, we evaluate the use of micro-RNA (miRNA) mediated silencing to effectively limit over-expression of cytotoxic transgenes during packaging as a method of increasing vector yield for ocular gene therapy applications.

Methods: Production of AAV serotype 2, 5 and 8 vectors packaging either a non-toxic fluorescent reporter (mCherry) or cytotoxic transgene (HSV-tk, ND4 or Bax) upstream of a quadruple repeated miRNA recognition site (mmu-miR-122-5p, mmu-miR-367-3p or hsa-miR-373-5p) was carried out in HEK293T cells by standard plasmid co-transfection. Transgene silencing was achieved during production by over-expression of the paired mature miRNA from either a separate expression plasmid or the adenoviral helper plasmid (pHelper-MIR). The titer (vg/ml) was compared to concurrently produced vector preparations without silencing by a Picogreen DNA assay.

Results: Over-expression of paired mature miRNA during vector production led to a significant (p<0.01) increase in viral titer for all serotypes when packaging the cytotoxic HSV-tk, ND4 and BAX transgenes. Vector yield was not substantially increased when packaging the mCherry construct. Importantly, the addition of the miRNA recognition site to the transgene cassette did not affect the ability of the packaged transgene to be expressed in the rodent eye.

Conclusions: The significant increase in vector yield observed following over-expression of miRNA indicates that transgene silencing during production is an effective method of increasing vector yields for ocular gene therapy applications. This finding may have significant economic and logistical implications for the production of AAV vectors for research and clinical use.