Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells

Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells

Accepted Manuscript Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal g...

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Accepted Manuscript Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells Christophe J. Langouet-Astrie, Zhiyong Yang, Sraavya M. Polisetti, Derek S. Welsbie, William W. Hauswirth, Donald J. Zack, Shannath L. Merbs, Raymond A. Enke PII:

S0014-4835(16)30210-X

DOI:

10.1016/j.exer.2016.07.019

Reference:

YEXER 6993

To appear in:

Experimental Eye Research

Received Date: 3 June 2016 Revised Date:

27 July 2016

Accepted Date: 28 July 2016

Please cite this article as: Langouet-Astrie, C.J., Yang, Z., Polisetti, S.M., Welsbie, D.S., Hauswirth, W.W., Zack, D.J., Merbs, S.L., Enke, R.A., Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells, Experimental Eye Research (2016), doi: 10.1016/j.exer.2016.07.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells Christophe J. Langouet-Astriea, Zhiyong Yangc, Sraavya M. Polisettia, Derek S. Welsbied, William W. Hauswirthi, Donald J. Zackd,e,f,g,h Shannath L. Merbsd, Raymond A. Enkea,b,j a

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Department of Biology, 951 Carrier Drive, MSC 7801, James Madison University, Harrisonburg, VA, 22807 b Center for Genome & Metagenome Studies, 951 Carrier Drive, MSC 7801, James Madison University, Harrisonburg, VA, 22807 c Department of Surgery, University of California San Diego, 4150 Regents Park Row, La Jolla, CA 92037 d Department of Ophthalmology, Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD 21287 e Department of Neuroscience, Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD 21287 f Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD 21287 g Institute of Genetic Medicine, Johns Hopkins University School of Medicine, 400 N. Broadway, Baltimore, MD 21287 h Institute de la Vision, Université Pierre et Marie Curie, 17 Rue Moreau, Paris, France, 75012 i Department of Ophthalmology, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610 j Corresponding author: [email protected]

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Abstract

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Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adenoassociated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 109 vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs.

Keywords

Retina, Cre-mediated mutation; Cre toxicity, Retinal ganglion cell, AAV2, Intravitreal injection, Optic neuropathy, Glaucoma

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Abbreviations

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RGCs, retinal ganglion cells; AAV2, Adeno-associated virus 2; R26, Rosa 26; Vg, vector genomes; GFP, green fluorescent protein; Tuj1, βIII-tubulin; ANOVA, analysis of variance; Y, Tyrosine; F, phenylalanine; INL, inner nuclear layer; ONL, outer nuclear layer; DLK, Dual Leucine Zipper Kinase; GCL, ganglion cell layer; sc, sclera; photoreceptors, PR

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1. Introduction Processing and transmitting visual information from the retina to the brain is achieved by projection neurons known as retinal ganglion cells (RGC) (Welsbie et al., 2013). Injury to these

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neurons results in neurodegenerative diseases such as glaucoma, a leading cause of irreversible blindness worldwide (Quigley and Broman, 2006; Welsbie et al., 2013).

Transcriptome and functional genomic studies in animal models of glaucoma have been critical

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in characterizing novel molecular pathways related to human disease (Howell et al., 2011;

Steele et al., 2006; Welsbie et al., 2013; Yang et al., 2007). These studies have uncovered

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many disease-related candidate genes, which require robust molecular analysis for further characterization. A common technique for studying gene-specific function in mice is targeted mutation using the Cre/loxP recombinase system (Le et al., 2006; Lee et al., 2006). Originating from a P1 bacteriophage, the 38 kDa Cre protein catalyzes recombination by forming homodimers between two 34 bp target loxP sequences thereby excising the intervening

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sequence (Le et al., 2006; Lee et al., 2006; Schmidt et al., 2000). Limited expression of Cre recombinase in retinal neurons can be achieved by fusing tissue-specific regulatory sequences to the Cre coding sequence (Nasonkin et al., 2013; Thanos et al., 2012). Alternatively, tissue-

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specific expression of Cre in the retina can be achieved using a recombinant viral delivery

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method (Le et al., 2006; Petrs-Silva et al., 2011, 2009).

Adeno-associated virus (AAV) viral vectors have been used in gene therapy and conditional gene targeting applications within the retina. AAV serotype 2 (AAV2) has demonstrated broad trophism for retinal cell types making it an effective serotype for retinal applications (Day et al., 2014). Furthermore, technical issues such as slow expression of viral genes and the host cell immune response have been addressed with genetically engineered AAV2 viral particles (McCarty et al., 2001; Petrs-Silva et al., 2011, 2009). AAV vectors with naturally occurring capsid proteins are known to have delayed onset of transgene expression in part limited by

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host-mediated degradation of virions. To circumvent these issues, triple tyrosine-tophenylalanine (Y-F) capsid mutant AAV2 vectors at residuals 444, 500, 730 demonstrated an increased ability to evade host cell degradation and displays a significant increase in

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transduction efficiency relative to non-mutant AAV2 in the murine retina (Petrs-Silva et al., 2011, 2009). Previous studies have demonstrated a 30-fold increase in transgenic GFP expression using triple capsid mutant vectors compared to wild type vectors in ganglion cell layer (Petrs-

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Silva et al., 2009). Intravitreal injection of capsid mutant AAV2 particles is particularly useful for

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targeting the inner most GCL retinal neurons (Day et al., 2014; Petrs-Silva et al., 2009).

Prolonged and/or high levels of Cre expression in mammalian cells have the potential to induce adverse effects independent of loxP sites (De Alboran et al., 2001; Lee et al., 2006; Loonstra et al., 2001; Schmidt et al., 2000; Silver and Livingston, 2001). Cre toxicity has been reported in various cell types both in vitro and in vivo, including cultured mammalian cells (Loonstra et al.,

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2001) and pancreatic β-cells in murine models (Lee et al., 2006). Specifically in the eye, Cre expression in the Trp1-Cre transgenic mouse leads to RPE dysfunction, disorganization and atrophy as well as retinal photoreceptor dysfunction (Thanos et al., 2012). Cre toxicity in RGCs

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has not been previously characterized to our knowledge. Our current study focuses on determining dosage parameters for intravitreal delivery of triple capsid mutant AAV2 vector to

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mouse GCL retinal neurons as well as characterizing toxic effects to RGCs. Collectively, we demonstrate that intravitreal delivery of capsid mutant AAV2 particles encoding Cre recombinase can be used as an effective short-term strategy for inducing targeted gene mutations in murine RGCs.

2. Materials and Methods 2.1. Production of recombinant AAV2 vectors.

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Self-complementary, capsid-mutant (Y444, 500, 730F) AAV2 viral particles were created as previously described (Petrs-Silva et al., 2011, 2009). Briefly, site-directed mutagenesis of surface-exposed tyrosine residues on AAV2 VP3 was performed (Petrs-Silva et al., 2011,

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2009). Vector preparations were produced by the plasmid co-transfection method (Petrs-Silva et al., 2011, 2009). The crude iodixanol fraction was further purified and concentrated by column chromatography on a 5 ml HiTrap Q Sepharose column using a Pharmacia AKTA FPLC system

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(Amersham Biosciences, Piscataway, NJ). The vector was eluted from the column using 215 mM NaCl, pH 8.0, and the vector containing fractions were collected, pooled, concentrated and

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buffer exchanged into Alcon BSS with 0.014% Tween 20, using a Biomax 100 K concentrator (Millipore, Billerica, MA). The titer of DNase-resistant vector genomes was measured by realtime PCR relative to a standard. Finally, the purity of the vector was validated by silver-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis, assayed for sterility and lack of

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endotoxin and then aliquoted and stored at −80°C.

2.2. Animals

All animal experiments were conducted with the approval of the Johns Hopkins Animal Care

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and Use Committee and in accordance with the National Institutes of Health guide for the care and use of Laboratory animals. 6-12 week old female C57BL/6 mice and R26 LacZ floxed mice

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in a B/6 background (Mus musculus; The Jackson Laboratory, Bar Harbor, ME) were used in this study. Mice were maintained in standard conditions and fed a standard rodent diet.

2.3. Intravitreal injection

Mice were anesthetized with ketamine/xylazine and intravitreally injected using 1 µL of mutant AAV2 viral particles expressing either the coding sequence of Cre recombinase or green fluorescent protein (GFP) from the chicken β-actin promoter. Intraocular injections were done under a dissecting microscope with a Harvard Pump Microinjection System and pulled glass

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micropipettes as previously described (Mori et al., 2001). Individual mice were injected with AAV2-Cre in one eye and AAV2-GFP control vector in the fellow eye with vector genome (vg) titers ranging from 105 vg-1010 vg. Following injection, animals were monitored until awakening.

St. Joseph, MO) exposure followed by cervical dislocation.

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2.4. Histology

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Animals were euthanized at time intervals of 2-15 weeks post-injection using IsoSol™ (VEDCO,

Eyes and companion optic nerves were harvested, fixed and assayed for RGC survival or viral

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transduction. Viral transduction in the retina of R26 LacZ floxed mice was assayed using an Xgal assay as previously described (Thanos et al., 2012). RGC survival in the retina was assayed by flat mount immunostaining using rabbit anti-βIII-tubulin (Covance, 1-15-79 clone) and goat anti-Brn3 (Santa Cruz, C-13 epitope; detects Brn-3a, Brn-3b and Brn-3c) primary antibodies in conjunction with fluorescently labeled secondary antibodies as previously described (Welsbie et

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al., 2013). Retinas were imaged with a Nikon Eclipse TE2000-5 fluorescence microscope and Plan-fluor 40×/0.6 objective. For each retina, technical quadruplicate images were acquired from each of the superior, inferior, temporal, and nasal quadrants, 1 mm from the optic disk for a

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total of 16 images per eye. Each time point was assayed in biological triplicate with the exception of the exception of the 15 week cohort, which consisted of 2 mice. RGCs were

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counted manually from each image and technical and biological replicates were averaged. Companion optic nerves from the same cohort of animals were sectioned and assayed for βIIItubulin (Tuj1) to assess RGC axon survival and GFP expression to assess AAV2 transgene expression. Fluorescent nerve cross-section images were obtained using a Nikon Digital Eclipse C1Si fluorescence microscope and a Plan-fluor 20×/0.5 objective. For quantitative RGC axon survival assessment, average pixel intensity was calculated from 3 separate nerves (biological triplicates) as well as 3 serial sections per nerve (technical triplicates) as previously described using ImageJ software (Schneider et al., 2012). Cre expression was assayed in retinal cross

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sections using mouse an anti-Cre primary antibody (Millipore, MAB3120) in conjunction with fluorescently labeled secondary antibody. No primary control staining was conducted to assay autofluorescence as well as non-specific staining of the mouse anti-Cre antibodies. Detailed

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information on all antibodies used in this study is listed in Table S1.

2.5. Statistical Analysis

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Animal cohorts for each experiment consisted of 3 mice bilaterally injected with AAV2-Cre and AAV2-GFP respectively with the exception of the 15 week cohort, which consisted of 2 mice. All

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data are expressed as mean ± standard error. Group means were compared by two-way analysis of variance (ANOVA) followed by Holm-Sidak multiple-comparisons test. Analysis of all data was performed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA); p < 0.05 was considered statistically significant.

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3. Results & Discussion 3.1. Experimental Design Overview

One goal of this study was to determine an effective experimental dosage of an intravitreally-

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delivered tyrosine capsid mutant AAV2 viral vector encoding Cre recombinase to target murine GCL retinal neurons. Figure 1 outlines the experimental overview used to achieve this goal.

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Capsid mutant AAV2 viral vectors harboring Cre recombinase or GFP coding sequences were intravitreally injected into wt C57BL/6 or R26 LacZ floxed mice on day 1 (wk 0). Ocular tissues were subsequently harvested beginning week 2 post-injection until week 15 post-injection then assessed for viral transgene expression, Cre recombinase activity and RGC survival. Animal cohorts for each experiment consisted of 3 mice bilaterally injected with AAV2-Cre and AAV2GFP respectively with the exception of the 15 week cohort, which consisted of 2 mice. Eyes and companion optic nerves were harvested from the same animal for experiments assaying RGC cell bodies in the retina as well as axons in the optic nerve.

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3.2. Determination of Viral Vector Dosage Point mutagenesis of three self complementary AAV2 capsid tyrosine (Y) residues

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(Y444,500,730) to phenylalanine (F) enhances the intravitreally-delivered vector transduction efficiency in RGCs cells by more than 30-fold within two weeks post infection (Petrs-Silva et al., 2011). A qualitative titer dilution experiment was used to determine the lowest possible dose of

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capsid mutant AAV2-Cre viral particles required to achieve a uniform infection and transduction of the RGC retinal layer following intravitreal delivery for subsequent experiments. Vector doses

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ranging from 105 to 1010 vg of AAV2-Cre or AAV2-GFP were intravitreally injected into fellow eyes of R26 LacZ floxed mice. Two weeks later, Cre activity was measured using a qualitative ß-galactosidase assay and retinal whole mount images were obtained for eyes in each sample group. Uniform Cre activity was observed throughout the retina two weeks post-injection in AAV2-Cre injected eyes until vector titers were diluted to 108 vg (Figure 2A-C, Figure S1).

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AAV2-Cre injections diluted to 108 vg resulted in patches of retina devoid of Cre activity (Figure 2C). Further dilution of AAV2-Cre vector resulted in dramatically less Cre activity (Figure 2D-F). Based on these qualitative observations a titer of 109 vg was chosen as a suitable dosage for

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subsequent experiments.

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3.3. Short and Long-term Expression of Viral Transgenes in RGCs Following Intravitreal Injection.

Cre recombinase and GFP expression analysis were conducted to characterize the onset of expression of virally delivered transgenes. Using the triple mutant AAV-Cre vector, uniform Cre expression is observed within the GCL as early as 2 weeks. Strong Cre expression was also observed in cells within the inner nuclear layer (INL). Sparse Cre expression was observed in the outer nuclear layer (ONL) of the retina (Figure 3A-3B). Transgene expression analysis was used to determine the distribution of retinal neurons infected and expressing virally delivered

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genes following intravitreal AAV2 injection. Serial sections without primary antibody incubation confirmed that staining of the RPE and choroid in the outermost portion of the tissue was due to autofluorescence and/or non-specific secondary antibody cross reactivity in these tissues

term axonal transgene GFP expression (Figure 3C-3D).

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(Figure S2). Cross sectioning and staining of the optic nerve demonstrates both short and long-

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3.4. Toxicity in RGC Associated with Long-term Expression of Cre Recombinase

Expression of Cre recombinase in mice and other mammalian cells lacking loxP recognition

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sites has been linked to Cre toxicity and cell death (De Alboran et al., 2001; Lee et al., 2006; Loonstra et al., 2001; Schmidt et al., 2000; Silver and Livingston, 2001). To assess the extent of Cre toxicity specifically in RGCs, 109 vg of capsid mutant AAV2-Cre and AAV2-GFP vectors were injected into fellow eyes of wild type mice and RGC survival was monitored at subsequent time points post-injection. RGC nuclei in retinal flat mounts were quantitated using the RGC-

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specific nuclear marker Brn3 as previously described (Welsbie et al., 2013). RGC axon viability in the retina and optic nerve were measured using a Tuj1 antibody as previously described (Soto et al., 2008; Welsbie et al., 2013). AAV2-GFP infected retina contained a similar amount

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of Brn3 positive viable RGCs in the retina from 2 weeks post-injection out to 15 weeks postinjection (Figure 4A-D+I). Retinas harvested from animals injected with AAV2-Cre maintained

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commensurate levels of viable RGCs out to 4 weeks post-injection; however, as early as 5 weeks post-injection, RGC viability decreased in eyes injected with AAV2-Cre compared to AAV2-GFP eyes (Figure 4E-H+I). Loss of Tuj1 positive viable RGC axons in the optic nerve following long-term expression of Cre recombinase but not GFP was also observed. While there was no observable loss of RGC axons 4 weeks post-delivery of either AAV-Cre or AAV-GFP vectors, a 35% decrease in RGC axons was observed 11 weeks post-delivery of AAV2-Cre, (Figure 5, S3). Collectively, these data demonstrate a previously undescribed sensitivity of RGCs to long-term expression of recombinant Cre recombinase. This is an important caveat for

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RGC studies employing Cre/lox transgenic mouse lines. Such studies should address this issue by using wild type mice containing no loxP sites as control strains for assessing toxic affects of

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Cre expression.

4. Conclusions

The Cre/loxP recombinase system is an immensely useful tool for studying genetic contributions

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of candidate genes in mouse models of disease, especially when embryonic lethality prevents the use of a traditional knockout lines. However, recent reports in the literature demonstrate

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loxP-independent toxicity in tissues constitutively expressing Cre recombinase (De Alboran et al., 2001; Lee et al., 2006; Loonstra et al., 2001; Schmidt et al., 2000; Silver and Livingston, 2001; Thanos et al., 2012). This toxicity can be confounding and can lead to misinterpretation of results. Subsequently, Cre-targeted mutation experiments should be carefully controlled for loxP-independent toxicity when conducting targeted mutation experiments. Typically, these

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controls experiments would be conducted using parallel animal or cell lines that express Cre to the same extent and duration as experimental lines, but contain no genetically engineered loxP sites. Furthermore, observed toxicity suggests there is a need to explore alternative strategies

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for tissue-specific expression of Cre recombinase. Introduction of viral vectors encoding Cre recombinase is an effective alternative to constitutively expressing or induced expression Cre

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animals or cell lines. Viral transduction of Cre recombinase activity allows for optimal temporal and dosage control that is particularly useful for targeting cells and tissues in adult animals or long-term cell cultures. The use of intravitreal vector delivery of Cre recombinase as well as loxP-independent Cre toxicity within the retina have not been sufficiently characterized to date. Our findings in this study demonstrate 1) effective timing and dosage regiment for in vivo targeting of capsid mutant AAV2-Cre viral particles to murine GCL retinal neurons, 2) unintended loxP-independent toxicity in RGCs with long-term Cre expression and 3) an short-

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term Cre expression strategy for targeting adult murine GCL retinal neurons that has no measurable impact on RGC survival.

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Previous titer and retinal tropism experiments comparing intravitreal injection of wild-type AAV2 vectors to capsid mutant vectors in murine retinas demonstrated a dramatic improvement of transgenic expression in the respective capsid mutants due to evasion of host cell innate

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immunity (Day et al., 2014; Petrs-Silva et al., 2009). This current study focuses on determining sufficient dosage parameters for effective targeting of adult murine RGCs following intravitreal delivery of the triple mutant AAV2 viral vectors to floxed mice (Figure 2+3, S1). While our

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experiments demonstrate that 108 and 109 vg are sufficient for effective RGC targeting, more refined titer optimization between these doses is needed to pinpoint the optimal dosage. Additionally, the use of alternative transgene promoters is a strategy that may be further explored in future experiments to dampen observed Cre toxicity. In our subsequent experiments

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injecting 109 vg, loxP-independent toxicity levels were not present until the 5th week postinjection compared to mice injected with GFP control vector (Figure 4). These findings demonstrate a window of opportunity to use capsid mutant AAV2-Cre vectors in a short-term

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timeframe to study adult murine RGCs (Figure 6). However, it should be noted that global molecular changes associated with Cre-induced cell death during the first 4 weeks of viral

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exposure were not investigated in this study and could potentially be a confounding factor using this strategy. Although the mechanism of Cre toxicity observed here as well as in other studies is unknown, one suggested mechanism is promiscuity of Cre recombinase activity on endogenous lox-like genomic sequences. These cryptic endogenous loxP sites have been previously associated with increased sister chromatid recombination and chromosomal anomalies resulting in reduced cell survival rate (Loonstra et al., 2001; Silver and Livingston, 2001). Future studies further refining the minimal effective vector dose as well as the use of

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alternative promoters to the strong chicken-beta actin promoter used to drive transgene expression in this study, may help to elucidate strategies to dampen observed Cre toxicity.

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The accessibility of RGCs in the inner most portion of the retina using intravitreal injection can be exploited in studies investigating glaucoma and other optic neuropathies. Determining the effects of Cre-toxicity in RGCs is paramount to developing effective murine models for studying

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these disorders. This study demonstrates that experimental strategies for targeted Cre mutation in RGCs can be effectively employed within a 4 week window prior to the onset of detectable

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cell toxicity. Cre expression studies can be conducted in tandem with short-term axonal injury rodent models of optic neuropathy such as optic nerve crush, axotomy, and translimbal laser photocoagulation. Each of these rodent injury models has been previously demonstrated to incur RGC damage within a two week timeframe (Welsbie et al., 2013; Yang et al., 2007). Figure 6 outlines an experimental design that could be effectively employed to introduce RGC-

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specific targeted mutations within the context of a mouse injury model of glaucoma, such as our previous study characterizing the role of the Dual Leucine Zipper Kinase (DLK) in mediating

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RGC death (Welsbie et al., 2013).

Acknowledgments

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We thank Vince A. Chiado and Sanford L. Boye in the University of Florida Department of Ophthalmology for their assistance with viral vector production. We also thank Sophia L. Brown in the James Madison University Department of Biology and Julia VanBuskirk in the Johns Hopkins University Department of Ophthalmology for their assistance with immunohistochemistry analysis. This work was supported by the BrightFocus Foundation (grant #G2012033 awarded to S. Merbs and R. Enke), the Commonwealth Health Research Board

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(grant #216-05-15 awarded to R. Enke); and a James Madison University 4-VA Grant awarded

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to R. Enke.

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Figure 1. Schematic of experimental design. Capsid mutant AAV2-Cre or AAV2-GFP viral particles were intravitreally injected into C57BL/6 or R26 LacZ floxed mice. Mice were euthanized and tissues were harvested at the indicated time intervals ranging from 2 weeks (wk) out to 15 wk post-injection. An AAV2-Cre vector titer was determined in R26 LacZ floxed mice harvested at 2 wk post-injection. Viral transgene expression (Ex) was assessed in wt mice at 2, 4, and 11 wk post-injection. Retinal (ret) and axonal (ON) RGC survival was assessed at the indicated time points. Figure 2. Optimization of capsid mutant viral vector titer for GCL delivery. Representative images of a dilution series of AAV2-Cre ranging from 105 - 1010 vg intravitreally injected into R26 LacZ floxed mice. Eyes were harvested and flat-mounted 2 weeks post-injection and assayed for Cre activity using an X-gal assay. Figure 3. Early and persistent viral transgene expression in GCL retinal neurons. Eyecup cross sections were prepared from wt mice 2 weeks post-injection with 109 vg AAV2-Cre and stained with an αCre antibody (red) and DAPI nuclear stain (blue) (A) 40X (B) 200X. Optic nerve cross section were prepared from wt mice 4 and 11 weeks post injection with 109 vg AAV2-GFP to visualize expression of transgenic GFP (green) (C-D). Abbreviations: sc=sclera, PRs=photoreceptors, INL=inner nuclear layer, GCL=ganglion cell layer, GFP=Green Fluorescent Protein.

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Figure 4. Cre toxicity of RGCs in the retina. Eyes were harvested 2-15 weeks post-injection with 109 vg AAV2-Cre or AAV2-GFP and stained with αBrn3 (green) and αTuj1 (red) antibodies, both markers of viable RGCs. Eyecups were flat mounted and fluorescence was visualized using confocal microscopy (A-H). RGC cell body viability was quantified by counting Brn3+ RGCs in the retina (I). Eyes infected with AAV2-Cre vector showed significant decrease in Brn3 starting at 5 wk post-injection with greater significance at 6 wk to 15 wk post-injection (I). #, P<0.05; *, P<0.0001.

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Figure 5. Cre-associated RGC axon loss in the optic nerve. Representative images of optic nerves harvested 4 and 11 weeks post-injection with 109 vg AAV2-Cre or AAV2-GFP and stained with αTuj1 antibody marker of viable RGC axons. Nerves were cross sectioned and signal was visualized using fluorescence microscopy (A-D). RGC axon viability was quantified from replicate nerve images using ImageJ software. Eyes injected with AAV2-Cre vector showed significant decrease in axons when comparing 4wk to 11wk post injection (E). #, P<0.05.

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Figure 6. Proposed experimental design for studying axonal injury models in targeted Cre/lox RGC mutant mice. Following intravitreal injection of 109 capsid mutant AAV2-Cre vg copies, a viral incubation period of 1-2 weeks is sufficient for recombinant Cre expression and target gene knockout. Following the incubation period, experimental acute optic nerve injury can be employed in knockout GCL retinal neurons. Affect of genetic knockout on RGC survival can be measured within 4 weeks post injection prior to the onset of Cre-mediated RGC toxicity.

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Figure S1. Uniform distribution of Cre activity in GCL following delivery of 109 vg AAV2Cre dosage. Replicate images of flat mounted retina following intravitreally injection of 109 vg copies of AAV2-Cre into R26 LacZ floxed mice. Eyes were harvested and flat-mounted 2 weeks postinjection and assayed for Cre activity using an X-gal assay.

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Figure S2. Non-specific secondary antibody cross reactivity in sclera and RPE Serial retinal cross sections of eyes injected with 109 vg copies of AAV2-Cre without primary antibody incubation confirmed that staining of the sclera and RPE in the outermost portion of the retina was due to autofluorescence and/or non-specific secondary antibody cross reactivity in these tissues. DAPI nuclear counterstain appears as blue staining in panel A.

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Figure S3. Cre-associated RGC axon loss in the optic nerve. Full collection of optic nerves images used to calculate RGC axon loss in figure 5. Nerves harvested 4 and 11 weeks postinjection with 109 vg AAV2-Cre or AAV2-GFP and stained with an αTuj1 antibody markers of viable RGC axons. Nerves from 3 animals per treatment group were serial sectioned and 3 serial section technical replicates per nerve were visualized using fluorescence microscopy. RGC axon viability was quantified from nerve images using ImageJ software.

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An effective timing and dosage regiment for in vivo targeting of capsid mutant AAV2-Cre viral particles to murine ganglion cell layer retinal neurons is demonstrated. Unintended loxP-independent toxicity is observed in RGCs with long-term Cre expression. Short-term Cre expression strategies targeting adult murine RGCs have no measurable impact on RGC survival within a 4 week time frame. Short-term Cre expression studies can be conducted in tandem with axonal injury rodent models for genetic analysis of optic neuropathies.

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