Systemic and local immune responses to intraocular AAV vector administration in non-human primates

International audiencePositive clinical outcomes in adeno-associated virus (AAV)mediated retinal gene therapy have often been attributed to the low immunogenicity of AAVs and immune privilege of the eye. However, several recent studies have shown potential for inflammatory responses. The current understanding of the factors contributing to inflammation, such as the preexistence of serum antibodies against AAVs and their contribution to increases in antibody levels post-injection, is incomplete. The parameters that regulate the generation of new antibodies in response to the AAV capsid or transgene after intraocular injections are also insufficiently described. This study is a retrospective analysis of the pre-existing serum antibodies in correlation with changes in antibody levels after intraocular injections of AAV in non-human primates (NHPs) of the species Macaca fascicularis. In NHP serums, we analyzed the binding antibody (BAB) levels and a subset of these called neutralizing antibodies (NABs) that impede AAV transduction. We observed significantly higher pre-existing serum BABs against AAV8 compared with other serotypes and a dose-dependent increase in BABs and NABs in the serums collected post-injection, irrespective of the serotype or the mode of injection. Lastly, we were able to demonstrate a correlation between the serum BAB levels with clinical grading of inflammation and levels of transgene expression

Norbixin Protects Retinal Pigmented Epithelium Cells and Photoreceptors against A2E-Mediated Phototoxicity In Vitro and In Vivo.

The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9'-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9'-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial

Systemic administration of the di-apocarotenoid norbixin (BIO201) is neuroprotective, preserves photoreceptor function and inhibits A2E and lipofuscin accumulation in animal models of age-related macular degeneration and Stargardt disease

International audienceAtrophic A\age-related macular degeneration (AMD) and Stargardt disease (STGD) are major blinding diseases affecting millions of patients worldwide, but no treatment is available. In dry AMD and STGD oxidative stress and subretinal accumulation of N-retinylidene-N-retinylethanolamine (A2E), a toxic by-product of the visual cycle, causes retinal pigment epithelium (RPE) and photoreceptor degeneration leading to visual impairment. Acute and chronic retinal degeneration following blue light damage (BLD) in BALB/c mice and aging of Abca4-/- Rdh8-/- mice, respectively, reproduce features of AMD and STGD. Efficacy of systemic administrations of 9'-cis-norbixin (norbixin), a natural di-apocarotenoid, prepared from Bixa orellana seeds with anti-oxidative properties, was evaluated during BLD in BALB/c mice, and in Abca4-/- Rdh8-/- mice of different ages, following three experimental designs: "preventive", "early curative" and "late curative" supplementations. Norbixin injected intraperitoneally in BALB/c mice, maintained scotopic and photopic electroretinogram amplitude and was neuroprotective. Norbixin chronic oral administration for 6 months in Abca4-/- Rdh8-/- mice following the "early curative" supplementation showed optimal neuroprotection and maintenance of photoreceptor function and reduced ocular A2E accumulation. Thus, norbixin appears promising as a systemic drug candidate for both AMD and STGD treatment

In vivo optogenetic stimulation of the primate retina activates the visual cortex after long-term transduction

International audienceOver the last 15 years, optogenetics has changed fundamental research in neuroscience and is now reaching toward therapeutic applications. Vision restoration strategies using optogenetics are now at the forefront of these new clinical opportunities. But applications to human patients suffering from retinal diseases leading to blindness raise important concerns on the long-term functional expression of optogenes and the efficient signal transmission to higher visual centers. Here, we demonstrate in non-human primates continued expression and functionality at the retina level $20 months after delivery of our construct. We also performed in vivo recordings of visually evoked potentials in the primary visual cortex of anesthetized animals. Using synaptic blockers, we isolated the in vivo cortical activation resulting from the direct optogenetic stimulation of primate retina. In conclusion, our work indicates long-term transgene expression and transmission of the signal generated in the macaque retina to the visual cortex, two important features for future clinical applications

Effect of BLD on <i>Abca4^-/- Rdh8^-/-</i> and <i>Abca4^+/+ Rdh8^+/+</i> mice carrying the Rpe65-Leu450 and the <i>rd8</i> mutations.

<p>(A) A2E quantification in eyes from young (7–8 weeks) <i>Abca4^+/+ Rdh8^+/+</i> (+/+) and <i>Abca4</i>^<i>-/-</i> <i>Rdh8</i>^<i>-/-</i> (-/-) mice. Bars represent mean ± s.e.m. of 3 eyes. ****<i>p</i><0.0001 (Student’s t Test). (B) BLD was induced in +/+ and -/- during 1 hour and ERGs were recorded 7 days later. Non-illuminated +/+ mice were used as controls. A- and b-wave amplitudes are presented for the four groups and expressed as percentage of the non-illuminated <i>+/+</i> control. Bars represent mean ± s.e.m. of two separate experiments with <i>n</i> = 3–6. **<i>p</i><0.01 ****<i>p</i><0.0001 (One-way ANOVA, Tukey’s post-test). (C) Representative cryosection pictures showing Hoechst 33342 staining of the retinal cell nuclei one week after BLD. NI: non-illuminated. Scale bars = 20 μm. OS: outer segment; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.</p

Dose-dependent effect of bixin, norbixin, lutein, zeaxanthin and crocetin on RPE cell survival after A2E-induced blue-light phototoxicity.

<p>Molecules were tested as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167793#pone.0167793.g002" target="_blank">Fig 2A</a>. The concentrations of the substances are in μM. The positive control (cont–A2E) represents cells treated with DMSO alone. The negative control (cont + A2E) represents cells treated with A2E, but not with substances. Bars represent mean +/- s.e.m. of five independent experiments with <i>n</i> = 4. ***<i>p</i><0.001; ****<i>p</i><0.0001 compared to cont + A2E (One-way ANOVA, Dunnett’s post test).</p

A2E quantification after norbixin water supplementation.

<p>Two-month-old <i>Abca4</i>^<i>-/-</i> <i>Rdh8</i>^<i>-/-</i> mice were treated for 3 months with drinking water supplemented with 0.5 mg/mL norbixin or vehicle. Eyes from five-month old and two-month old <i>Abca4</i>^<i>-/-</i> <i>Rdh8</i>^<i>-/-</i> mice were also analysed and served as controls. NT: non treated. Data represent the mean +/- s.e.m. of 2 separate experiments with <i>n</i> = 5. ****<i>p</i><0.0001 compared to norbixin-treated mice (One-way ANOVA, Dunnett’s post test).</p

Effect of norbixin on ERG and retinal phototoxicity after BLD in the rat.

<p>ERG from rat non-induced or light-exposed which received vehicle, norbixin or PBN. Recordings were on recovery day 7. (A) A- and b-wave amplitudes are presented for the six groups studied. (B) Representative pictures of retinal sections stained with Trichrome-Masson showing cell nuclei one week after BLD. Scale bars = 30 μm. (C) Morphometric analysis of retina. Photoreceptor cell nuclei were measured from the optic nerve to the superior and inferior side of the retina. Data from (A) and (C) represent the mean ± s.e.m. of two separate experiments with <i>n</i> = 3. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001 compared to vehicle-treated group (One-way ANOVA, Dunnett’s post test).</p

Norbixin Protects Retinal Pigmented Epithelium Cells and Photoreceptors against A2E-Mediated Phototoxicity <i>In Vitro</i> and <i>In Vivo</i> - Fig 4

<p><b>Comparative pharmacokinetics of bixin (A) and norbixin (B) given orally.</b> (A) bixin or (B) norbixin were given at 50 mg/kg. Plasma analyses—note that bixin is efficiently converted into norbixin (values are the mean of three different animals). <b>(</b>C) HPLC-MS/MS analysis of a mouse plasma sample following oral administration of 9’-<i>cis</i>-norbixin. Both norbixin isomers and glucuronide conjugates are observed. DAD: diode-array detector; MRM: multiple reaction monitoring.</p

Effect of norbixin on ERG and retinal phototoxicity after BLD in the <i>Abca4</i>^<i>-/-</i> <i>Rdh8</i>^<i>-/-</i> mouse.

<p>(A) ERG from <i>Abca4</i>^<i>-/-</i> <i>Rdh8</i>^<i>-/-</i> mice injected in one eye with either norbixin or vehicle and light-exposed were recorded after 7 days. A- and b-wave amplitudes are presented for the five groups studied. non-induced: non-injected and non-illuminated; i.e.: injected eye; n.i.e.: non-injected eye. (B) Representative cryosection pictures showing Hoechst 33342 staining of the retinal cell nuclei one week after BLD in norbixin- or DMSO-injected or non-injected eyes and compared to non-induced eyes. OS: outer segment; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. Scale bars = 25 μm. (C) Graph showing the number of photoreceptor layers measured along the retina each 200 μm from the optic nerve. *: norbixin i.e. compared to norbixin n.i.e.; ^#: norbixin i.e. compared to DMSO i.e. Data from (A) and (C) represent the mean ± s.e.m. of four separate experiments with <i>n</i> = 3–4. (A): *<i>p</i><0.05, **<i>p</i><0.01 (One-way ANOVA, Tukey’s post test). (C) ^# <i>p</i><0.05, ** or ^##<i>p</i><0.01, *** or ^###<i>p</i><0.001, **** or ^####<i>p</i><0.0001 (One-way ANOVA, Dunnett’s post test).</p