METHODS FOR PREVENTING INDUCTION OF IMMUNE RESPONSES TO THE TRANSDUCED CELLS EXPRESSING A TRANSGENE PRODUCT AFTER OCULAR GENE THERAPY

Abstract

Despite the eye's immune-privileged status, a secondary loss of vision in some patients treated with AAV led the inventors to question the immunogenicity of AAV vectors after a subretinal injection. The inventors thus characterized anti-transgene and anti-capsid immune responses induced in the periphery after the subretinal AAV injection. Different doses of AAV8 encoding reporter proteins fused with the HY male antigen were injected at day 0 into the subretinal space of adult immunocompetent C57BL/6 female mice. Subretinal AAV injection induced a dose-dependent proinflammatory immune response to the transgene product, correlated with local transgene expression. In order to trigger a subretinal-associated immune inhibition (SRAII) mechanism, some mice were co-injected subretinally at day 0 with AAV and HY peptides. Interestingly, this subretinal co-injection of AAV8 with peptides of the transgene product modulated the anti-transgene T-cell immune response, even at high dose of vector (5.10.sup.10 vg). This immunodulation was also confirmed in a pathophysiological murine model of retinal degeneration. The inventors also demonstrated that injection of AAV8 in the subretinal space induces proinflammatory peripheral immune responses to the transgene and the capsid that could be counteracted y co-injection with transgene peptides. Accordingly, the object of the present invention is to provide methods for preventing induction of immune responses to the transgene product and the AAV capsid after ocular gene therapy.

Claims

1. A method for preventing a secondary vision loss in a patient who received an ocular gene therapy with a vector containing a transgene comprising administering to the patient a therapeutically effective dose of at least one peptide that derives from a transgene product or the vector, simultaneously with gene therapy, thereby preventing induction of immune responses to the transduced cells expressing the transgene product.

2. The method of claim 1 wherein the immune response is a cellular cytotoxic response.

3. A method for expressing a transgene of interest in the retina of a patient comprising injecting into a subretinal space of the patient a therapeutically effective amount of a vector containing a transgene of interest in combination with a therapeutically effective amount of at least one peptide that derives from a product of the transgene or the vector.

4. A method of treating a retinal disease in a patient in need thereof, comprising injecting into the subretinal space of the patient an amount of a vector containing a transgene of interest in combination with a therapeutically effective amount of at least one peptide that derives from a product of the transgene or the vector.

5. The method of claim 1, to wherein the patient suffers from a retinal acquired disease that is macular degeneration or diabetic retinopathies.

6. The method of claim 1, wherein the patient suffers from an inherited retinal disease selected from the group consisting of retinitis pigmentosa, Leber's congenital amaurosis, X-linked retinoschisis, autosomal recessive severe early-onset retinal degeneration (Leber's Congenital Amaurosis), congenital achromatopsia, Stargardt's disease, Best's disease, Doyne's disease, cone dystrophy, retinitis pigmentosa, X linked retinoschisis, Usher's syndrome, age related macular degeneration, atrophic age related macular degeneration (AMD), neovascular AMD, diabetic maculopathy, proliferative diabetic retinopathy (PDR), cystoid macular oedema, central serous retinopathy, retinal detachment, intra-ocular inflammation, glaucoma, posterior uveitis, choroideremia, and Leber hereditary optic neuropathy.

7. The method of claim 1, wherein the transgene product is a polypeptide that enhances the function of a retinal cell.

8. The method of claim 1, wherein the transgene product is an endonuclease that provides site-specific knock-down of gene function.

9. The method of claim 1, wherein the vector containing the transgene is selected from the group consisting of viral and non-viral vectors.

10. The method of claim 9, wherein the vector is an adenoviral vector (AVV).

11. The method of claim 10 wherein the AAV vector is an AAV8 vector.

12. The method of claim 1, wherein the peptide is an immunodominant peptide that derives from the transgene product or vector.

13. The method of claim 12 wherein the vector is an AAV vector and the immunodominant peptide derives from a capsid protein of the AAV vector.

14. The method of claim 13 wherein the immunodominant peptide derives from the VP1, VP2, or VP3 capsid protein of the AAV vector.

15. The method of claim 12 wherein the immunodominant peptide derives from the transgene product.

16. The method of claim 1, wherein the vector is injected in the subretinal space simultaneously with 2, 3, 4, 5, 6, 8, 9 or 10 immunodominant peptides.

17. The method of claim 12 wherein the vector is injected with at least one immunodominant peptide comprising a MHC-class I restricted epitope and/or at least one immunodominant peptide comprising a MHC-class II restricted epitope.

18. A pharmaceutical composition comprising a vector containing the transgene of interest, at least one peptide that derives from the transgene product or vector and a pharmaceutically acceptable carrier, diluent, excipient, or buffer.

19. The method of claim 5, wherein the macular degeneration is age related macular degeneration.

Description

FIGURES

[0083] FIG. 1. Correlation Analysis Between Ocular Transgene Expression Levels and Peripheral Anti-Transgene T-Cell Immune Response in Wild Type C57BL/6 Mice.

[0084] PBS, HY peptides, or different doses (4.10.sup.8 to 10.sup.11 vg) of AAV8-Luc2-HY were injected in the subretinal (SR) space of C57BL/6 female mice at day 0. Two weeks later, the immune response was challenged by subcutaneous immunization (SC) of either PBS:CFA or HY:CFA. The immune response of total splenocytes re-stimulated in vitro by HY peptides was assessed 1 week after immunization by IFN-γ ELISpot. In parallel, bioluminescent imaging every 3-4 days monitored the transgene expression level (5 mice/group). (A) AAV dose-dependent quantification of transgene expression by bioluminescence in the periphery at day 20. (B) Kinetic study of the loco-regional transgene expression by bioluminescence. (C) Correlation between ocular transgene expression level at day 20 and IFN-γ secretion at day 21 after in vitro anti-HY T-cell stimulation.

[0085] FIG. 2. Inhibition of Peripheral Anti-Transgene T-Cell Pro-Inflammatory Immune Response By a Subretinal Co-Injection of HY Peptides and Different Doses of AAV8 in Wild Type C57BL/6 Mice.

[0086] PBS, HY peptides, and two doses (2.10.sup.9 or 5.10.sup.10 vg) of AAV8-GFP-HY or AAV8-GFP-HY+HY peptides were injected in the subretinal space of C57BL/6 female mice atday 0. Two weeks later, the immune response was challenged by subcutaneous immunization of either PBS:CFA or HY:CFA. The immune response of total splenocytes re-stimulated in vitro by HY peptides was assessed 1 week after immunization by IFN-γ ELISpot. The number of spot-forming units (SFUs) from mice receiving PBS in the eye and immunized with HY peptides (positive control of anti-HY immune response) was indexed to 100 and SFUs for other mice were proportionally calculated. Bars correspond to mean+/−SEM. Data were obtained from 9 independent experiments.

[0087] FIG. 3. Inhibition of In Vivo Anti-Transgene Cytotoxicity By a Subretinal Co-Injection of HY Peptides and a High Dose of AAV8 in Wild Type C57BL/6 Mice.

[0088] PBS, a high dose (5.10.sup.10 vg) of AAV8-GFP-HY alone, or AAV8-GFP-HY+HY peptides were injected in the subretinal space of C57BL/6 female mice at day 0. Two weeks later, the immune response was challenged by subcutaneous immunization with HY:CFA. At day 17, a mixture of 3.10.sup.6 CD45.1.sup.+ CD45.2.sup.−CTV.sup.low male and 3.10.sup.6 CD45.1.sup.−CD45.2.sup.+CTV.sup.high female spleen cells from C57BL/6 wild type mice were injected intravenously. At day 20, blood was harvested and leucocytes were stained for flow cytometry with an anti-CD45.1-PE mAb to analyse the male cell survival in vivo. Data were obtained from 1 experiment. CTV: Cell Trace Violet.

[0089] FIG. 4. Inhibition of Peripheral Anti-AAV8 T-Cell Immune Response By a Subretinal Co-Injection of HY Peptides and High Dose of AAV8 in Wild Type C57BL/6 Mice.

[0090] PBS (Neg ctrl), HY peptides, and 5.10.sup.10 vg of AAV8-GFP-HY or AAV8-GFP-HY+HY peptides were injected in the subretinal space of C57BL/6 female mice at day 0. Two weeks later, the immune response was challenged by subcutaneous immunization of either PBS:CFA (Neg ctrl) or HY:CFA. The immune response of total splenocytes re-stimulated in vitro by AAV8 capsids was assessed 1 week after immunization by IFN-γ ELISpot and displayed as the number of spot-forming units (SFUs) per well. Data were obtained from 1 experiment.

[0091] FIG. 5. Inhibition of Peripheral Anti-Transgene T-Cell Immune Response By a Subretinal Co-Injection of HY Peptides and Different Doses of AAV8 in rd10 Mice.

[0092] PBS, HY peptides, and two doses (2.10.sup.9 or 5.10.sup.10 vg) of AAV8-GFP-HY or AAV8-GFP-HY+HY peptides were injected in the subretinal space of rd10 female mice at day 0. Two weeks later, the immune response was challenged by subcutaneous immunization of either PBS:CFA or HY:CFA. The immune response of total splenocytes re-stimulated in vitro by HY peptides was assessed 1 week after immunization by IFN-γ ELISpot. The number of spot-forming units (SFUs) from mice receiving PBS in the eye and immunized with HY peptides (positive control of anti-HY immune response) was indexed to 100 and SFUs for other mice were proportionally calculated. Bars correspond to mean+/−SEM. Data were obtained from 5 independent experiments.

EXAMPLE 1

[0093] Materials & Methods

[0094] Animals

[0095] Wild-type six- to eight-week-old C57BL/6 female mice (H-2.sup.b) were purchased from Charles River Laboratories (L'Arbresle, France). Animals were anesthetized either by intraperitoneal injection of 120 mg/kg ketamine (Virbac, Carros, France) and 6 mg/kg xylazine (Bayer, Lyon, France) or by inhalation of isoflurane (Baxter, Guyancourt, France). They were euthanized by cervical elongation. All mice were housed, cared for, and handled in accordance with the European Union guidelines and with the approval of the local research ethics committee (CEEA-51 Ethics Committee in Animal Experimentation, Evry, France; authorization number 2015102117539948).

[0096] AAV Vectors

[0097] AAV8-PGK-GFP-HY was produced by INSERM unit U1089 in Nantes, France. They used the tri-transfection technique in 293T cells cultured in CF10. AAV8-PGK-Luc2-HY was produced by Vector Core in Généthon, Evry, France. They used the tri-transfection technique in 293T cells cultured in roller bottles (Liu et al., 2003). Endotoxin levels were below 6 E.U/mL.

[0098] Peptides

[0099] The DEAD Box polypeptide 3 Y-linked (DBY) and Ubiquitously Transcribed tetratricopeptide repeat gene Y-linked (UTY) peptides, NAGFNSNRANSSRSS and WMHHNMDLI respectively, were synthesized by Genepep (Montpellier, France) and shown to be more than 95% pure.

[0100] Subretinal Injections

[0101] The eye was protruded under microscopic visualization and perforated with a 27G bevelled needle. A blunt 32G needle set on a 10 μL Hamilton syringe was inserted in the hole and 2 μL of PBS or UTY+DBY and/or AAV vector was injected into the subretinal space. The quality of the injection was verified by checking the detachment of the retina.

[0102] Subcutaneous Injections

[0103] PBS or UTY+DBY were emulsified in Complete Freund's Adjuvant (Sigma, Lyon, France) at a 1:1 ratio, and 100 μL of the preparation (200 μg of UTY+DBY/mouse) was injected at the base of the tail.

[0104] Cell Extraction From Spleen

[0105] After euthanasia, spleens were removed and crushed with a syringe plunger on a 70-μm filter in 2 mL of RPMI medium. Red blood cells were lysed by adding ACK buffer (8.29 g/L NH4Cl, 0.037 g/L EDTA, and 1 g/L KHCO3) for one min. Lysis was stopped by addition of complete RPMI medium (10% FBS, 1% penicillin/streptomycin, 1% glutamine, and 50 μM β-mercaptoethanol). After centrifugation, cells were counted, and the concentration was adjusted in complete RPMI medium.

[0106] Inguinal lymph nodes were crushed with a syringe plunger in 2 mL of RPMI medium. Debris were eliminated by transferring the supernatants into new tubes. After centrifugation, cells were counted and the concentration was adjusted in complete RPMI medium.

[0107] ELISpot Assay

[0108] IFN-γ Enzyme-Linked Immunospot plates (MAHAS45, Millipore, Molsheim, France) were coated with anti-IFN-γ antibody (eBiosciences, San Diego, Calif.) overnight at +4° C. Stimulation media (complete RPMI, UTY (2 μg/mL), DBY (2 μg/mL), UTY+DBY (2 μg/mL) or Concanavalin A (Sigma, Lyon, France) (5 μg/mL) were plated and 5.10.sup.5 splenocytes/well were added. After 24 hours of culture at +37° C., plates were washed and the secretion of IFN-γ was revealed with a biotinylated anti-IFN-γ antibody (eBiosciences), Streptavidin-Alcalin Phosphatase (Roche Diagnostics, Mannheim, Germany), and BCIP/NBT (Mabtech, Les Ulis, France). Spots were counted with an AID ELISpot iSpot Reader system ILF05 and AID ELISpot Reader v6.0 software.

[0109] Bioluminescence Imaging

[0110] Mice were injected intraperitoneally with luciferin (250 mg/kg of mice) and anesthetized with isoflurane for imaging. Ten minutes after luciferin injection, mice were placed in the imager for measurements. The imaging process used IVIS Lumina equipment and Living Image software.

[0111] In Vivo Cell Cytotoxicity Assay

[0112] Spleen cells from CD45.1.sup.+ CD45.2.sup.− male and CD45.1.sup.− CD45.2.sup.+ female C57BL/6 wild type mice were harvested as described above, and stained with Cell Trace Violet cell proliferation kit (Molecular Probes) in PBS at different concentration: 2 μM for male and 20 μM for female cells for 20 min at 37° C. in the dark. The reaction was quenched by addition of cold complete RPMI medium containing 10% FBS. Cells were incubated for 5 min in complete RPMI medium at 37° C. and then washed with PBS 1×. A mixture of 3.10.sup.6 male cells and the same number of female cells in 200 μL was injected intravenously in the experimented (CD45.1.sup.−CD45.2.sup.+) female C57BL/6 mice at day 17 of the protocol. Three days after injection, blood was harvested, red blood cells were lysed by adding ACK buffer, washed in PBS 1×, and leucocytes were stained for flow cytometry. First, cells were resuspended in 50 μL of Fc block solution (Pharmingen, BD Biosciences) diluted to 1.7 μg/mL in PBS containing 1% BSA and incubated for 10 min at 4° C. Next, 50 μL of anti-CD45.1-PE (Pharmingen, BD Biosciences) at 5 μg/mL in PBS 1% BSA was added. The cells were then incubated for 20 min at 4° C. As a control, some cells were stained in the same conditions with a the corresponding isotype antibody: mouse IgG2a,κ-PE (Pharmingen, BD Biosciences). Data were acquired on a CytoFLEX LX flow cytometer (Beckman Coulter) and analyzed with the CytExpert software (Beckman Coulter).

[0113] Statistical Analysis

[0114] Statistical analyses were performed with GraphPad Prism V6.0. After ANOVA, Tukey's test was performed. P-value<0.05: *, <0.01: **, <0.001: ***, <0.0001: ****.

[0115] Results

[0116] High Doses of Subretinal AAV8 Vectors Induce Anti-Transgene Proinflammatory T-Cell Immune Responses

[0117] To evaluate the possibility that subretinal injection of AAV8 induces anti-transgene cellular immune responses, wild-type mice were injected with PBS, UTY+DBY (HY) peptides, or different doses of AAV8 encoding for GFP fused with HY peptides. Two weeks later, these mice were subcutaneously immunized with PBS or HY peptides. Spleen cells were harvested on day 21 and stimulated in vitro with HY peptides for ELISpot quantification of IFN-γ secretion by HY-specific T cells (FIG. 1). The challenge on day 14 makes it possible to observe the induction of subclinical immune responses or immune inhibition (Vendomèle et al., 2018).

[0118] As a positive control for anti-HY immune response, mice received PBS in the subretinal space on day 0 and HY peptides subcutaneously on day 14. In this case, 150 to 250 spot forming units (SFU) were counted in response to HY peptides, corresponding to IFN-γ-secreting spleen cells. To normalize the data from the different experiments, the index of IFN-γ secretion of the positive control was set to 100 (FIG. 1, black line). As a negative control (not shown), some mice received PBS in the subretinal space, and the immune response was challenged by subcutaneous immunization by PBS:CFA. No significant IFN-γ secretion was detected in this group (25 SFUs/10.sup.6 cells). We have previously reported (Vendomèle et al., 2018) that subretinal injection of HY peptides induces inhibition of T-cell immune responses (proliferation, polarization, and cytokine secretion). Thus, we used the injection of HY peptides in the subretinal space on day 0 followed by an immunization with the same peptides on day 14 as a control for immune modulation: the IFN-γ secretion index for these mice was inhibited by 65% (+/−13%) compared to the positive control. We next assessed the capacity of a wide range of AAV8-PGK-GFP-HY doses to induce an anti-transgene immune response. Low and medium doses of AAV (10.sup.3 to 2.10.sup.9 vg) induced levels of IFN-γ secretion similar to that of the positive control. High doses of AAV (10.sup.10 to 5.10.sup.10 vg), however, induced a two-fold increase of IFN-γ secretion compared to the positive control. Taken together, these data show that low and medium doses of AAV8 injected in the subretinal space neither induced immune modulation nor increased Th1 immune response to the transgene product. Conversely, high subretinally-injected doses of AAV8 (10.sup.10 to 5.10.sup.10 vg) induced an anti-transgene proinflammatory T-cell immune response in the periphery.

[0119] Peripheral Anti-Transgene T-Cell Immune Response is Closely Correlated with Loco-Regional Transgene Expression Levels

[0120] After demonstrating that subretinal injection of a high dose of AAV8 induced peripheral T-cell immune responses to the transgene product, we assessed the impact of the transgene expression level on the anti-transgene immune response. Mice were injected with PBS, HY peptides, or different doses of AAV8 encoding for Luciferase (Luc2) fused with HY peptides; two weeks later, they were subcutaneously immunized with PBS or HY peptides. Spleen cells were harvested on day 21 and stimulated in vitro with HY peptides to quantify IFN-γ secretion by HY-specific T cells with ELISpot. In parallel, bioluminescence imaging of the mice every three days enabled detection of Luc2 expression. We quantified transgene (Luc2) expression by the luminoscore method described elsewhere (Cosette et al., 2016). For each mouse, dorsal and ventral views were acquired, and for each view, 2 regions of interest (ROI) drawn. Local-regional (head of each mouse) transgene expression was calculated as: Head.sup.dorsal view+Head.sup.ventral view (blue ROIs) whereas peripheral transgene expression was calculated as: (Body.sup.dorsal view+Body.sup.ventral view)−(Head.sup.dorsal view+Head.sup.ventral view) (red ROI−blue ROIs).

[0121] Control mice (negative, positive, and HY-injected) were imaged but obviously no Luc2 expression was detected locally. Medium (4.10.sup.8 to 2.10.sup.9 vg) and high (5.10.sup.10 to 10.sup.11 vg) doses of AAV8 induced dose-dependent transgene expression from 3 days post-injection; this expression remained stable over 3 weeks (FIG. 1A). High doses of AAV8 induced transgene expression from 3 days that increased until day 13 and then declined until day 20 (p-value <0.01 between day 13 and day 20) (FIG. 1B). Note that the local-regional expression of the transgene was restricted to the eye, and there was no evidence of expression in the ipsilateral cervical lymph node through 21 days, regardless of the AAV dose. On day 21, an IFN-γ ELISpot assay was performed on spleen cells stimulated in vitro with HY peptides. FIG. 1C shows a plot of each mouse according to its transgene expression level on day 20 and the number of its SFUs (ELISpot). Results show that the IFN-γ secretion was correlated with local-regional (head) transgene expression (p-value=0.0056). Nonetheless, according to the coefficient of determination (r.sup.2=0.5123), this transgene expression in the eye explains only 51% of the immune response (FIG. 1C). Taken together, these data show that the transgene expression level in the eye was tightly correlated to the dose of AAV8 injected subretinally, and to the systemic anti-transgene immune response.

[0122] Subretinal-Associated Immune Inhibition Can Be Induced By a Simultaneous Injection of Peptides From the Transgene Product and AAV8 in the Retina, Even With High Doses of AAV

[0123] We have shown that subretinal injection of high doses of AAV induces proinflammatory anti-transgene immune responses that are not observed with low or medium doses. We have previously pointed out that the subretinal injection of HY peptides leads to peripheral immune inhibition (Vendomèle et al., 2018). Accordingly, we tested the possibility of using this mechanism as an immune-modulatory tool in subretinal AAV gene transfer, by co-injecting peptides from the transgene together with the AAV. Mice were injected with PBS, HY peptides, 2.10.sup.9 or 5.10.sup.10 vg of AAV8-PGK-GFP-HY or the same AAV8 doses plus HY. Two weeks later, mice were subcutaneously immunized with PBS or HY peptides. Spleen cells were harvested on day 21 and stimulated in vitro with HY peptides to quantify IFN-γ secretion by HY-specific T-cells by IFN-γ ELISpot assay (FIG. 2). Our results show that IFNγ secretion was inhibited by 40.5% in the mice that received HY peptides subretinally, compared with the positive control. Interestingly, co-injecting HY peptides with 2.10.sup.9 vg of AAV8, compared to the 2.10.sup.9 vg of AAV8 alone, reduced IFN-γ secretion significantly (p=0.0007), by half In the same way, co-injection of HY peptides with the high dose of AAV8 decreased by 52.9% IFN-γ secretion by HY-specific T cells. Taken together, these data show that co-injection of immunodominant peptides from the transgene together with different doses of AAV8 inhibited the T-cell pro-inflammatory cytokine secretion in response to the transgene.

[0124] Anti-Transgene Cell Cytotoxicity Can Be Inhibited By a Simultaneous Injection of Peptides From the Transgene Product and AAV8 in the Retina

[0125] Since we have demonstrated that a simultaneous subretinal injection of high doses of AAV and peptides from the transgene product can lead to peripheral inhibition of the secretion by T-cells of pro-inflammatory cytokines such as IFNγ, we investigated the potentiality to inhibit the anti-transgene in vivo cytotoxicity. PBS, a high dose (5.10.sup.10 vg) of AAV8-GFP-HY alone, or AAV8-GFP-HY+HY peptides were injected in the subretinal space of C57BL/6 female mice on day 0, and two weeks later the immune response was challenged by subcutaneous immunization with HY:CFA. At day 17, a mixture of 3.10.sup.6 CD45.1.sup.+ CD45.2.sup.− CTV.sup.low male and 3.10.sup.6 CD45.1.sup.− CD45.2.sup.+ CTV.sup.high female spleen cells from C57BL/6 wild type mice were injected intravenously. At day 20, leucocyte analysis showed that the same proportion of male HY.sup.+ (CTV.sup.low) and female HY.sup.− (CTV.sup.high) cells survived in the PBS-injected control group (FIG. 3A, 3B). As expected, very few male cells survived in the AAV-GF-HY injected group (5.2% male vs 94.8 female cells), in contrast to the AAV+HY peptides immunomodulatory group (26.4% male vs 73.6 female cells). Thus, co-injection of immunodominant peptides from the transgene together with a high dose of AAV8 is able to inhibit in vivo anti-transgene cell cytotoxicity.

[0126] A Bystander Inhibition of Peripheral AAV8 Capsid T-Cell Immune Responses Can Be Obtained By a Simultaneous Injection of Peptides From the Transgene Product and AAV8 in the Retina

[0127] Since a subretinal injection of high doses of AAV and peptides from the transgene product can lead to peripheral inhibition of the pro-inflammatory cytokine secretion by T-cells and anti-transgene in vivo cytotoxicity, we wondered whether SRAII could also affect anti-capsid specific T-cell immune responses that are usually triggered by an AAV injection. For this purpose, PBS (negative control group), HY peptides (SRAII control group), and 5.10.sup.10 vg of AAV8-GFP-HY, or AAV8-GFP-HY+HY peptides were injected in the subretinal space of C57BL/6 female mice at day 0. Two weeks later, the immune response was challenged by subcutaneous immunization of either PBS:CFA (negative control group) or HY:CFA. The T-cell immune response was assessed by in vitro re-stimulation with AAV8 capsids 1 week after immunization by IFN-γ ELISpot assay (FIG. 4). Our results show that IFNγ secretion was inhibited by 68.9% in mice receiving AAV8+HY peptides subretinally, compared with mice injected with AAV8 alone. Interestingly, since the AAV8 capsid did not contain HY peptides, it indicates a bystander immunosuppression directed against the anti-capsid (AAV8) T-cell responses that were generated simultaneously with the anti-transgene (HY) specific T-cell activation. Hence, these data show that co-injection of immunodominant peptides from the transgene together with high doses of AAV8 can also inhibit anti-capsid specific T-cell pro-inflammatory cytokine secretion.

[0128] Discussion:

[0129] AAV-mediated gene transfer in the retina has advanced enormously over the past 20 years, from the proof-of-concept in 1996 (Ali et al., 1996) to clinical trials in the 2000s. Despite initially promising results, long-term follow-up in some clinical trials have revealed a secondary loss of vision after the initial AAV-induced improvement (Bainbridge et al., 2015; Jacobson et al., 2015). This led us to explore the possibility of subclinical anti-transgene immune response. Actually patients enrolled in clinical trials received immunosuppressive treatments, either locally and/or systemically during the first few days after AAV injection, which probably limited, delayed, or masked the induction of immune responses. Because transgene expression continues to be expressed after the treatment, however, an immune response to the transgene product can be induced over the long term.

[0130] Several studies have highlighted the immune-privileged status of the eye. Delayed-type hypersensitivity measurements have shown that subretinal injection of ovalbumin induces inhibition of the Thl profile in the periphery (Wenkel and Streilein, 1998), and McPherson et al. showed that regulatory T cells specific to retinal antigens are generated there (McPherson et al., 2011). We further characterized systemic immune responses associated with the subretinal space and showed that subretinal injection of HY, a male antigen, induced SRAII, that inhibited the proliferation and polarization of T cells,) (Vendomele et al., 2018).

[0131] Since the antigenic load is closely correlated with immune responses, as vaccine or AAV vector studies have shown (Gu et al., 2018; Khabou et al., 2018), and since immune responses are likely to be dependent on the AAV dose (Ramachandran et al., 2016), we wondered whether they are also correlated with the transgene expression level. Bioluminescence imaging revealed dose-dependent transgene expression in the eye. It is nonetheless important to bear in mind that transgene peptides might also be processed by retinal antigen-presenting cells (such as microglia) that could then migrate to the periphery (e.g., spleen, cervical lymph nodes) and trigger a systemic anti-transgene immune response. A study of local immune responses and retinal structure would now be of major interest, to determine the existence of an association between the transgene expression level and immune response in the eye and in the periphery. Furthermore, the patients in ocular AAV-mediated clinical trials received local and/or systemic immunosuppressive treatments (e.g., prednisolone) before and for a few days after their injection. This kind of approach enables non-specific inhibition of the immune response, which can be deleterious for the patient. Moreover, its effect is only transient, while the transgene is expressed over the long term. We therefore sought to partially inhibit the anti-transgene and the anti-capsid immune responses induced in our context by exploiting the SRAII mechanism. The co-injection of immunodominant peptides from the transgene with medium doses of AAV8 induced both the inhibition of the immune response to the transgene product and the AAV capsid. The role of transgene- and bystander capsid-specific modulation that we began to study by co-injection of immunodominant peptides from the transgene should be examined in greater depth in further studies.

[0132] All the experiments in our study were performed in wild-type C57BL/6 female mice, which enabled us to highlight and decipher the subclinical immune mechanisms involved in AAV-mediated ocular gene transfer. A useful question to be further examined for gene therapy applications is how these mechanisms would be influenced by the presence of various ocular pathologies. Several ocular pathologies affect the blood-retinal barrier (Milam et al., 1998; Vinores et al., 1995; Wang et al., 2011) and the local environment is inflammatory (Chen and Xu, 2015; Yoshida et al., 2013). In particular, the use of retinal degeneration models such as rd10 mice would enable new insights. Although we might hypothesize that proinflammatory immune responses would also be induced in that context, their potential for modulation by co-injection is uncertain. The possibility of inducing the SRAII mechanism in a context of retinal degeneration should be explored (cf Example 2 below).

[0133] Over the long term, these results could lead to improvement in the safety and effectiveness of AAV-mediated gene transfer for patients. Our work opens a new avenue of investigation in the field of immune responses in AAV-mediated subretinal gene transfer, and may provide insights for transgene- and capsid-specific immune modulation in a larger context.

EXAMPLE 2

[0134] To confirm the relevance of the present claim in a pathophysiological context, experiments were done in the rd10 murine model of retinal degeneration, aiming to prevent induction of T-cell immune responses to the transgene product after ocular gene transfer. The retinal degeneration 10 (rd10) murine model is characterized by is a spontaneous missense point mutation in Pde6b (cGMP phosphodiesterase 6B, rod receptor, beta polypeptide) gene. The rd10 phenotype has a late onset and mild retinal degeneration and provide a good experimental drug therapy model for retinitis pigmentosa.

[0135] Different doses (2.10.sup.9 or 5.10.sup.10 vg) of AAV8 encoding the GFP reporter protein fused with the HY male antigen, under PGK promoter, were injected at day 0 into the subretinal space of adult immunocompetent rd10 female mice. The mice were subcutaneously immunized at day 14 with or without HY peptides, and their T-cell immune responses in the spleen were analyzed at day 21 by an IFN-γ ELISpot assay after in vitro restimulation with HY peptides. Data showed that subretinal injection of AAV8 induced an anti-transgene proinflammatory T-cell immune response (FIG. 5). Subretinal co-injection at day 0 with AAV8 and HY peptides leaded to a modulation (at least 50% inhibition) of the anti-transgene T-cell immune response, even at high dose of vector (5.10.sup.10 vg) (FIG. 5).

[0136] Taken together, these data confirm that a subretinal co-injection of a vector and peptides of the transgene product can counteract the proinflammatory peripheral immune responses to the transgene induced by the AAV introduction in the eye, even in pathophysiological conditions.

REFERENCES

[0137] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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