IMPROVED METHODS FOR TREATING OCULAR DISEASES BY GENE THERAPY

Abstract

The invention relates to a pharmaceutical composition comprising a recombinant adeno-associated virus (r AAV) vector carrying a nucleic acid sequence encoding a functional gene under the control of regulatory sequences which express the product of said gene in the retinal cells, for use in a method for preventing or treating an inherited retinal degenerative disorder associated with mutations in said gene, wherein the pharmaceutical composition is administered during the same operative period by at least one subretinal injection in each quadrant of retina of the patient in need thereof and wherein said quadrants consist of infero-temporal retina, supero-temporal retina, infero-nasal retina and supero-nasal retina.

Claims

1-15. (canceled)

16. A method for preventing or treating an inherited retinal degenerative disorder associated with mutations in a gene in a patient in need thereof, the method comprising administering to the patient a pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) vector carrying a nucleic acid sequence encoding the functional gene under the control of regulatory sequences which express the product of said gene in the retinal cells, wherein the pharmaceutical composition is administered during the same operative period by at least one subretinal injection in each quadrant of retina of the patient, and wherein said quadrants consist of infero-temporal retina, supero-temporal retina, infero-nasal retina and supero-nasal retina.

17. The method according to claim 16, wherein preventing or treating an inherited retinal degenerative disorder comprises preventing, arresting progression or ameliorating vision loss associated with the inherited retinal degenerative disorder associated with mutations in said gene.

18. The method according to claim 16, wherein preventing or treating an inherited retinal degenerative disorder comprises enhancing retinal cell survival, including photoreceptor cell survival and retinal pigment epithelium (RPE) survival.

19. The method according to claim 16, wherein said inherited retinal degenerative disorder is retinitis pigmentosa (RP).

20. The method according to claim 16, wherein said inherited retinal degenerative disorder is Leber congenital amaurosis (LCA).

21. The method according to claim 16, wherein said functional gene is RLBP1 or RPE65.

22. The method according to claim 16, wherein said rAAV is AAV2/5 or AAV2/4 serotype.

23. The method according to claim 16, wherein the retinal cells in which the functional gene is expressed are RPE cells.

24. The method according to claim 16, wherein the pharmaceutical composition is administered before disease onset.

25. The method according to claim 16, wherein the pharmaceutical composition is administered after initiation of photoreceptor loss.

26. The method according to claim 16, wherein the pharmaceutical composition is administered when less than 50% of photoreceptors are functioning or remaining.

27. The method according to claim 16, wherein the pharmaceutical composition is administered at a concentration between 10.sup.9 and 10.sup.12 vector genomes per milliliter (vg/mL).

28. The method according to claim 16, wherein the pharmaceutical composition is administered at a concentration of about 5.10.sup.10 vg/mL.

29. The method according to claim 16, wherein the pharmaceutical composition is administered in a volume of 450 L.

30. The method according to claim 16, wherein the pharmaceutical composition is administered in a volume of 750 L or 800 L.

Description

FIGURES

[0055] FIG. 1: Inflammation evaluation after subretinal injection of rAAV2/4.hrpe65.rpe65. (A) Graph of the laser flare meter measure at D-90, D-1, D+4, D+14, D+60, D+180, D+360. (B) Value of the laser flare meter for the three patients who present a modification of the value at D+4 and D+14 in Ph/ms.

[0056] FIG. 2: Sensorial and oculomotricity evaluation. (A) ETDRS visual acuity results in injected eye and uninjected eye before injection and at the last visit one year post injection, presence of nystagmus, presence of exotropia. (B) Variation of the visual acuity mean after the surgery in the untreated eye and in the treated for the nine patients. (C) Variation of the visual acuity mean after the surgery in the untreated eye and in the treated for the nystagmic patients. LP: light perception; TE: treated eye; UE, untreated eye.

[0057] FIG. 3: Follow up of visual field based on injected surface. (A) For the three columns: on the left a composite photograph of patient retina; the area exposed to the vector is note with the dashed line, on the right the goldmann visual field; in clear, V4 surface before injection, in dark the V4 surface at one year post injection. (B) Variation of the mean visual field surface after the surgery for treated eye in dark and for untreated eye in bright for night patients. (C) Variation of the visual field average gain based on the injected dose of the vector.

EXAMPLE: SAFETY AND EFFICACY OF GENE TRANSFER WITH A AAV4 FOR RPE65 LEBER'S CONGENITAL AMAUROSIS

[0058] Material & Methods

[0059] The Trial:

[0060] This clinical trial (NCT01496040) is a Phase I/II study that was approved by the Tours-Ouest 1 Ethics Committee on 4 Mar. 2011 and by Afssaps on 1 Sep. 2011. After information had been given to the patient or legal guardian, consent to participate was obtained. Patients were divided into three groups according to the dose of virus injected and their age. Adult patients in the first group were given the lowest dose of viral vector (up to 400 l) and the other two groups were given higher doses, up to 800 l of solution for adults in the second group and children in the third. An independent safety data monitoring board appointed to monitor the study was convened between patients 1 and 2, 3 and 4, 4 and 5, 6 and 7, 7 and 8 to gauge the safety and tolerance of AAV2/4.rpe65.rpe65.

[0061] Patients:

[0062] Patients included in this study all carried two mutations in the rpe65 gene (checked at baseline) (Table 1):

TABLE-US-00002 TABLE 1 Demographic and genetic characteristics of the patients. Age DNA allele 1 Protein 1 DNA allele 2 Protein 2 CG01 28 Y c.700C > T p.Arg234X c.1067delA Asn356Methfs*17 BJ03 27 Y c.544C > G p.His182Asp c.726 2A > G fs* MM04 35 Y c.444G > C p.Glu148Asp c.1451G > A p.Gly484Asp MR05 42 Y c.74C > T p.Pro25Leu c.1301C > A p.Ala434Glu HM06 22 Y c.843_858 + 7del23 p.Asn282fs* c.843_858 + 7del23 p.Asn282fs* HT07 20 Y 440_441delCA p.Thr147Argfs*9 c.1448_1450delATG p.Asp483del AM08 19 Y 246 11A > G fs* c.615_616delCA p.Ile206Cysfs*27 HM09 15 Y c.989 G > A p.Cys 330 Tyr c.843_858 + 7del23 p.Cys 330 Tyr LC10 9 Y c.11 + 5G > A fs* c.1039C > T p.Arg347Cys DNA, desoxynucleotidic acid; Y, year

[0063] Vector Production:

[0064] The pAAV-hRPE65 vector plasmid carries the transgene expression cassette flanked by AAV serotype 2 inverted terminal repeats (ITRs). The expression cassette contains the human RPE65 coding sequence (NCBI RefSeq NM_000329) under control of a human RPE65 promoter fragment (positions 1359 to +23 relative to the transcription start site), and a bovine growth hormone polyadenylation signal.

[0065] For production of the rAAV-2/4.hRPE65 vector, pAAV-hRPE65 plasmid was transfected into HEK293 cells together with pDP4-Kana helper plasmid, which provides both AAV serotype 4 rep and cap genes and adenovirus helper genes (VA RNA, E2A and E4). The vector was purified by ion-exchange chromatography and formulated in a saline solution specific for ocular surgery.

[0066] The rAAV-2/4.hRPE65 vector was filled in 0.5 mL aliquots into 1.2 mL cryovials. Concentration of the final drug product was 610.sup.10 vector genomes per mL, as titered by dot blot hybridization.

[0067] Surgery and Peroperative Treatment:

[0068] Sub-retinal injection was performed under general anesthetic into the eye with the worst visual function. Vitrectomy (20 gauge, three channels) was performed before injection using a 41G cannula. The patient was kept still for 20 minutes after the surgery to promote contact between the viral vector and EPR cells.

[0069] Since retinal detachment varied from one patient to the next, different volumes were injected into each patient, between 200 l and 800 l, corresponding to 1.22. 10.sup.10-4,8. 10.sup.10 vector genomes (Table 2). The number of sub-retinal injection sites was between two and four in each operation (Table 2) with the sites chosen to favor treatment of the peripheral, extramacular retina.

TABLE-US-00003 TABLE 2 Patients injection characteristics. Vol Vector genom Injection number First cohort CG01 330 L 2.01 .Math. 10.sup.10 2 BJ03 200 L 1.22 .Math. 10.sup.10 3 MM04 300 L 1.83 .Math. 10.sup.10 3 Second cohort MR05 700 L 4.27 .Math. 10.sup.10 5 HM06 770 L 4.7 .Math. 10.sup.10 2 HT07 530 L 3.23 .Math. 10.sup.10 4 Third cohort AM08 700 L 4.27 .Math. 10.sup.10 4 HM09 800 L 4.8 .Math. 10.sup.10 4 LC10 770 L 4.7 .Math. 10.sup.10 3 Vol, volume; L, microliters

[0070] A week after injection, patients were given oral prednisolone ( mg/kg/day) then 1 mg/kg/day for a week after surgery. This dose was then stepped down over the next month. Topical postoperative treatment consisted of dexamethasone-tobramycin eye drops (three times a day for a month) together with 1% atropine eye drops in the operated eye (once daily for seven days).

[0071] Assessment of Dissemination of the Viral Vector:

[0072] After surgery, patients were kept in a confinement chamber from D0 to D+3. Biodissemination of the AAV2/4.RPE 65 vector was analyzed in serum, nasal discharge and urine before injection of the viral vector and then one, two and three days after injection. Tests were carried out by qPCR PREMIX EX TAQ (Perfect Real Time) TAKARA (Sigma), Fluo: FAM/TAMRA (Eurogentec). The Kit QlAamp Viral RNA mini kit (QIAGEN) was used for extraction: 1 cycle at 95 C. for 10 minutes, 4515-second cycles at 95 C., 4530-second cycles at 62 C. The limit of detection was 25 copies and the limit of quantitation was 100 copies.

[0073] Safety:

[0074] A routine ophthalmologic examination was carried out with microscopic inspection of the anterior chamber and vitreal cavity. Retinal inflammation was scored on the Nussenblatt scale combined with a Tyndall protein measurement in the anterior chamber using a laser flare meter (Kowa FM700). Chorioretinal tolerance was assessed on photographs of the retina according to the ETDRS method using non-mydriatic retinography (TOPCON TRC-NW6S) after dilatation of the pupil (tropicamide, Ciba Vision Faure, Novartis, Annonay, France). Macular thickness, retinal structure and nerve fibre thickness were analyzed by spectral domain OCT (Heidelberg Engineering, Spectralis HRA-OCT). The thickness of the external nuclear layer was measured manually by two different observers at the fovea then at points 300 m and 1000 temporal and nasal to the fovea (Heidelberg Engineering, Spectralis HRA-OCT). Angiography (Heidelberg Engineering, Spectralis HRA-OCT) with fluorescein (5 mL fluorescein sodium) and Indocyanin Green (Infracyanine, SERB) was carried out to observe vascular and retinal changes following vector injection. Physical examinations, blood chemistry and hematological tests were carried out before and after sub-retinal injection.

[0075] Patients filled in a safety questionnaire on eye pain, ocular discomfort and blurred vision after surgery.

[0076] An Immunological Study:

[0077] Humoral Responses to AAV4 Vector: The analyses were performed in INSERM 1089 laboratory under the control of our quality management system that is approved by Lloyd's Register Quality Assurance LRQA to meet requirements of international Management System Standards ISO 9001:2008.

[0078] The detection of anti-AAV4 IgG antibodies in patient sera was performed using an Enzyme Linked Immuno Sorbent Assay (ELISA) with a method validated according to the ICH(Q2 R1) quality guideline. Briefly, patient sera were serially diluted in PBS-Tween 0.1% buffer and incubated in 96 well plates pre-coated with recombinant AAV2/4 viral particles. The reaction was revealed after incubation with peroxidase conjugated donkey anti-human IgG F(ab)2 fragment (Jackson Immunoresearch), and TMB substrate (BD Biosciences). Optical densities were read (450 nm-570 nm) using a microplate spectrophotometer reader (MultScan GO, Thermo). For each dilution, the threshold of positivity was determined as the mean of optic densities+3SD obtained independently with 19 negative serum from healthy donors. For positive samples, IgG titer was defined as the last serum dilution with an optical density remaining above the threshold curve.

[0079] Neutralizing factors against AAV4 were detected using a neutralization assay. The assay is based on the inhibition of Cos cell line transduction in the presence of serial serum dilutions using an AAV4 vector expressing the Green Fluorescent Protein (GFP) reporter gene. Percentages of GFP positive cells were determined by flow cytometry 72 hours after cell infection. The neutralizing titer was defined as the highest serum dilution inhibiting the AAV transduction by 50% in comparison with the transduction control without serum.

[0080] Cellular immune responses to AAV4 vector and RPE65 transgene product: Cellular immune responses against AAV4 capsid and RPE65 gene product were evaluated using IFN ELISpot assays, and were performed at the immunology platform of Nantes University Hospital and when necessary, for some sample second runs, at INSERM 1089 laboratory. Briefly, frozen PBMC were plated in anti-INF precoated 96-well ELIspot plates (human INF ELISpot plus kit, Mabtech) and stimulated in the presence of an overlapping peptide library at the final concentration of 2 g/ml (Pepscreen, Sigma) covering either the sequence of AAV4 VP1 capsid protein (divided in 3 pools), or the sequence of RPE65 protein (divided in 2 pools). The reaction was revealed 24 hours after cell stimulation according to the manufacturer instruction (human INF ELISpot plus kit, Mabtech). The results were expressed as spot-forming units (SFC)/10.sup.6 cells. A positive response to any peptide pool was arbitrarily defined as a SFC/10.sup.6 response >50 SFC/10.sup.6 cells and at least 3 times higher than the number of spots recorded with non-activated cells (medium alone).

[0081] Efficacy:

[0082] Distant visual acuity was scored on the ETDRS scale and near visual acuity on the Parinaud scale. Color perception was assessed with a monocular, saturated 15-hue test. When visual acuity was better than 20/200, changes in visual field were assessed using an automatic perimeter visual field (Octopus 101 perimeter, Haag-streit Inc, Koeninz, Switzerland) coupled to semi-static Goldmann analysis in V4. Visual field areas were analyzed using Allplan 2015 software with statistical analysis by R software (Version 3.0, R Foundation for Statistical Computing, Vienna, Austria). Microperimetry with a 4-2 strategy was carried out after 10 minutes of dark adaptation using 200 ms stimuli u to a luminescence of 127 cd/m.sup.2 (Nidek MP1 microperimeter-NAVIS software version 1.7.1, Nidek Technologies, Padova, Italy). Broad-field ERG according to the ISCEV protocol was carried out on a vision monitor (Monpack3, Metrovision, Perenchies, France). When fixation was good enough, multifocal ERG was carried out on a RETIscan system (Roland Consult, Wiesbaden, Germany) with RETIscan software (version 3.15) in line with ISCEV recommendations. Dynamic pupillometry was used to measure pupil size and rates of dilatation and contraction in response to a series of flashes was measured using a Vision Monitor Pupillometry device (Metrovision, Perenchies, France). In order to assess changes in patients' capacity for displacement after sub-retinal injection, a mobility test was carried out. The displacement time for patients with either the operated eye or the other one covered up was measured in milliseconds. For this test, patients had to move round a maze with two different light levels (4 lux and 240 lux) with the path chosen randomly. The test was repeated in triplicate for each eye and in each lighting condition. A questionnaire about the patients' impressions of their vision was administered after surgery.

[0083] Functional MRI:

[0084] The inventors use a block design study, one run consisting in three 30 second conditions presented alternatively 4 times: [0085] condition 1: rest in darkness, without any visual stimulation. [0086] condition 2: white uniform screen flickering (5 Hz). Luminance will be constant during the 30 second presentation, but will be modified from low to high level between the 4 repetitions. [0087] condition 3: black and white full screen checkerboard flickering (5 Hz). Luminance will be constant during all the presentation, but the checkerboard contrast will be modified from low to high level between the 4 repetitions.

[0088] Each subject will undergo 3 runs during the fMRI session. Comparing recorded activities between conditions 1 and 2 will show cortical responses to luminance modulations; Activity between conditions 1 and 3 will be related to contrast modulations.

[0089] Visual stimulations will be generated with specific software to control images luminance and contrast. Functional acquisitions will be made with a 1.5 Tesla Magnetic Resonance system and a standard head coil. Functional data will be acquired with T2*-weighted gradient-Echo Planar Image (EPI) sequences. T1 weighted three-dimensional anatomical acquisitions (MP-RAGE) will be recorded at the end of the session. Individual MRI data will be analyzed with SPMS software package (Wellcome Department of Cognitive Neurology, London, U.K.).

[0090] Results

[0091] The patients were between 15 and 42 years of age at the time of surgery (Table 1). All carried mutations in the rpe65 gene. Since retinal detachment varied from one patient to the next, different volumes were injected into each patient, between 200 l and 800 l, corresponding to 1.22. 10.sup.10-4.8. 10.sup.10 vector genomes (Table 2). The number of sub-retinal injection sites was between two and four in each operation (Table 2) with the sites chosen according to either the residual visual field prior to the operation or peroperative retinal detachment.

[0092] During the year of follow-up, no systemic adverse effects were reported following sub-retinal injection of the AAV2/4-Rpe65-Rpe65 vector in any of the nine treated patients. Pre- and post-operative ocular inflammation was measured with a Laser Flare meter.

[0093] Increased inflammation was observed on D+4 in three patients (HT07, HM09 and LC10) with return to normal 14 days after sub-retinal injection (FIG. 1A). For patient HT07, topical anti-inflammatory treatment was stepped up to six instillations a day from D+3 and continued throughout the hospital stay, resulting in rapid regression of the inflammation. More severe inflammation was observed in the other two patients with a Laser Flare readings of 125.78.5 ph/ms and 153.613.9 ph/ms on D+4 followed by normalization by D+14 (FIG. 1B). These two patients received doses of 800 L and 770 L of viral vector suspension and sub-retinal injection bubbles had been observed during the operation, especially in the vitreous with a bubble that spread out somewhat over the surface. For patient LC10, the bubble took over four days to disappear with a sheet of vector fluid still visible in the OCT examination performed four days after injection.

[0094] Ophthalmologic monitoring did not detect any adverse effects during the year of follow-up, i.e. no retinal detachment or cataract. No adverse systemic effects were reported with no changes in hematological parameters or blood chemistry results at a series of different time points. The safety questionnaire revealed some itching and pain at the suture points immediately after surgery and lasting a few days. Angiography did not detect postoperative inflammatory or vascular abnormalities. The only significant facts were a mask effect at the spots where the cannula had been inserted into the retina for the injection, which left a scar.

[0095] In the distribution analysis, the viral vector was mostly detected in postoperative samples of nasal discharge. In four patients (BJ03, HM06, HT07 and LC10), between 4 and 201 copies were measured with peak leaching of the viral vector around D+2. Only in BJ03 and LC10 were viral load readings above the limit of detection and, in LC10 above the limit of quantitation with a peak of 204 copies measured in the tears on D+2. Between D0 and D+2, virus was only detected in the blood of one patient, HM06: this was temporary and low-level (24 and 19 copies). No virus was ever detected in urine. Patients were able to leave the confinement chamber on D+3.

[0096] Six of the nine included patients had nystagmus (FIG. 2A) and four of them have divergent strabismus (FIG. 2A). Patient HM06 saw his director eye change with preferential fixation with the treated eye following surgery. Patient HT07 reported preferring to use his treated eye which had originally been the weaker one for near vision with installation of alternating fixation according to distance. MR05 reported a change in sensation of modification of ocular dominance following surgery. Visual acuity increased by 2.5 EDTRS letters after surgery in the treated eye and by 1 EDTRS letter in the other one (FIG. 2B). There was a difference in visual acuity gain between patients with and without nystagmus. In those with nystagmus, the gain was +7.6 EDTRS letters in the treated eye and +1.6 letters in the other one (FIG. 2C). This difference approaches significance (p=0.05855). Patients HT07 and HM08 showed the strongest gain in visual acuity (+15 and +12 EDTRS letters).

[0097] Change in visual field varied from one subject to the next. It improved in patients CG01, BJ03, HM06, HT07, AM08 and LC01, it remained unchanged in MR05 (the oldest patient) and it decreased in MM04 and HM09 (FIGS. 3A and 3B). Some patients like HM06 and BJ03 saw the gain in visual field area multiplied respectively by factors of 4.2 and 2.8 (FIG. 3C). In contrast, visual field shrunk in MM04 and HM09 by respective factors of 0.9 and 0.65 (FIG. 3C). Visual field recovery was greatest in patients injected with the highest dose with a mean loss of 5.32667 in patients injected with the lowest dose compared with a mean gain of 11.293167 in those injected with the highest dose. Recovery seems to correlate with the dose of vector injected although the result for this small sample is insignificant (p=0.381).

[0098] No change in electroretinographic pattern was observed after sub-retinal injection of the AAV2/4.rpe65 vector.

[0099] The efficacy questionnaire revealed improved detail perception in four out of nine patients, improved fixation in three and, in one patient each, improved color vision, reduced photophobia and less visual fatigue.

Discussion

[0100] The safety of sub-retinal injection of the retinal epithelium-specific AAV2/4-Rpe65-Rpe65 vector was evaluated in patients with Leber's congenital amaurosis due to a defective rpe65 gene. No adverse systemic or ophthalmologic effects were reported in any of the nine patients treated.

[0101] Several sub-retinal injections with 2-4 retinotomies in the course of this study did not lead to any adverse effects in the retina. No retinal detachment was observed immediately after surgery or in one year of follow-up. Multiple injections mean that a greater retinal surface area can be treated, adapted to the preoperative state of the retina. Monitoring of postoperative ocular inflammation showed that some patients experience transient and moderate inflammation as measured using a Flare Meter. This increase was observed in three patients injected with the highest dose of vector in the D+4 examination but not on D+14. In these patients, we had observed that the sub-retinal injection bubble was dominant in the vitreous during surgery with slower disappearance (over 24 hours). It is likely that diffusion of the vector into the vitreous happens some time after injection which meant that we saw peak inflammation after four days. Nevertheless, vitrectomy alone without viral vector injection induces a rise in Laser Flare reading with a peak within a week of surgery, e.g. vitrectomy for rhegmatogenous retinal detachment (Hoshi) and all the more so because patients with pigmentary retinopathy have a modified hematoretinal barrier (Murikami).

REFERENCES

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