GENE THERAPY FOR BARDET-BIEDL SYNDROME

Abstract

There is provided a vector for treating retinal degeneration associated with Bardet-Biedl Syndrome (BBS), wherein the vector comprises a promoter operably linked to a BBS1 gene, wherein the promoter is selected from a rhodopsin kinase (RK) promoter, a cytomegalovirus immediate-early (CMV) promoter and a CAG promoter, and wherein the vector is selected from an AAV2/8 vector, an AAV2/7m8 vector and an AAV9 vector. Also disclosed is a pharmaceutical composition comprising the vector, and use of the vector in a method of treating retinal degeneration associated with BBS comprising administering a therapeutically effective amount of the vector to a patient suffering from BBS, wherein the vector is administered directly to the eye of the patient.

Claims

1. A vector for treating retinal degeneration associated with Bardet-Biedl Syndrome (BBS), wherein the vector comprises a promoter operably linked to a BBS1 gene, wherein the promoter is selected from a rhodopsin kinase (RK) promoter, a cytomegalovirus immediate-early (CMV) promoter and a CAG promoter, and wherein the vector is selected from an AAV2/8 vector, an AAV2/7m8 vector and an AAV9 vector.

2. (canceled)

3. A vector according to claim 1, wherein the promoter is a human RK promoter.

4. A vector according to claim 3, wherein the RK promoter comprises the sequence of SEQ ID NO. 7.

5. A vector according to claim 1, wherein the promoter is a CMV promoter.

6. A vector according to claim 5, wherein the CMV promoter comprises a nucleotide sequence selected from SEQ ID NO. 5 and SEQ ID NO. 6.

7. A vector according to claim 1, wherein the promoter is a CAG promoter.

8. A vector according to claim 7, wherein the CAG promoter comprises a nucleotide sequence selected from SEQ ID NO. 3 and SEQ ID NO. 4.

9.-11. (canceled)

12. A vector according to claim 1, wherein the BBS1 gene encodes a functional human BBS1 protein comprising the amino acid sequence of SEQ ID NO. 2 or an amino acid sequence with at least 80% sequence identity thereto.

13. A vector according to claim 1, wherein the BBS1 gene encodes a wild type human BBS1 protein.

14. A vector according to claim 1, wherein the BBS1 gene comprises the nucleotide sequence of SEQ ID NO. 1 or a nucleotide sequence with at least 70% sequence identity thereto, and encodes a functional human BBS1 protein.

15. A vector according to claim 14, wherein the BBS1 gene comprises the nucleotide sequence of SEQ ID NO. 1.

16. A vector according to of claim 1, wherein the BBS1 gene comprises the nucleotide sequence of SEQ ID NO. 8 or 9.

17. A vector according to claim 1, wherein: 1) the BBS1 gene comprises the nucleotide sequence of SEQ ID NO: 1, and the promoter is a human RK promoter; 2) the BBS1 gene comprises the nucleotide sequence of SEQ ID NO. 8, and the promoter is a human RK promoter; 3) the BBS1 gene comprises the nucleotide sequence of SEQ ID NO. 9, and the promoter is a human RK promoter; 4) the BBS1 gene comprises the nucleotide sequence of SEQ ID NO. 1, and the promoter is a CMV promoter; 5) the BBS1 gene comprises the nucleotide sequence of SEQ ID NO. 8, and the promoter is a CMV promoter; 6) the BBS1 gene comprises the nucleotide sequence of SEQ ID NO. 9, and the promoter is a CMV promoter; 7) the BBS1 gene comprises the nucleotide sequence of SEQ ID NO. 1, and the promoter is a CAG promoter; 8) the BB gene comprises the nucleotide sequence of SEQ ID NO. 8, and the promoter is a CAG promoter; or 9) the BB gene comprises the nucleotide sequence of SEQ ID NO. 9, and the promoter is a CAG promoter.

18.-19. (canceled)

20. A vector according to claim 1, wherein: 1) the vector is an AAV2/8 vector, the promoter is a rhodopsin kinase (RK) promoter, and the BBS1 gene encodes a functional human BBS1 protein; 2) the vector is an AAV2/8 vector, the promoter is a cytomegalovirus immediate-early (CMV) promoter, and the BB S1 gene encodes a functional human BBS1 protein; 3) the vector is an AAV2/8 vector, the promoter is a CAG promoter, and the BBS1 gene encodes a functional human BBS1 protein; 4) the vector is an AAV2/7m8 vector, the promoter is a rhodopsin kinase (RK) promoter, and the BBS1 gene encodes a functional human BBS1 protein; 5) the vector is an AAV2/7m8 vector, the promoter is a cytomegalovirus immediate-early (CMV) promoter, and the BB S1 gene encodes a functional human BB S1 protein; 6) the vector is an AAV2/7m8 vector, the promoter is a CAG promoter, and the BB S1 gene encodes a functional human BB S1 protein; 7) the vector is an AAV9 vector, the promoter is a rhodopsin kinase (RK) promoter, and the BBS1 gene encodes a functional human BBS1 protein; 8) the vector is an AAV9 vector, the promoter is a cytomegalovirus immediate-early (CMV) promoter, and the BBS1 gene encodes a functional human BBS1 protein; or 9) the vector is an AAV9 vector, the promoter is a CAG promoter, and the BBS1 gene encodes a functional human BB S1 protein.

21.-22. (canceled)

23. A vector according to claim 1, wherein the vector is an AAV2/7m8 vector, the promoter is a cytomegalovirus immediate-early (CMV) promoter, and the BBS1 gene encodes a functional human BBS1 protein.

24. A pharmaceutical composition comprising the vector according to claim 1 and one or more pharmaceutically acceptable excipients.

25. A method of treating retinal degeneration associated with Bardet-Biedl Syndrome (BBS) comprising administering a therapeutically effective amount of a vector according to claim 1 to a patient suffering from BBS, wherein the vector is administered directly to the eye of the patient.

26. The method of claim 25, wherein the vector is administered subretinally or intravitreally.

27. The method of claim 25, wherein the vector is administered by subretinal injection.

28. The method of claim 25, wherein the vector is administered by intravitreal injection.

29.-34. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The invention will now be described in detail by way of example only with reference to the figures which are as follows:

[0050] FIG. 1: BBS1 mutation leads to degeneration of the photoreceptors layers in Bbs1.sup.M390R/M390R mice. Histological analysis show the degeneration of the outer nuclear layer and photoreceptor numbers 6 month after birth. This degeneration is also observed by the reduction of both photopic and scotopic amplitude at 6 months of age of Bbs1.sup.M390R/M390R mice compare with wild-type littermates.

[0051] FIGS. 2A-2C: AAV2/8.RK.hBBS1 supplementation of BBS1 only in photoreceptors is not a long term efficient therapeutic strategy in Bbs1.sup.M390R/M390R mice. FIG. 2A shows that delivery of the AAV2/8.RK.hBBS1 vector achieve a small short term recovery of the ERG responses 3 months after delivery but is not able to sustain the recovery long-term when compared with the contralateral control eyes as seen after 11 months of treatment. FIG. 2A shows scotopic a-wave amplitude (uV). FIG. 2B shows scotopic b-wave amplitude (uV). FIG. 2C shows photopic b-wave amplitude (uV). Blue points indicate responses of eyes injected with AAV2/8.RK.hBBS1 and red points indicate responses from control contralateral eyes. Error bars are Standard Deviation (SD).

[0052] FIGS. 3A-3C: Subretinal delivery of AAV2/8.CMV.hBBS1 results in variability in its efficiency to rescue retinal degeneration in Bbs1.sup.M390R/M390R mice. We found there is high variability in the ERG responses of animal injected with the AAV2/8.CMV.hBBS1 vector. Statistical analysis show rescues at different light stimulus for the scotopic and photopic b-wave for P7-9 and P30 injected retinae up to 6 months post injection. FIG. 3A shows scotopic a-wave amplitude (uV). FIG. 3B shows scotopic b-wave amplitude (uV). FIG. 3C shows photopic b-wave amplitude (uV). Blue points indicate responses of eyes injected with AAV2/8.CMV.hBBS1 and red points indicate responses from control contralateral eyes. Error bars are Standard Deviation (SD).

[0053] FIGS. 4A and 4B: Subretinal delivery of AAV2/8.CMV.hBBS1 completely halts retinal degeneration in Bbs1.sup.M390R/M390R in some treated animals for a year. FIG. 4A shows that half of the P7-9 treated retinae achieve complete retinal degeneration rescue up to 11 month after injection of a- and b-wave scotopic and b-wave photopic responses up to 11 months after treatment for all light intensity tested. Blue points indicate responses of eyes injected with AAV2/8.RK.hBBS1 and red points indicate responses from control contralateral eyes. Error bars are Standard Deviation (SD). FIG. 4B shows histological analysis of treated retinae show complete recovery of the outer nuclear layer with maintenance of a nuclei in the layer. Controlateral control untreated eyes show complete lost of all photoreceptors and cell nuclei.

[0054] FIG. 5: Overexpression of BBS1 is not toxic in wild-type (WT) or in Bbs1.sup.M390R/M390R mice. We tested overexpression of BBS1 transgene in the photoreceptors of wild-type P30 animals with low and high doses of AAV2/8.RK-hBBS1 and photopic and Scotopic ERG responses were measured. TUNEL staining was carried out to look for any increase in apoptosis in injected eyes. No TUNEL staining was seen in any of the sections tested from three controls and three injected retinae suggesting no apoptosis is occurring. A western blot was carried out using total retinae protein extracts. The top panel shows the blot probed with an antibody specific to the activated form of caspase 3 (marked with an arrow). The second panel shows a β-Actin loading control. Titer injected in: A; 1×10.sup.11, B; 8×10.sup.11 and C; 4×10.sup.12 vg/ml.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The inventors have developed an ocular gene therapy to halt and further prevent the retinal degeneration phenotype associated with BBS1 deficiency. The inventors found that although Bbs1.sup.M390R/M390R mice presented mainly a photoreceptor degeneration of the outer nuclear layer as seen in patients, supplementation of human BBS1 only in photoreceptors cells alone was not always efficient or sufficient to prevent retinal degeneration. The inventors then explored the therapeutic effects of delivering BBS1 to also target the retinal pigmented epithelium (RPE) cells, highly ciliated and crucial for photoreceptor survival. Expression of the BBS1 transgene in both photoreceptors and RPE was able to prevent completely the degeneration of the photoreceptor layers. Variability in the treatment effect between different treated individuals was observed. The complexity of the function of the BBS1 gene as part of a multi-complex unit might explain the variability on the treatment effect observed.

[0056] The inventors demonstrated that supplementing BBS1 in photoreceptors and RPE leads to functional and cellular rescue of the retinal degeneration in Bbs1.sup.M390R/M390R animals.

Materials and Methods

[0057] A construct has been produced where human BBS1 cDNA (SEQ ID NO. 1—NM_024649.4) has been cloned under the control of the human rhodopsin kinase (RK) promoter in an AAV2 viral plasmid. For virus production, usual methods were used to produce an AAV2/8 virus. 4000 cm.sup.2 of HEK293T cell monolayer cells were transfected with the RK-BBS1-AAV-ITR containing plasmid, AAV2 Rep-Cap plasmid and the helper plasmid. Once showing cytopathic effects, cells were harvested and lysed to release the virus. The adeno-associated virus was purified by Sephacryl 5300 column followed by anion exchange chromatography using a POROS 50 HQ column. The final product was titered by quantitative real-time PCR using AAV specific probe and a SV-40 probe as a standard.

Virus Administration and Titer

[0058] Time matings were prepared between Bbs1.sup.M390R/+ males and Bbs1.sup.M390R/− females. Pups were genotyped for Bbs1 genotype. The adenoviral-associated vector was given via subretinal unilaterally injection in P7-9 and P30 1×10.sup.12 vg/ml (vector genomes/ml). The inventors injected 2 different groups of animals; Bbs1.sup.M390R/M390R animals, wild-type. Contralateral eyes serve as controls for each group. A total of n=10 animals/group were used. Treated animals do not show any physical or behaviour distress after 12 months post-injection.

Results

[0059] FIG. 1: BBS1 mutation leads to degeneration of the photoreceptors layers in Bbs1.sup.M390R/M390R mice. Histological analysis show the degeneration of the outer nuclear layer and photoreceptor numbers 6 month after birth. This degeneration is also observed by the reduction of both photopic and scotopic amplitude at 6 months of age of Bbs1.sup.M390R/M390R mice compare with wild-type littermates.

[0060] FIGS. 2A-2C: AAV2/8.RK.hBBS1 supplementation of BBS1 only in photoreceptors is not a long term efficient therapeutic strategy in Bbs1.sup.M390R/M390R mice. Delivery of the AAV2/8.RK.hBBS1 vector achieves a small short term recovery of the ERG responses 3 months after delivery but is not able to sustain the recovery long-term when compared with the contralateral control eyes as seen after 11 months of treatment. Blue points indicate responses of eyes injected with AAV2/8.RK.hBBS1 and red points indicate responses from control contralateral eyes. Error bars are Standard Deviation (SD).

[0061] FIGS. 3A-3C: Subretinal delivery of AAV2/8.CMV.hBBS1 results in variability in its efficiency to rescue retinal degeneration in Bbs1.sup.M390R/M390R mice. We found there is high variability in the ERG responses of animal injected with the AAV2/8.CMV.hBBS1 vector. Statistical analysis show rescues at different light stimulus for the scotopic and photopic b-wave for P7-9 and P30 injected retinae up to 6 months post injection. Blue points indicate responses of eyes injected with AAV2/8.CMV.hBBS1 and red points indicate responses from control contralateral eyes. Error bars are Standard Deviation (SD).

[0062] FIGS. 4A and 4B: Subretinal delivery of AAV2/8.CMV.hBBS1 completely halts retinal degeneration in Bbs1.sup.M390R/M390R in some treated animals for a year. Half of the P7-9 treated retinae achieve complete retinal degeneration rescue up to 11 month after injection of a- and b-wave scototopic and b-wave photopic responses up to 11 months after treatment for all light intensity tested. Blue points indicate responses of eyes injected with AAV2/8.RK.hBBS1 and red points indicate responses from control contralateral eyes. Error bars are Standard Deviation (SD). Histological analysis of treated retinae show complete recovery of the outer nuclear layer with maintenance of a nuclei in the layer. Controlateral control untreated eyes show complete loss of all photoreceptors and cell nuclei.

[0063] FIG. 5: Overexpression of BBS1 is not toxic in wild-type (WT) or in Bbs1.sup.M390R/M390R mice. We tested overexpression of BBS1 transgene in the photoreceptors of wild-type P30 animals with low and high doses of AAV2/8.RK-hBBS1 and photopic and Scotopic ERG responses were measured. TUNEL staining was carried out to look for any increase in apoptosis in injected eyes. No TUNEL staining was seen in any of the sections tested from three controls and three injected retinae suggesting no apoptosis is occurring. A western blot was carried out using total retinae protein extracts. The top panel shows the blot probed with an antibody specific to the activated form of caspase 3 (marked with an arrow). The second panel shows a β-Actin loading control. Titer injected in: A; 1×10.sup.11, B; 8×10.sup.11 and C; 4×10.sup.12 vg/ml.

[0064] These results demonstrate that the vector has advantageous properties compared to the vector used in Seo et al. (Invest Ophthalmol Vis Sci. 54(9):6118-32 (2013)) in which overexpression of BBS1 protein was shown to be toxic to the retina in mice.

SEQUENCES

[0065] SEQ ID NO. 1—Human Bardet-Biedl syndrome 1 (BBS1) nucleotide sequence (WT), cDNA (NM_024649.4)

[0066] SEQ ID NO. 2—Human BBS1 full protein sequence (Q8NFJ9)

[0067] SEQ ID NO. 3—CAG promoter sequence

[0068] SEQ ID NO. 4—Alternative CAG promoter sequence

[0069] SEQ ID NO. 5—Cytomegalovirus (CMV) immediate-early promoter sequence

[0070] SEQ ID NO. 6—Alternative CMV promoter sequence

[0071] SEQ ID NO. 7—Rhodopsin kinase promoter sequence

[0072] SEQ ID NO. 8—Codon optimised nucleotide sequence encoding human BBS1 protein (referred to as COSEQ1-BBS1)

[0073] SEQ ID NO. 9—Codon optimised nucleotide sequence encoding human BBS1 protein (referred to as COSEQ2-BBS1)

[0074] SEQ ID NO. 10—Construct comprising CMV promoter (nt 52-256) and wild type BBS1 nucleotide sequence (nt 324-2108)

[0075] SEQ ID NO. 11—Construct comprising CMV promoter (nt 367-570) and COSEQ1-BBS1 nucleotide sequence (nt 630-2411)

[0076] SEQ ID NO. 12—Construct comprising CMV promoter (nt 367-570) and COSEQ2-BBS1 nucleotide sequence (nt 630-2411)

[0077] SEQ ID NO. 13—Construct comprising CAG promoter (nt 35-562) and wild type BBS1 nucleotide sequence (nt 712-2493)

[0078] SEQ ID NO. 14—Construct comprising CAG promoter (nt 35-562) and COSEQ1-BBS1 nucleotide sequence (nt 716-2497)

[0079] SEQ ID NO. 15—Construct comprising CAG promoter (nt 35-562) and COSEQ2-BBS1 nucleotide sequence (nt 716-2497)

[0080] SEQ ID NO. 16—Construct comprising RK promoter (nt 16-255) and wild type human BBS1 nucleotide sequence (nt 546-2330)

[0081] SEQ ID NO. 17—AAV2/8 construct comprising RK promoter (nt 16-255) and wild type human BBS1 nucleotide sequence (nt 546-2330)

[0082] SEQ ID NO. 18—AAV2/8 construct comprising CMV promoter (nt 52-256) and wild type human BBS1 nucleotide sequence (nt 324-2108)