Trans-splicing ribozyme targeting rhodopsin transcript and uses thereof

Abstract

A trans-splicing ribozyme capable of splicing a rhodopsin transcript at a target splicing site and containing a sequence that is capable of complementarily binding to a target binding site of the rhodopsin transcript is disclosed. The trans-splicing ribozyme may further containing a desired rhodopsin transcript at 3′-end. The trans-splicing ribozyme may further contains an antisense sequence that is complementary to a region downstream the target binding site of the rhodopsin transcript. A nucleotide molecule encoding the trans-splicing ribozyme is also disclosed. Delivery systems to delivery the nucleotide molecule and/or the trans-splicing ribozyme to target tissue or cells as well as uses of the trans-splicing ribozyme, the nucleotide molecule, delivery systems, or pharmaceutical compositions containing any of them are also disclosed.

Claims

1. A recombinant nucleic acid molecule comprising: (a) a trans-splicing ribozyme comprising a ribozyme sequence capable of splicing at a target splicing site of a target rhodopsin transcript with an internal guide sequence (IGS) complementarily binding to a binding region of the target rhodopsin transcript containing the target splicing site, wherein the binding region comprises 5 to 10 consecutive nucleotides; or (b) a recombinant DNA encoding the trans-splicing ribozyme of (a), wherein the IGS comprises a consecutive nucleotide sequence of 5-10 nt in length and is capable of complementarily binding and forming G/U wobble base pairs at the target splicing site of the target rhodopsin transcript, wherein the target splicing site comprises a nucleotide at the following positions: +30, +35, +42, +43, +52, +54, +55, +59, +75, +97, +116, +121, +123, +127, +132, +140, +154, +165, +171, +187, +191, +207, +215, +222, +230, +232, +235, +244, +256, +262, +273, +298, +308, +381, +403, +661 or +688, wherein the positions are identified with reference to SEQ ID NO: 1.

2. The recombinant nucleic acid molecule according to claim 1, wherein the trans-splicing ribozyme has a structure of 5′-IGS-ribozyme*-3′, wherein the ribozyme* is a ribozyme sequence without an IGS.

3. The recombinant nucleic acid molecule according to claim 2, wherein the trans-splicing ribozyme further comprises an exon sequence at a position downstream of the ribozyme.

4. The recombinant nucleic acid molecule according to claim 1, wherein the target rhodopsin transcript comprises a mutation.

5. The recombinant nucleic acid molecule according to claim 4, wherein the mutation in the target rhodopsin transcript is mutation at one or more positions corresponding to position 1 to position 1142 of SEQ ID NO: 1.

6. The recombinant nucleic acid molecule according to claim 5, wherein the mutation is one or more selected from the group consisting of: L328P, T342M, Q344R/P/ter, V345L/M, A346P, P347A/R/Q/L/S/T, ter349/Q/E, N15S, T17M, V20G, P23A/H/L, Q28H, G51R/V, P53R, T58R/M, V87D/L, G89D, G106R/W, C110F/R/S/Y, E113K, L125R, W161R, A164E/V, C167R/W, P171Q/L/S, Y178N/D/C, E181K, G182S/V, C185R, C187G/Y, G188R/E, D190N/G/Y, H211R/P, C222R, P267R/L, S270R, K296N/E/M, R135G/L/P/W, T4K, T17M, M39R, N55K, G90V, M44T, V137M, G90D, T94I, A292E, A295V, F45L, V209M, F220C, P12R, R21C, Q28H, L40R, L46R, L47R, F52Y, F56Y, L57R, Y60ter, Q64ter, R69H, N78I, L79P, L88P, T92I, T97I, V104F, G109R, G114D/V, E122G, W126L/ter, S127F, L131P, Y136ter, C140S, T160T, M163T, A169P, P170H/R, S176F, P180A/S, Q184P, S186P/W, Y191C, T193M, M207R/K, V210F, I214N, P215L/T, M216R/L/K, R252P, T289P, S297R, A298D, K311E, N315ter, E341K, S343C, and Q312ter in the rhodopsin protein encoded by SEQ ID NO: 1.

7. The recombinant nucleic acid molecule according to claim 1, wherein the recombinant DNA encoding the trans-splicing ribozyme comprises the sequence of SEQ ID NO: 2, 3, or 4.

8. A non-viral gene carrier comprising nucleic acid molecule of claim 1.

9. The non-viral gene carrier according to claim 8, which is a lipid bilayer nanoparticle or liposome.

10. The recombinant nucleic acid molecule according to claim 3, wherein the exon sequence is a polynucleotide encoding a normal wild-type rhodopsin protein, a polynucleotide encoding a reporter protein, and/or a combination thereof.

11. The recombinant nucleic acid molecule according to claim 10, wherein the trans-splicing ribozyme of (a) further comprises an antisense sequence at a position upstream of the IGS, said antisense sequence being complimentary to a portion of the target rhodopsin transcript sequence.

12. A gene construct comprising the recombinant DNA of claim 1 and a promoter sequence operably linked the recombinant DNA to express the trans-splicing ribozyme.

13. A gene construct comprising the recombinant DNA of claim 3 and a promoter sequence operably linked the recombinant DNA to express the trans-splicing ribozyme.

14. A recombinant expression vector comprising the gene construct according to claim 12.

15. A recombinant virus comprising the gene construct according to claim 13.

16. A non-viral gene carrier comprising the recombinant nucleic acid molecule of claim 10.

17. The recombinant virus of claim 15, wherein the virus is selected from the group consisting of adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes simplex virus, and vaccinia virus.

18. The recombinant virus of claim 15, wherein the virus is a recombinant adeno-associated virus (AAV) comprising a polynucleotide encoding the trans-splicing ribozyme, wherein the polynucleotide is operably linked to a promoter sequence; and wherein the recombinant AAV is derived from a native or artificial adenovirus serotype, an isolate thereof, or a clade thereof.

19. A pharmaceutical composition comprising any of the following (i)-(vii): (i) the recombinant nucleic acid molecule according to claim 1; (ii) a non-viral gene carrier comprising nucleic acid molecule of claim 1; (iii) a gene construct comprising the recombinant DNA of claim 1 and a promoter sequence operably linked the recombinant DNA to express the trans-splicing ribozyme; (iv) a recombinant expression vector comprising the gene construct (iii); (v) a recombinant virus comprising the gene construct (iii) or the recombinant expression vector (iv); (vi) a non-viral gene carrier comprising the gene construct (iii) or the recombinant expression vector (iv); (vii) a combination of one or more of (i)-(vi), as an active ingredient, and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of claim 19, wherein the trans-splicing ribozyme further comprises an exon sequence at a position downstream of the ribozyme.

21. The pharmaceutical composition of claim 19, wherein the target rhodopsin transcript comprises a mutation.

22. A method of treatment or prevention of retinitis pigmentosa in a subject in need thereof, comprising administering the pharmaceutical composition of claim 19 to the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 is a schematic diagram showing the mechanism of trans-splicing ribozyme targeting RHO transcript according to an embodiment of the present invention.

(3) FIG. 2 is a schematic diagram showing a basic configuration of a gene construct comprising a ribozyme according to an embodiment of the present invention containing a promoter, trans-splicing ribozyme sequence, and normal (WT) rhodopsin gene sequence.

(4) FIG. 3A is a schematic diagram showing the process of selection of target sites on RHO RNA through in vitro mapping and intracellular mapping and FIG. 3B shows the in vitro and in vivo (intracellular) mapping results.

(5) FIG. 4 is a schematic diagram showing optimization of a gene construct comprising a trans-splicing ribozyme according to an embodiment of the present invention targeting +59 site (the base at position 59) of the Rho transcript (hereinafter referred to as “RHO targeting ribozyme”). In FIG. 4, the sequence cagcauucUUGGGUGGGagcagccac is represented by SEQ ID NO: 20 (underlined U″ is at the position 59), the IGS sequences 5′-gcccaa-3′ is represented by SEQ ID NO: 18. Kozak sequence 5′-cccacc-3′ is represented by SEQ ID NO: 17. P1 sequences 5′-cccgcccaa-3′ and 5′-uccgcccaa-3′ are represented by SEQ ID NOs: 14 and 21, respectively, and P10 sequences 5′-cguacuc-3′ and 5′-gaguacg-3′ are represented by SEQ ID NOs: 15 and 16, respectively.

(6) FIG. 5 is a schematic diagram of a vector including a RHO targeting ribozyme according to an embodiment of the present invention.

(7) FIG. 6A-FIG. 6D depict the results of comparison of RHO targeting ribozyme constructs in 293A cells.

(8) FIG. 7A and FIG. 7B show the results of an experiment confirming in vitro efficacy of the RHO targeting ribozyme using cell lines stably expressing the P23H mutant hRHO gene. FIG. 7A is nucleotide sequence analysis result confirming that trans-splicing (t/s) occurred at the target site. FIG. 7B is fluorescent microscopic photos showing the expression of ribozyme in the cells using an anti-RHO antibody and a secondary fluorescent-labeled antibody, which confirm that in the cells transfected with the RHO targeting ribozyme, RHO proteins were localized to the cell membrane, whereas the RHO proteins with P23H mutation were dispersed in cytosol.

(9) FIG. 8 shows the results of an experiment confirming in vitro efficacy of RHO-targeting ribozymes against various RHO mutations.

(10) FIG. 9 is a schematic diagram of a recombinant AAV expression vector comprising a RHO targeting ribozyme according to an embodiment of the present invention.

(11) FIG. 10A and FIG. 10B are microscopic photos of the eyeballs of hP23H-RFP ADRP mouse model, in which cells of the eyeballs were stained with DAPI. The photos confirm the efficacy of ribozyme in a mouse model using an AAV expression vector comprising a RHO targeting ribozyme according to an embodiment of the present invention. FIG. 10A shows the result 2 weeks after administration, and FIG. 10B shows the result 5 weeks after administration.

(12) FIG. 11A and FIG. 11B show immune and inflammatory responses in serum after administration of an AAV vector containing RHO targeting ribozyme in an hP23H-RFP ADRP mouse model according to an embodiment of the present invention. (FIG. 11A is the result 2 weeks after administration, FIG. 11B is the result 5 weeks after administration).

(13) FIG. 12A-FIG. 12C show experimental results confirming the efficacy of the RHO targeting ribozyme in retina and retinal pigment epithelium (RPE) in a hP23H-RFP ADRP mouse model after administration of the AAV vector according to an embodiment of the present invention.

(14) FIG. 13 shows the in vivo distribution of RHO target ribozyme in a hP23H-RFP ADRP mouse model after administration of an AAV vector comprising the ribozyme according to an embodiment of the present invention.

(15) FIG. 14 is microscopic photos of H&E stained eyes of normal mice, showing the result of analyzing the toxicity after administration of the AAV vector containing a RHO targeting ribozyme in a normal mouse according to an embodiment of the present invention.

(16) FIG. 15 shows the result of performing an electroretinogram test after administration of the AAV vector containing a RHO targeting ribozyme in a hP23H-RFP ADRP mouse model according to an embodiment of the present invention.

(17) FIG. 16A shows the results of the analysis of serum and FIG. 16B shows the results of ribozyme activity, after administration of the AAV vector containing a RHO targeting ribozyme according to an embodiment of the present invention in a hP23H-RFP ADRP mouse model.

(18) FIG. 17 is a schematic diagram of various recombinant AAV vectors containing RHO targeting ribozymes according to an embodiment of the present invention.

(19) FIG. 18A and FIG. 18B show experimental result confirming the trans-splicing activity of RHO targeting ribozyme in a hP23H mouse-RFP ADRP mouse model at 3 weeks after administration of the recombinant AAV vector containing the ribozyme according to an embodiment of the present invention.

(20) FIGS. 19 and 20 show electroretinograms of a hP23H mouse-RFP ADRP mouse model, after administration of a recombinant AAV vector containing the RHO targeting ribozyme according to an embodiment of the present invention.

(21) FIG. 21 shows ribozyme distribution and ribozyme RNA expression in the retina after administration of a recombinant AAV vector containing the RHO targeting ribozyme according to an embodiment of the present invention.

MODES FOR CARRYING OUT THE EMBODIMENTS

(22) The present invention has various embodiments which may include various modifications. Hereinafter, specific embodiments illustrated in the drawings are described in detail. However, this is not intended to limit the invention to specific embodiments. All modifications, equivalents and substitutes within the spirit and scope of the present invention should be understood to be included. When a detailed description of the technology may obscure the essence of the present invention, such description may be omitted.

Example 1: Design of Rhodopsin (RHO) RNA Targeting Trans-Splicing Ribozyme

(23) Sequences of RHO RNA variants were analyzed to select a target RNA site. Design of RHO targeting trans-splicing ribozyme is shown in FIG. 2.

Example 2: Selection of Target Site of Rhodopsin (RHO) RNA by In Vitro Mapping and Intracellular Mapping

(24) WT RHO RNA or P23H RHO RNA and ribozyme RNA library was used to perform in vitro mapping and intracellular mapping. A reaction was carried out by mixing the random ribozyme library RNA with Rhodopsin RNA in test tubes for in vitro mapping, RT-PCR was performed and then trans-splicing was performed through sequencing of PCR DNA bands expected as products. The target rhodopsin RNA site was identified.

(25) More specifically, rhodopsin RNA is synthesized by in vitro transcription and trans-splicing ribozyme library RNA with a random sequence (G) at the 5′ end is constructed. In vitro transcription was performed as follows: 0.1 pmol of rhodopsin RNA was added to 1× in vitro trans-splicing reaction Buffer (50 mM HEPES (pH 7.0), 150 mM NaCl, 5 mM MgCl2) to make a total of 10 μL. 1 pmole of ribozyme library RNA was added to mix with in vitro trans-splicing reaction buffer (1×) to make a total of 9 μL, then 1 μL of 1 mM GTP was added for the final concentration of 0.1 mM GTP in a separate tube. The two separate reactions were incubated at 95° C. for 1 minute and then at 37° C. for 3 minutes, respectively. The two reactions were then mixed and incubated at 37° C. for 3 hours. RNA was recovered from the reaction mix by phenol extraction/EtOH precipitation, and RT-PCR was performed.

(26) A ribozyme-specific RT primer (5′-ATGTGCTGCAAGGCGATT-3′) (SEQ ID NO: 10) was added, and cDNA was synthesized by reverse transcription in a total volume of 20 μL 35 cycles of PCR were carried out using 2 uL of cDNA, 10 pmol of rhodopsin RNA specific 5′ primer (5′-CTACTCAGCCCCAGCGGAGG-3′) (SEQ ID NO: 11), and 10 pmol of ribozyme specific 3′ primer (RY-TS) (5′-TGTAAAACGACGGCCAGTG-3′) (SEQ ID NO: 12) under the following cycling conditions: 95° C. 30 seconds, 55° C. for 30 seconds and 72° C. for 30 seconds. PCR products were separated by electrophoresis on a 2% TBE agarose gel.

(27) All identified PCR products were subjected to phenol extraction & ethanol precipitation process. After buffer change, a sequencing was performed to determine the site(s) where the trans-splicing reaction occurs in the rhodopsin RNA. Sequencing analysis of trans-splicing reaction sites revealed that most efficient targeting occurred at 59.sup.th site that is the 5′ UTR region of RHO RNA (FIG. 3A and FIG. 3B).

(28) Intracellular co-transfection was performed by mixing random ribozyme library RNA with rhodopsin RNAs in test tube, to carry out intracellular mapping. Specifically, the 293 cells were seeded in a 35 mm culture dish at a density 1×10.sup.5 and were maintained at 37° C. incubator with 5% CO.sub.2. The random ribozyme library RNA and rhodopsin RNA were collected in a 1.5 ml tube and mixed with OPTI-MEM™ 100 μl. LIPOFECTAMIN™ 2000 and OPTI-MEM™ 100 μl were added and incubated at room temperature for 5 minutes. Then, the contents of the two tubes were mixed and incubated at room temperature for 20 minutes to allow for liposome complex formation. After 20 minutes, the tubes were centrifuged for 10 seconds and the liposome complex was added on the cells for co-transfection. After 4 hours, the medium was replaced with a new medium. The cells were kept in the incubator at 37° C. with 5% zCO.sub.2 for 24 hours. After incubation, the cells were washed with 1×PBS, and RNA was extracted by treatment with trizol 500 ul. 5 μg of the extracted RNA was treated with 1 μl of DNase I to remove gDNA.

(29) 1 μg of RNA was mixed with the ribozyme-specific RT primer (5′-ATGTGCTGCAAGGCGATT-3′) (SEQ ID NO: 10), and cDNA was synthesized through reverse transcription in a volume condition of 20 μL. 2 μL of synthesized cDNA was amplified with 10 pmol each of rhodopsin RNA specific 5′ primer (5′ CTACTCAGCCCCAGCGGAGG-3′) (SEQ ID NO: 11), ribozyme specific 3′ primer (RY-TS) (5′-TGTAAACGACGGCCAGTG-3′) (SEQ ID NO: 12) using the following cycling conditions: 35 cycles of 30 sec at 95° C., 30 sec at 55° C., 30 sec at 72° C. PCR products were separated on a 2% TBE agarose gel. PCR products were isolated by phenol extraction & ethanol precipitation. The nucleotide sequence analysis was performed to determine where the trans-splicing reaction occurred within RHO RNA.

Example 3: Preparation of Trans-Splicing Ribozyme

(30) A gene construct containing a nucleotide encoding normal wild-type rhodopsin (“3’ exon’ in FIG. 4) and trans-splicing ribozyme targeting the base (U) at position 59 of the RHO transcript (position numbering is based on SEQ ID NO: 1) was manufactured and optimized (FIG. 4). The target region comprises the sequence cagcauucUUGGGUGGGagcagccac (SEQ ID NO: 20) and the target splicing site is U at position 59 (underlined) of RHO RNA, and the construct includes IGS (5′-GCCCAA-3′: SEQ ID NO: 18), at the 5′ end, wherein the IGS is capable of partially complementarily binding to the above target region. To analyze the trans-splicing efficiency of the ribozyme targeting RHO RNA +59 base (“RHO target ribozyme”) in cells, an improved RHO targeting ribozyme construct was prepared. It is known that Group I introns having 6-nt IGS alone are, when expressed in mammalian cells, either inactivated or exhibit non-specific activity.

(31) To prepare an improved RHO targeting ribozyme construct, a complementary oligonucleotide containing an extended P1 (5′-CCCGCCCAA-3′ (SEQ ID NO: 14) and/or 5′-UCCGCCCAA-3′ (SEQ ID NO: 21)) and 7-nt long P10 helix (5′-CGUACUC-3′ (SEQ ID NO: 15) and/or 5′-GAGUACG-3′ (SEQ ID NO: 16)) was inserted to the upstream of IGS of the ribozyme.

Example 4: Optimization of Trans-Splicing Ribozymes

(32) To confirm that RHO targeting ribozyme was optimized for trans-splicing, vectors were constructed with RHO targeting ribozyme of the first, second and third design prepared in Example 3 (FIG. 5).

(33) In FIG. 5, AS150 is SEQ ID NO: 6; SD/SA is SEQ ID NO: 7; sequence of part of 5′UTR is SEQ ID NO: 8. Linker sequence T2A, YFP and polyA sequences are well known in the art and their specific nucleotide sequences are omitted.

(34) The effect of the three designs of RHO targeting ribozyme was investigated. Comparative verification was performed in vitro (in mammalian cells). The results are shown in FIG. 5. Design 1 was compared to design 2 and it was confirmed that the trans-splicing efficiency of design 2 was better (FIG. 6A). Comparison of designs 2 and 3 demonstrated that the trans-splicing efficiency of design 2 was better (FIG. 6B). Antisense sequences of different lengths were compared to optimize the specificity and effectiveness of the ribozyme. Antisense sequence of 150 nucleotides in length (SEQ ID NO: 6) was found to be the most effective (FIG. 6C). Sequencing of trans-splicing products was performed to confirm that trans-splicing was performed accurately. It was confirmed that trans-splicing occurred at the target site (FIG. 6D).

Example 5: Assay for Trans-Splicing Activity In Vitro

(35) The in vitro efficacy of RHO-targeting ribozyme was confirmed using stable cells. A YFP-tagged wild-type RHO (wild Rho-YFP) was used as a positive control. A 293A cell line stably expressing WT or P23H RHO was prepared, and functional analysis was performed in this cell line. Normal rhodopsin proteins migrate to and localizes at the cell membrane. However, rhodopsin with P23H mutation does not fold properly and does not localize at the cell membrane but stays in the endoplasmic reticulum. Therefore, by identifying the location of the protein, the functional change of the protein can be confirmed.

(36) The RHO targeting ribozyme expression vector was transfected into stable cells. To confirm the trans-splicing effect, RNA was extracted, and RT-PCR was carried out.

(37) Specifically, 1×10.sup.5 stable cells were seeded in a 35 mm culture dish and grown in an incubator at 37° C. with 5% CO.sub.2. 2 μg of RHO-targeting ribozyme expression vector and 100 μl of OPTI-MEM™ were mixed in a 1.5 ml tube. 2 μl of LIPOFECTAMINE® 2000 and 100 μl of OPTI-MEM™ were mixed in another 1.5 ml tube and incubated at room temperature for 5 minutes. After that, the contents of the tubes were combined and mixed and incubated at room temperature for 20 minutes to allow the formation of a liposome complex. After 20 min, the tube was centrifuged for 10 sec. The mixture was added to plated cells for transfection and incubated for 4 hours, after which the transfection medium was replaced with fresh medium.

(38) The cells were then placed in an incubator for 48 hours at 37° C. with 5% CO.sub.2. After 48 hours, the cells were washed with 1×PBS and RNA was extracted with 500 μl of trizol. 5 μg of extracted RNA was treated with 1 μl of DNase I to remove gDNA. 1 μg of treated RNA was reverse transcribed to obtain cDNA. Using the synthesized cDNA, the trans-splicing product was amplified by PCR and purified. It was confirmed that the trans-splicing action occurred at the target site by nucleotide sequence analysis (FIG. 7A).

(39) In addition, after transfection of the RHO targeting ribozyme expression vector as described above, the cells were stained using an anti-RHO antibody and a secondary fluorescently labeled antibody. The intracellular distribution of RHO protein was analyzed by a fluorescence microscopy. It was confirmed that in the cells transfected with the RHO targeting ribozyme, RHO proteins were localized to the cell membrane, whereas the RHO proteins with P23H mutation were dispersed in cytosol (FIG. 7B).

(40) In addition, in vitro efficacy of RHO targeting ribozymes specific for various mutations was demonstrated in 293A cells by co-transfection (FIG. 8). Specifically, 0.5 μg of the RHO-targeting ribozyme expression vector and 2 μg of expression vectors with various RHO mutant transcripts were mixed with 100 μl of OPTI-MEM™ in a 1.5 ml tube. 2.5 μl of LIPOFECTAMIN™ 2000 and 100 μl of OPTI-MEM™ were mixed in another 1.5 ml tube and incubated at room temperature for 5 minutes. Cell transfection and RT-PCR were performed as described above. It was confirmed that mutant RHO transcripts were successfully replaced with normal RHO RNA by RHO-targeting ribozymes. Therefore, a trans-splicing ribozyme according to embodiments of the present disclosure can correct a specific RHO mutation by replacing the mutation site with WT sequence regardless of the type of mutation.

Example 6. Recombinant Virus Construction and Confirmation of Trans-Splice Efficacy in Animal Models of RHO-adRP

(41) 6-1: Recombinant Virus Construction

(42) An expression vector was constructed using an Adeno-associated viruses (AAV) vector as a backbone, a RHO-targeting ribozyme of the second design of Example 4, and a CMV promoter (FIG. 9). 20 μg of the backbone pAAV plasmid was digested with EcoRI and eluted on an agarose gel. After reacting DNA construct containing ribozyme with WT RHO synthesized using an IN-FUSION® HD cloning kit, transformed colonies were obtained. Each colony was cultured, plasmid was isolated and sequenced, and then clones with the correct cloned sequence were selected. The isolated plasmid was digested with EcoRV and XbaI, separated on the agarose gel and eluted. Synthesized YFP DNA was inserted using an IN-FUSION® HD cloning kit. Bacteria were transformed with the resulting construct. Plasmids were extracted from resulting colonies and correct cloning was confirmed by sequencing. In order to add the antisense sequence, selected constructs were digested with EcoRI, separated on an agarose gel and eluted. The AS150 sequence obtained by PCR is reacted using an IN-FUSION® HD cloning kit to obtain colonies. After sequencing the colonies in the same way, the final AAV-cRib-YFP plasmid was obtained.

(43) HEK-293T cells were triple transfected with the Helper vector, RHO ribozyme expression vector and Rep2Cap5 vector to produce an AAV viral vector. The transfected cells were lysed 72 hours post-transfection and supernatant was harvested using PEG precipitation. AAV viral vector was purified by density gradient ultracentrifugation using iodixanol.

(44) 6-2: Confirmation of the Efficacy of Trans-Splicing Ribozymes in Animal Models of RHO-adRP

(45) The efficacy of ribozyme was confirmed in hP23H-RFP mouse model of RHO-adRP using the AAV expression vector of Example 6-1. hP23H mouse-RFP mouse model is a knocked-in model carrying an RFP-tagged human P23H RHO. It is a model widely used in RHO P23H studies. 1 μl of AAV expression vector was injected into the subretinal space of each mouse eye. The injection was performed using the IO kit with 34G needle and 1 μl of the vector was injected per eye into both eyes by scleral puncture. Upon completion of administration, antibiotics were injected into the eyes of the mouse and eye drops were used to prevent infection. The success or failure of administration was determined by confirming the formation of a bleb by FP/OCT imaging.

(46) At 2 or 5 weeks after administration, the eyeballs were removed, and tissue section slides were prepared. Cell nuclei were stained by DAPI. Staining of RHO protein itself was not performed because RHO was labeled with YFP. FIG. 10 shows the results of 2 weeks after administration (FIG. 10A) and 5 weeks after administration (FIG. 10B).

(47) AAV5-YFP (fluorescence positive) and AAV-WT hRho-YFP (gene, fluorescence positive) were used as a control for AAV5-cRib-YFP. Fluorescence of AAV5-YFP and AAV-WT hRho-YFP were observed also in retinal pigment epithelium (RPE), indicating that these were not introduced in a photoreceptor specific manner. On the other hand, in the case of AAV5-cRib-YFP carrying the RHO targeting ribozyme, no expression was observed in RPE and its expression was observed in photoreceptors only.

Example 7: Assessment of Immune Reaction and Inflammatory Reaction after Administration of Trans-Splicing Ribozyme in Animal Model of RHO-adRP

(48) The results of assessing the immune and inflammatory responses in the serum of hP23H-RFP mice after administration of a trans-splicing ribozyme are shown in FIG. 11 (FIG. 11A: 2 weeks after administration; FIG. 11B: 5 weeks after administration).

(49) An increase in IL-6, IL-17A, TNF-α, INF-γ, and IL-10 levels was observed at 2 weeks after AAV5-cRibYFP administration, and no changes in IL-4 and IL-2 levels were observed. Such changes were not observed in the AAV5-YFP and AAV-WT hRho-YFP-treated control groups, indicating that these changes are not attributed to viral vectors. Cytokines, which were increased at 2 weeks, returned (decreased) to levels of the control groups at the 5 weeks, indicating that inflammation and immune response are increased at the early subretinal administration and then returned to the original level at 5 weeks.

Example 8: Confirmation of Activity of Ribozyme in Retina and Retinal Pigment Epithelium (RPE) Tissue in an Animal Model of RHO-adRP after Administration of Trans-Splicing Ribozyme

(50) Occurrence of trans-splicing reaction through targeting RHO RNA in the photoreceptor was confirmed in the animal model after administration of a trans-splicing ribozyme. For this, the mouse eyeball was removed, retina and retinal pigment epithelial cells (RPE/choroid) were isolated, RNA was extracted and RT-PCR was performed to observe and confirm the production of trans-splicing products. The results are shown in FIG. 12.

(51) The amplified trans-splicing product PCR band could not be identified in a sample extracted from RPE. Retina-specific trans-splicing products were identified (FIG. 12A). Sequencing was performed on the amplified trans-splicing product to confirm that the mutant RHO RNA was targeted and substituted with normal RHO RNA (FIG. 12B).

(52) In addition, when the AAV ITR region was amplified after extracting gDNA from the retina and retinal pigment epithelial (RPE) cells, higher levels were detected in retinal pigment epithelial cells than in the retina (FIG. 12C), which indicates that AAV vectors are well delivered to RPE. However, transgene expression controlled by ribozyme was photoreceptor-specific.

Example 9: Distribution of Ribozymes In Vivo after Administration of Trans-Splicing Ribozyme in Mouse Model of RHO-adRP

(53) 2 weeks after administration of ribozyme to hP23H-RFP mice, the distribution of ribozymes in organs was confirmed (FIG. 13). The mice were sacrificed at 2 weeks after administration, whole organs were obtained, and gDNA was isolated and purified. Real-time PCR was performed using 1 μg of gDNA from each organ with a ribozyme-specific primer. Ribozymes were detected within a detection range in the liver of all animals administered with the trans-splicing ribozyme expression vector. In some animals, ribozymes were detected within a detection range in the lung, small intestine, and prostate. Ribozymes were not detected in other organs and tissues.

Example 10: Toxicity Analysis after Administration of Trans-Splicing Ribozyme to Normal Mice

(54) Toxicity after administration of trans-splicing ribozymes was assessed in normal mice (C57BL/6J). Images of fundus and OCT images were taken each week for 4 weeks after administration, and H&E staining was performed (FIG. 14). It was confirmed that the bleb formed in the test group at the site of subretinal administration (solid line in FIG. 14). Corneal inflammation and vitreous bleeding occurred in some individuals (dotted line). Lens degeneration, retinal degeneration and outer retina were observed in the groups treated with 3×10.sup.9 GC/eye by H&E staining. The occurrence of atrophy and histological inflammation of the choroid was also observed. No toxicity was observed at the concentration of less than 1×10.sup.9 GC/eye.

Example 11: Electroretinogram after Administration of Trans-Splicing Ribozyme in Mouse Model of RHO-adRP

(55) Electroretinogram (ERG) was performed at indicated time intervals after administration of trans-splicing ribozyme in 5 weeks-old hP23H-RFP mice (FIG. 15 and Table 1). Scotopic-ERG responses were evaluated and amplitude of B-wave were compared. Mice were anesthetized with ketamine for electroretinogram evaluation. After additional local anesthesia, pupils of eye were dilated with eye drops. Mice were placed on the stage and electrodes were placed on the tail end, glabella, and retina, and ERG responses were recorded simultaneously from both eyes. Full-filed ERG were recorded at a flash intensity of 0.9 log cds/m.sup.2 (10 responses/intensity). After the measurement was complete, one drop of antibiotic ophthalmic solution was administered into the mouse eye. ‘LabScribeERG (iWorx DataAcquisition Software)’ program was used for the analysis.

(56) The P23H mutation of the rhodopsin leads to loss of normal function and visional impairment by inducing the death of rod cells and cone cells. It impairs the ability of retire photoreceptor cells (rods and cones) to convert light energy into electrical signals. As the number and function of photoreceptor cells decreases, the electrical signals diminish. The evaluation of these electrical signals was performed through electroretinography (ERG) to assess the difference in visual function. In electroretinography, B-wave is an indicator that determines whether an electrical signal is transmitted. B-wave was significantly increased at 5 weeks after administration of RHO-targeting ribozyme (AAV5-cRib-YFP) and this effect persisted until 8 weeks. Thus, maintenance and improvement of visual function was observed in disease models treated with AAV5-cRib-YFP.

(57) TABLE-US-00001 TABLE 1 Scotopic B-wave C57BL/6J P23H-RFP (+/−), P23H-RFP (+/−), Amplitude PBS PBS AAV5-cRib- (μV) treatment treatment YFP treatment Week 2 after 471.6 ± 139.12 258.2 ± 62.68 251.1 ± 56.08 administration Week 5 after 500.5 ± 101.22 204.7 ± 39.73 308.1 ± 92.87 administration Week 8 after 462.7 ± 116.38 193.6 ± 55.66 318.6 ± 46.17 administration

Example 12: Analysis of Ribozyme Activity and Serum after Administration of Trans-Splicing Ribozyme in Mouse Model of RHO-adRP

(58) Serum analysis and ribozyme activity analysis were performed at 8 weeks after administration of trans-splicing ribozyme in hP23H-RFP mice (5 weeks old) (FIG. 16). At week 8, when comparing normal animals (G1) administered with PBS and the animal model (G2) group administered only with PBS, no change in cytokines was observed by administration of trans-splicing ribozyme (FIG. 16A). In Example 7, cytokine changes were observed at week 2 and then returned to a same level as normal control group animals at week 5. Thus, the results in this Example and results of Example 7 mean that the administration of the trans-splicing ribozyme does not affect cytokine changes even at week 8.

(59) To assess whether or not RHO RNA-targeting trans-splicing action is maintained at week 8, RNA was extracted from the retina of the mice and presence of trans-splicing products was confirmed by RT-PCR (FIG. 16B). The expression and action of AAV5-cRib-YFP was still observed at week 8. Thus, successful delivery of the recombinant AAV vector and maintenance of the ribozyme expression and activity in the retina was confirmed even at week 8.

Example 13: Recombinant Viral Vector Optimization

(60) The ability of the various recombinant AAV vectors to infect and express in the retina was assessed (FIG. 17). As shown in FIG. 17, splicing donor/acceptor (SD/SA) sequence is linked to ribozyme at the 5′ end and WPRE (Woodchuck hepatitis virus Posttranscriptional Regulatory Element) is linked at the 3′ end. SD/SA and WPRE sequences are represented by SEQ ID NO: 7 and SEQ ID NO: 9, respectively. Various recombinant AAV serotypes and different promoters were used to construct different recombinant viral vectors. For example, as promoters, CMV promoter and the RHO promoter were used. And, to evaluate efficacy of the final candidate, YFP sequence was removed, and to increase expression, WPRE was inserted.

(61) 13-1: Assessment of Ribozyme Distribution and Trans-Splicing Efficiency

(62) 3 weeks after administration of the prepared recombinant AAV vector in 5 weeks old hP23H-RFP mice, ribozyme distribution and trans-splicing were evaluated (FIG. 18).

(63) The configurations of G1 to G7 are shown in FIG. 18 and in Table 2 below.

(64) TABLE-US-00002 TABLE 2 Group Animal Tested Substance G1 P23H- PBS G2 RFP(+/−) AAV2/5-cRib-YFP AAV serotype 5, CMV Promoter G3 AAV2/2 cRib-WPRE AAV serotype 2, CMV Promoter G4 AAV2/2 RL-Rib-WPRE AAV serotype 2, RHO Promoter G5 AAV2/5 cRib-WPRE AAV serotype 5, CMV Promoter G6 AAV2/5 RL-Rib-WPRE AAV serotype 5, RHO Promoter G7 AAV2/8 cRib-WPRE AAV serotype 8, CMV Promoter

(65) In order to check the infection rate for each serotype, the eyeballs of treated mice were removed 3 weeks after injection. Retina and RPE/choroid were separated and RNA and gDNA were extracted. When the extracted gDNA was compared, AAV serotype 5 showed more efficient transduction to both the retina and the RPE, than serotype 2 or 8. Parallel RNA analysis was performed by RT-PCR. The trans-splicing ribozyme was highly expressed in RPE as well under the CMV promoter regulation, while, in the retina, the ribozyme was highly expressed under the RHO promoter regulation compared to the ribozyme expression under the CMV promoter regulation (FIG. 18A).

(66) The presence of trans-splicing products through the trans-splicing action of the ribozyme in the retina was detected and confirmed by PCR. Then, the PCR bands of trans-splicing products formed by AAV serotype 5 under the RHO promoter regulation were dense (FIG. 18B).

(67) The results indicate that AAV serotype 5 RL-Rib-WPRE showed excellent delivery and expression in the retina as well as excellent trans-splicing efficiency.

(68) 13-2: Electroretinogram after Administration of Trans-Splicing Ribozyme in Mouse Model of RHO-adRP

(69) Electroretinogram examination was conducted at 6 weeks after administration of trans-splicing ribozyme in hP23H-RFP mice (5 weeks of age). The results are shown in FIG. 19 and Table 3.

(70) TABLE-US-00003 TABLE 3 Tested B-wave Group Animal Substance (μV) G0 C57BL/6J PBS 428.7 ± 65.15 (mRHO/mRHO) G1 P23H-RFP(+/−) PBS 240.1 ± 39.67 G2 AAV2/5-cRib-YFP 272.1 ± 56.75 G3 AAV2/2 cRib-WPRE 263.7 ± 40.46 G4 AAV2/2 RL-Rib-WPRE 284.9 ± 64.47 G5 AAV2/5 cRib-WPRE 263.4 ± 37.69 G6 AAV2/5 RL-Rib-WPRE 318.1 ± 26.35 G7 AAV2/8-cRib-WPRE 296.2 ± 57.86

(71) B-wave amplitude of P23H-RFP mice (G1) was significantly decreased compared to the normal wild-type mice (G0). This result is consistent with the photoreceptor cells damage in P23H-RFP mice caused by the RHO mutation resulting impaired signal transmission.

(72) When the electroretinogram was taken after administration of each AAV to the hP23H-RFP mice, it was confirmed that the average scotopic B-wave amplitude in all the test groups increased compared to the PBS-treated group (G1). Comparison between promoters showed that B-wave amplitude was significantly higher when RHO promoter was used than when CMV promoter was used (G3<G4, G5<G6), compared to the control group administered with PBS (G1). Comparison between serotypes, both AAV2/5 serotypes (G5, G6) and the AAV2/8 group (G7) showed significantly increased B-wave amplitude, compared to the PBS administration group (G1). The AAV2/5 RL-Rib-WPRE administration group exhibited the most significantly increased B-wave, indicating that subretinal administration of AAV2/5 RL-Rib-WPRE could show the highest effect of improving the visual function of the disease model.

(73) Electroretinogram was performed at 4 weeks after administration of the following recombinant AAV vectors to 5 weeks-old hP23H-RFP mice: AAV2/5 RL-Rib-WPRE and AAV2/8 RL-Rib-WPRE, recombinant AAV vectors of serotype 5 and 8 with RHO promoter, respectively. The results in FIG. 20 and Table 4.

(74) TABLE-US-00004 TABLE 4 Tested B-wave (μV) B-wave (μV) Group Animal Substance (0 week) (4 week) H1 C57BL/6J PBS 240.3 ± 52.45 267.8 ± 33.37 (mRHO/mRHO) H2 P23H-RFP(+/−) PBS 173.7 ± 50.60 146.8 ± 41.18 H3 AAV2/5 176.1 ± 44.53 206.2 ± 33.59 RL-Rib-WPRE H4 AAV2/8 191.5 ± 47.24 235.8 ± 36.06 RL-Rib-WPRE

(75) As shown in Table 4 and FIG. 20, the B-wave amplitude at 4 weeks after administration was significantly higher in AAV2/5 RL-Rib-WPRE (H3) group and AAV2/8 RL-Rib-WPRE group (H4), compared to PBS-treated group (H2). Considering that AAV2/8 RL-Rib-WPRE group (H4) showed higher B-wave before administration (0 week) than the PBS control group (H2) and the AAV2/5 RL-Rib-WPRE administration group (H3), we speculate that B-wave increasing ability of the AAV2/5 RL-Rib-WPRE administration group (H3) and the AAV2/8 RL-Rib-WPRE administration group (H4) were similar. Therefore, administration of AAV2/5 RL-Rib-WPRE and AAV2/8 RL-Rib-WPRE significantly improved visual function in this mouse model compared to the PBS-administered control group (H2).

(76) 13-3: Distribution and Trans-Splicing Activity of Ribozyme in Retinal after Trans-Splicing Ribozyme Administration in Mouse Model of RHO-adRP

(77) The distribution of ribozyme in the retina and trans-splicing activity was assessed 6 weeks after administration of ribozyme to 5-week-old hP23H-RFP mice as shown in FIG. 21 and Table 5.

(78) TABLE-US-00005 TABLE 5 Group Animal Tested Substance G0 C57BL/6J PBS (mRHO/mRHO) G1 P23H-RFP(+/−) PBS G2 AAV2/5-cRib-YFP G3 AAV2/2 cRib-WPRE G4 AAV2/2 RL-Rib-WPRE G5 AAV2/5 cRib-WPRE G6 AAV2/5 RL-Rib-WPRE G7 AAV2/8-cRib-WPRE

(79) After confirming the functional efficacy in mouse model, the retinal gDNA and RNA was isolated and analyzed. AAV serotype 5 showed the same results as those seen at week 3 in Example 13-1 and it was confirmed that infection of the retina was successful. RNA analysis showed good ribozyme expression with AAV serotype 5 with RHO promoter. Delivery and expression of AAV2/5 RL-Rib-WPRE in the retina was stable at week 6.

(80) Summarizing the above results, it was confirmed that AAV serotypes 5 and 8 was efficiently delivered into both retinal and RPE and the trans-splicing ribozyme regulated by RHO promoter was specifically expressed in the retina in the hP23H disease model.

(81) Expression of trans-splicing ribozyme according to the present invention was maintained from week 3 of the initial administration to 6 weeks after administration, and improvement of visual function was observed as measured by electroretinography at weeks 4 and 6 after administration.

(82) Aspects of the present invention has been described in detail above. Those of ordinary skill in the art would understand that these specific techniques are merely some of the preferred implementations. It is to be understood that the description above does not limit the scope of the present invention. The substantial scope of the present invention is defined by the claims and includes the equivalents.