Gene therapy for FAM161A-related retinopathy and other fiberopathy

Abstract

The present invention relates to a vector comprising an FCBR1-F0.4 promoter or an IRBP-GRK1 promoter and a nucleic acid sequence encoding a cilia protein, or a fragment or variant thereof. In particular, the present invention relates to methods of treating and preventing retinal ciliary cases, such as retinitis pigmentosa 28.

Description

Technical Field

[0001] The present invention relates to the field of production of vectors encoding ciliary proteins, and to methods for treating and / or preventing retinal ciliary diseases. In particular, the present invention relates to a method for treating and / or preventing FAM161A-related retinopathy, such as retinitis pigmentosa 28 induced by FAM161A protein deficiency. Background Art

[0002] Retinitis pigmentosa (RP) is a group of inherited diseases involving progressive degeneration of the retina that affects its ability to respond to light, leading to a slow loss of vision. RP is genetically and clinically heterogeneous and is characterized by night blindness, progressive degeneration of photoreceptors that leads to a gradual loss of peripheral vision and subsequently central vision, often ultimately leading to blindness.

[0003] Currently, mutations in more than 60 genes are associated with nonsyndromic RP, of which 41 genes have been reported to cause autosomal recessive (AR) disease, including FAM161A, which was originally discovered in patients in Israel, Palestine, India, North America, and Europe (Beryozkin et al., 2020, Scientific Reports, 10:15156). In particular, retinitis pigmentosa-28 (RP28) is an autosomal recessive retinitis pigmentosa (arRP) caused by pathogenic variants in the gene FAM161A (Langmann et al., 2010, Am J Hum Genet. 87(3): 376–381; Bandah-Rozenfeld et al., 2010, Am J Hum Genet. 87(3):382-91). Retinitis pigmentosa-28 (RP28) is characterized by a slow retinal degeneration process, but visual impairment is detected early in life and a rapid decline in retinal activity from the 40th to 50th decade of life (Beryozkin et al., 2020, supra). The FAM161A protein is localized at photoreceptors that connect cilia in humans, mice, and rats, and it is also present in the basal body of ciliated mammalian cells (Di Gioia et al., 2012, Hum Mol Genet. 21(23):5174-84; Di Gioia et al., 2015, Hum Mol Genet. 24(12):3359-71). To investigate the pathological mechanisms of RP28 disease progression and to aid in the design of therapeutic strategies, a knockout (KO) mouse model (Fam161a tm1b / tm1b), lacking major exon #3, which was replaced by a construct containing LacZ expressed under the Fam161a promoter (Beryozkin et al., 2021, Scientific Reports 11: 2030). So far, it has been shown that the loss of FAM161A alters the maintenance of ciliary structure, leading to progressive changes in photoreceptor outer segments, which are essential for photoreceptor function by converting light stimuli into electrical signals (Karlstetter et al., 2014 Hum. Mol. Gen. 23(19), 5197-5210; Mercey et al., 2022, PLoS Biol e3001649). Currently, there is no treatment for RP28 disease.

[0004] Therefore, there is a need for a method of preventing and / or treating retinitis pigmentosa-28. Summary of the invention

[0005] The present invention is based on the surprising discovery that a vector comprising the human FAM161A gene (both long (hFAM161A-L) or short (hFAM161A-S) isoforms) and comprising the human FCBR1-F0.4 promoter (FCBR, Crx binding region; F0.4, FAM161A core promoter) is particularly suitable for expressing human FAM161A protein (both long (hFAM161A-L) or short (hFAM161A-LS) isoforms) in the connecting cilia of photoreceptors (Table 1). Restoration of FAM161A protein expression and improvement of visual function were observed by ERG recordings. The FCBR2-F0.4 promoter with the human short (hFAM161A-S) isoform produced similar ERG improvements 2 months after injection (Table 1), but expression was not addressed only in the connecting cilia, but was present throughout the photoreceptors. On the other hand, the presence of the juxtaposed FCBR2-FCBR1 sequences of the core promoter that binds to the mouse ML1 (mouse long 1; mFam161a-L1) isoform increases the degradation process. The vector of the present invention was shown to be effective in the FAM161A knockout (KO) mouse model (Fam161a tm1b / tm1b Referred to as Fam161a in this article - / - ) to provide sufficient protein expression levels to restore photoreceptor anatomy and photoreceptor function (Table 1 and Figure 5 -7, 11-13). Therefore, the vector of the present invention is proposed to be used for treating disorders associated with FAM161A gene mutation, such as FAM161A-related retinopathy, in particular for treating retinitis pigmentosa-28 (RP28).

[0006] Since FAM161A is an example of a fimbrin, a protein found in the photoreceptor cilia connecting the inner and outer segments, vectors comprising in particular the FCBR1-F0.4 promoter (among other promoters) and a transgene encoding any fimbrin are proposed for the treatment of relevant ciliopathy.

[0007] The IRBP-GRK1 promoter was used to drive expression of at least the mouse L isoform (Table 1, Figure 5 -7), can achieve successful and effective protection of retinal integrity and functional preservation.

[0008] However, the IRBP-GRK1 promoter in combination with either the HL or HS isoforms did not improve the maintenance of mouse retinal viability, and co-administration of both HL+HS isoforms with the IRBP-GRK1 vector did not allow mouse retinal viability 3 months post-injection (mpi). In addition, the promoter was more efficient in driving expression of the mouse ML isoform (Figure 6) than the human isoforms (HS or HL, Figure 9).

[0009] One aspect of the present invention provides a vector comprising a FCBR1-F0.4 promoter and a nucleic acid sequence encoding a ciliary protein or a fragment or variant thereof, for example, wherein the FCBR1-F0.4 promoter is SEQ ID NO: 10 or SEQ ID NO: 66.

[0010] Another aspect of the present invention provides a vector comprising an IRBP-GRK1 promoter and a nucleic acid sequence encoding a ciliary protein or a fragment or variant thereof, for example, wherein the IRBP-GRK1 promoter is SEQ ID NO:14.

[0011] Another aspect of the present invention provides a vector as described herein, wherein the nucleic acid sequence encoding the ciliary protein is selected from the long isoform of the human FAM161A gene of SEQ ID NO: 1 and the short isoform of the human FAM161A gene of SEQ ID NO: 2, or a fragment or variant thereof.

[0012] Another aspect of the present invention provides a vector as described herein, wherein the nucleic acid sequence encoding a ciliary protein is selected from the group consisting of the POC5 gene of SEQ ID NO: 29, the NPHP4 gene of SEQ ID NO: 30, the ARL6 gene of SEQ ID NO: 31, the BBS1 gene of SEQ ID NO: 32, the BBS2 gene of SEQ ID NO: 33, the BBS9 gene of SEQ ID NO: 34, the PCARE gene of SEQ ID NO: 35, the CFAP418 gene of SEQ ID NO: 36, the CEP164 gene of SEQ ID NO: 37 (CEP164 isoform 1) or SEQ ID NO: 38 (CEP164 isoform 2), the CEP290 gene of SEQ ID NO: 39, the CLRN1 gene of SEQ ID NO: 40, the IFT140 gene of SEQ ID NO: 41, the IFT172 gene of SEQ ID NO: 42, the IQCB1 gene of SEQ ID NO: 43, the KIZ gene of SEQ ID NO: 44, the KIZ gene of SEQ ID NO: 45, the KIZ gene of SEQ ID NO: 46, the KIZ gene of SEQ ID NO: 47, the KIZ gene of SEQ ID NO: 48, the KIZ gene of SEQ ID NO: 49, the KIZ gene of SEQ ID NO: 50, the KIZ gene of SEQ ID NO: 51, the KIZ gene of SEQ ID NO: 52, the KIZ gene of SEQ ID NO: 53, the KIZ gene of SEQ ID NO: 54, the KIZ gene of SEQ ID NO: 55, the KIZ gene of SEQ ID NO: 56, the KIZ gene of SEQ ID NO: 57, the KIZ gene of SEQ ID NO: 58, the the LCA5 gene of SEQ ID NO:45, the MAK gene of SEQ ID NO:46, the NEK2 gene of SEQ ID NO:47, the OFD1 gene of SEQ ID NO:48, the OFD1 gene of SEQ ID NO:49, the RP1 gene of SEQ ID NO:50, the RP1L1 gene of SEQ ID NO:51, the RP2 gene of SEQ ID NO:52, the RPGR gene of SEQ ID NO:53 (RPGR isoform C) or SEQ ID NO:54 (RPGR isoform J), the RPGRIP1 gene of SEQ ID NO:55 (RPGRIP1 isoform 1) or SEQ ID NO:56 (RPGRIP1 isoform 3), the SPATA7 gene of SEQ ID NO:57, the TOPORS gene of SEQ ID NO:58, the TTC8 gene of SEQ ID NO:59, the USH2A gene of SEQ ID NO:60, the WDR19 gene of SEQ ID NO:61, The CFAP410 gene of NO:62, or a fragment or variant thereof.

[0013] Another aspect of the present invention provides a vector as described herein, further comprising a WPRE (Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element) post-transcriptional regulatory element of SEQ ID NO: 15, or a fragment or variant thereof.

[0014] Another aspect of the present invention provides a vector as described herein, which is a recombinant AAV vector having an AAV2 / 8 capsid protein or an AAV2 / 5 capsid protein.

[0015] Another aspect of the present invention relates to a pharmaceutical composition comprising at least one vector according to the present invention or a combination thereof and at least one pharmaceutically acceptable carrier.

[0016] Another aspect of the present invention relates to the use of a vector as described herein as a medicament.

[0017] Another aspect of the present invention relates to the use of a vector according to the present invention or a pharmaceutical composition thereof for altering, preventing, delaying, arresting the progression or ameliorating vision loss associated with retinal ciliopathy, such as FAM161A-related retinopathy.

[0018] Another aspect of the present invention relates to the use of a vector according to the present invention or a pharmaceutical composition thereof for treating a retinal ciliopathy, such as a FAM161A-related retinopathy, preferably retinitis pigmentosa 28.

[0019] Another aspect of the present invention relates to use of the vector according to the present invention or a pharmaceutical composition thereof for improving or maintaining the structure of photoreceptors.

[0020] Another aspect of the present invention relates to use of the vector according to the present invention or a pharmaceutical composition thereof for inducing stabilization of the photoreceptor cytoskeleton. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Several plasmid constructs designed as described in Example 1 are shown.

[0022] Figure 2 shows in vitro FAM161A protein expression as described in Example 2. A) shows human FAM161A protein isoforms detected by Western blotting in 661W cells (long and short isoforms, HL and HS, respectively). B) shows mouse FAM161A protein (mFAM161A) isoforms detected by Western blotting in HEK293T cells (long, short and short 1, ML, MS and MS1, respectively, only ML, MS and MS1 are shown in the figure). CG) shows immunocytochemistry of hFAM161A or mFAM161A isoforms and acetylated tubulin (Ac-tub) (C) HL; D) HS; E) ML; F) MS; G) MS1). hFAM161A or mFAM161A isoforms target different cellular compartments and induce tubulin stabilization. H) shows quantification of the percentage of stable acetylated tubulin of transfected cells.

[0023] Figure 3 shows the transgenic plasmids used to generate the following vectors described in Examples 3 and 5: A) AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE, B) AAV2 / 8-IRBP-GRK1-hFAM161A-S-WPRE, C) AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE, D) AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE.

[0024] Figure 4 FAF images (fundus autofluorescence) as described in Example 3 are shown. In Figure A (A.1-A.5), untreated Fam161 - / - Serial FAF images of the retina show narrowing of blood vessels and formation of patchy autofluorescent spots, indicating widespread degeneration of the retina at 1, 3, 4.5, 6, or 8 months (m) of age. Following the gene augmentation therapy shown in panels B (B.1-B.4) (injection of AAV2 / 8-IRBP-GRK1-mFam161a-L vector at 3-4 weeks of age), a significant reduction in the formation of autofluorescent spots can be seen in the treated areas at 3, 4.5, 6, or 8 months of age (2, 3.5, 5, and 7 months after injection, respectively) (left side of each image) compared to untreated retinal areas (right side).

[0025] Figure 5 OCT images as described in Example 3 are shown. OCT images of eyes treated with AAV2 / 8-IRBP-GRK1-mFam161a-L vector at 3, 6, and 8 months of age show that photoreceptors in the treated area survived for at least 8 months after gene therapy. The vector was injected at P21-29 when cilia had already changed. Note the rescued ONL thickness compared to the contralateral side (arrow).

[0026] Figure 6 shows dark-adapted (AE) ERG recordings at 3, 4.5, 6, and 8 months after injection of AAV2 / 8-IRBP-GRK1-mFam161a-L treatment compared to untreated eyes, as described in Example 3. The results show that functional rescue persists for at least 8 months after gene therapy stimulation (*p-value: 1<0.05, #p-value<0.01). (F) Visual acuity was significantly improved in the ML-treated group compared to the untreated eye control group (Figure F) (*p-value<0.05, ***p-value<0.01).

[0027] FIG7 shows histological analysis of representative eyes as described in Example 3. Eyes treated with AAV2 / 8-IRBP-GRK1-mFam161a-L gene therapy at P21-29 (8 months after vector injection) show significant preservation of ONL in treated areas compared to untreated areas. (A) Quantification of ONL thickness. (B) Left: Fam161a treated with vector. - / - Retinal sections.

[0028] Figure 8 shows that compared with untreated eyes, 10 10 gc / ul AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE ((1); AAV-IG-HL-W) or AAV2 / 8-IRBP-GRK1-hFAM161A-S-WPRE ((2); AAV-IG-HS-W) or control (AAV-IG-GFP-W) vectors were injected into Fam161 - / - Mice, ERG recordings at 1 month (A), 2 months (B) and 3 months (C) post-injection (pi), as described in Example 4.

[0029] Figure 9 shows a 0.5x10 10 gc / ul AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE ((1); AAV-IG-HL-W) with 0.5x10 10 gc / ul AAV2 / 8-IRBP-GRK1-hFAM161A-S-WPRE ((2); AAV-IG-HS-W) mixture or control (AAV-IG-GFP-W, 10 10 gc / ul) vector injected into Fam161 - / - Mice, ERG recordings at 1 month (A), 2 months (B) and 3 months (C) post-injection (pi), as described in Example 4.

[0030] Fig.10 The expression of Fam161a in wild type (WT, A) and cells injected with AAV2 / 8-IRBP-GRK1-hFAM161A-S-WPRE ((2); AAV-IG-HS-W, C) or AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE ((1); AAV-IG-HS-W, E) is shown. - / - Mouse retina, (B): Untreated Fam161a - / -Localization of FAM161A protein in the retina (Fig. 1A). In (A), the arrow points to normal FAM161A expression in the WT retina corresponding to the connecting cilia (CC). In (C), the arrow indicates ectopic expression of FAM161A protein. In (D and E), the arrows indicate the retinal areas protected by treatment. (F) Quantification of retinal thickness after AAV treatment is shown. For information, the thickness of the retina in WT (wild-type mice) is approximately 60 μM (not shown). ONL: outer nuclear layer (corresponding to photoreceptor cells).

[0031] Figure 11 shows the results of AAV2 / 8 FCBR1-F0.4-hFAM161A-L-WPRE (10) (A), FCBR1-F0.4-hFAM161A-S-WPRE (11) (B), and AAV2 / 8 FCBR1-F0.4-hFAM161A-S-WPRE + -F0.4-hFAM161A-L-WPRE (10+11) (C) vector injection into Fam161a - / - Mice, ERG recordings 3 months after injection, as described in Example 6. The six best responses from each group were analyzed.

[0032] Figure 12 shows Fam161a injected with AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE (11) (A) or AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE (10) (B), or both (C), or AAV2 / 8-FCBR2-F0.4-hFAM161A-S-WPRE (7) (E) - / - Immunohistochemistry of FAM161A in the retina. (D) shows an untreated area of ​​the treadretina treated with HL as above. In (A) and (E), white arrows indicate ectopic expression of FAM161A, while in (B and C), these white arrows indicate correct expression of FAM161A in the connecting cilia. (F) shows rescued retinal thickness from the optic nerve (0 point) to the periphery in mice treated with the above vectors. Note that the combination of HS+HL has the best rescue of retinal thickness.

[0033] Figure 13 shows ERG recordings (A) and retinal histology (B) 7 months after AAV2 / 8-FCBR1-F0.4-mouse-Fam161a-L-WPRE injection. During the treatment period, mice received a single injection (dorsal retina) or two injections (dorsal and ventral). The arrow in (B) indicates the rescued ONL in the treatment area. This representative mouse received a single injection. DETAILED DESCRIPTION

[0034] The term "photoreceptor" herein refers to specialized types of sensory neuron cells found in the retina that are capable of visual phototransduction, namely rods and cones. There are three known types of photoreceptor cells in the mammalian eye: rods (contribute to vision), cones (contribute to vision), and intrinsic photosensitive retinal ganglion cells (not directly contributing to vision). Rods contribute primarily to night vision (scotopic conditions), while cones contribute primarily to daytime vision (photopic conditions), but the chemical processes supporting phototransduction in each cell are similar. Two types of photoreceptor cells (rods and cones) are found on the outermost layer of the retina, and they both have the same basic structure. Closest to the visual field (and farthest from the brain) are the axon terminals, which release neurotransmitters (glutamate) to the bipolar cells. Further back are the cell bodies, which contain the cell's organelles. Further back are the inner segments (IS), which are specialized parts of the cell filled with mitochondria (which provide ATP (energy) for the sodium-potassium pump). Finally, closest to the brain (and farthest from the field of vision) is the outer segment (OS), the part of the photoreceptor that absorbs light to convert it into an electrical signal. The outer segment is actually a modified cilium that contains a disk filled with opsins (molecules that absorb photons), as well as voltage-gated sodium channels.

[0035] The term "connecting cilium" or "CC" (or plural cilia) or "transition zone" refers herein to a specific structure of rods and cones located between their inner and outer segments (IS and OS). The CC is a defined structure of the cilium, in which nine tubulin filaments are assembled by other proteins, such as FAM161A, to form a tubular body to position the opsins in the correct location. Thus, the CC is involved in the transport of molecules between the two compartments. "FAM161A" refers herein to the human gene encoding the FAM161A protein. The FAM161A gene produces two different mRNA isoforms in the retina. "FAM161A" is a human protein found in the CC of photoreceptors and is therefore a ciliary protein (Di Gioia et al., 2012, supra; Karlstetter et al., 2014, supra). In addition, FAM161A is also a component of the Golgi-centrosomal network (Di Gioia et al., 2015, supra). The function of the FAM161A protein is not fully defined. FAM161A is a microtubule-associated ciliary protein that is presumed to be involved in maintaining microtubule stability (Karlstetter et al., 2014, supra; Mercey et al., 2021, supra). "Fam161a" herein refers to the mouse gene encoding the mouse "Fam161a" protein.

[0036] The term "retinitis pigmentosa-28" or "RP28" refers to an autosomal recessive form of retinitis pigmentosa (arRP) caused by pathogenic variants in the gene FAM161A.

[0037] The term "FAM161A-related retinopathy" or "FAM161A-related RP" refers to a retinal ciliopathy (retinopathy) caused by pathogenic variants in the gene FAM161A. An example is RP-28. It is understood that some patients with mutations in the FAM161A gene will not have the phenotype of retinitis pigmentosa 28, but another phenotype.

[0038] The term "retinal ciliopathy" refers to a group of disorders related to the pathophysiology of photoreceptor cilia. Retinal ciliopathy includes, but is not limited to, retinitis pigmentosa, such as retinitis pigmentosa-28, FAM161A-related RP, RP, BBS (Bardet-Biedl syndrome), SLS (Senior-Loken syndrome), JBS (Joubert syndrome), MKS (Meckel-Gruber syndrome), USH (Usher syndrome), JATD (Jeune asphyxiating thoracic dysplasia), MZSDS (Mainzer–Saldino syndrome), OFD (oral-facial-digital syndrome), OMD (occult macular dystrophy), CRD (cone-rod dystrophy), LCA (Leber congenital amaurosis), CED (cranial ectodermal dysplasia, also known as Sensenbrenner syndrome) (Bujakowska et al., 2017, Cold Spring Harb Perspect Biol. 10: a028274).

[0039] The term "adeno-associated virus" or "AAV" refers to a virus that is primarily composed of a protein coat that surrounds and protects a small single-stranded DNA genome of approximately 4.8 kilobases (kb). The single-stranded genome of AAV contains three genes, Rep (replication), Cap (capsid), and aap (assembly), and these coding sequences are flanked by inverted terminal repeats (ITRs) required for genome replication and packaging. "Recombinant AAV" or "rAAV" refers to a virus that lacks viral DNA and is primarily a protein-based nanoparticle that is able to cross the cell membrane and ultimately can be transported and deliver its DNA cargo into the cell nucleus. rAAV, also known as "AAV vector," is engineered not to replicate. In the absence of Rep protein, the ITR-flanked transgene encoded within rAAV can form circular concatemers that exist as episomes in the nucleus of the transduced cell. Because the recombinant free DNA does not integrate into the host genome (or has very low efficacy), it will eventually be diluted over time as the cell undergoes repeated replication cycles. This will ultimately lead to the loss of the transgene and transgene expression, with the rate of transgene loss depending on the turnover rate of the transduced cells. These features make rAAV a good candidate for certain gene therapy applications, particularly for post-mitotic cells, such as neurons, including photoreceptors. Typically, in rAAV design, the sequences located between the ITRs typically include a mammalian promoter, a gene of interest (i.e., a transgene), and a terminator. Typically, the nucleic acid sequence encoding the gene of interest is a cDNA (complementary DNA) or shRNA (short hairpin RNA or small hairpin RNA). In addition, the inclusion of terminator / polyadenylation signal elements, post-transcriptional regulatory elements, and messenger RNA (mRNA) stability elements, as well as the presence of microRNA (miRNA) target sequences in the gene cassette (referred to as "cis-regulatory elements") may be considered. All elements between the ITRs may be referred to as "rAAV expression cassettes" or "transgene (expression) cassettes." "Post-transcriptional regulatory elements" refer to nucleotide sequences located upstream (5' non-coding sequences), internally, or downstream (3' non-coding sequences) of a coding sequence that affect RNA processing or stability, or translation of the associated coding sequence. Regulatory elements may include, but are not limited to, translation leader sequences, introns, and polyadenylation recognition sequences. Regulatory elements present on a recombinant DNA construct introduced into a cell may be endogenous to the cell, or they may be heterologous to the cell.The terms "regulatory element" and "regulatory sequence" are used interchangeably herein.

[0040] Recombinant AAV is based on AAV, wherein AAV is selected from a natural serotype (AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9)), or based on a hybrid AAV vector engineered using the genome of AAV serotype 2 and the capsid proteins of AAV serotypes 1-9 (e.g., AAV2 / 1, AAV2 / 2, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, and AAV2 / 9).

[0041] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting a nucleic acid sequence encoding a pilin protein that has been connected thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA fragments can be connected. Another type of vector is a viral vector, in which additional DNA fragments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, thereby replicating with the host genome. In addition, certain vectors are capable of directing the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors").

[0042] The term "protein fragment" or "functional protein fragment" refers to any fragment that retains the function of the full-length protein. Similarly, the term "protein variant" or "functional protein variant" refers to any protein variant that retains the function of the full-length protein. The functional protein fragment or variant of pilin or FAM161A can be determined by those skilled in the art according to known methods.

[0043] The term "gene fragment" or "gene functional fragment" refers to any fragment that retains the function of the full-length gene, although not necessarily having the same expression or activity level. Similarly, the term "gene variant" or "gene functional variant" refers to any variant that retains the function of the full-length gene, although not necessarily having the same expression or activity level. Functional fragments or variants of sequences encoding ciliary proteins or FAM161A can be determined by those skilled in the art according to known methods.

[0044] Ciliary proteins

[0045] The term "cillin" or "ciliary protein" refers to proteins found in specialized photoreceptor cilia, including FAM161A, POC5, NPHP4, ARL6, BBS1, BBS2, BBS9, PCARE, CFAP418, CEP164, CEP290, CLRN1, IFT140, IFT172, IQCB1, KIZ, LAC5, MAK, NEK2, OFD1, RAB28, RP1, RP1L1, RP2, RPGR, RPGRIP1, SPATA7, TOPORS, TTC8, USH2A, WDR19, CFAP410 (Bujakowska et al., 2017, Cold Spring Harb Perspect Biol. 10: a028274).

[0046] In one embodiment, the ciliary protein is selected from the list comprising:

[0047] FAM161A; wherein the sequence encoding the long isoform of the human FAM161A gene is, for example, SEQ ID NO: 1, and the sequence encoding the short isoform of the human FAM161A gene is, for example, SEQ ID NO: 2;

[0048] POC5 (protein name: centrosome protein POC5; gene: proteome of centriole 5 (POC5); alias C5orf37), wherein the sequence encoding POC5 is, for example, SEQ ID NO: 29; NPHP4 (protein name: kidney cyst protein 4; gene: NPHP4; alias POC10; SLSN4; KIAA067), wherein the sequence encoding NPHP4 is, for example, SEQ ID NO: 30;

[0049] ARL6 (protein name: ADP ribosylation factor, such as GTPase 6; gene: ARL6; aliases: BBS3, RP55), wherein the sequence encoding ARL6 is, for example, SEQ ID NO: 31;

[0050] BBS1 (protein name: Bardet-Biedl syndrome 1 protein; gene: BBS1; alias BBS2L2), wherein the sequence encoding BBS1 is, for example, SEQ ID NO: 32;

[0051] BBS2 (protein name: Bardet-Biedl syndrome 2; gene: BBS2; aliases: BBS, RP74), wherein the sequence encoding BBS2 is, for example, SEQ ID NO: 33;

[0052] BBS9 (protein name: Bardet-Biedl syndrome 9; gene: BBS9; aliases B1, C18, D1, PTHB1), wherein the sequence encoding BBS9 is, for example, SEQ ID NO: 34;

[0053] PCARE (protein name: photoreceptor ciliary actin regulator; gene: PCARE; aliases: RP54, C2orf71), wherein the sequence encoding PCARE is, for example, SEQ ID NO: 35;

[0054] CFAP418 (protein name: Cilia and Flagella-associated protein 418; gene: CFAP418, aliases: RP64, BBS21, MOT25, CORD16, FAP418, C8orf37, smalltalk), wherein the sequence encoding CFAP418 is, for example, SEQ ID NO: 36;

[0055] CEP164 (protein name: centrosomal protein 164; gene: CEP164, alias NPHP15), wherein the sequence encoding CEP164 is, for example, SEQ ID NO: 37 (CEP164 isoform 1) or SEQ ID NO: 38 (CEP164 isoform 2);

[0056] CEP290 (protein name: centrosomal protein 290; gene: CEP290; aliases: 3H11Ag, BBS14, CT87, JBTS5, LCA10, MKS4, NPHP6, POC3, SLSN6, rd16), wherein the sequence encoding CEP290 is, for example, SEQ ID NO: 39;

[0057] CLRN1 (protein name: clarin1; gene: CLRN1; aliases: RP61, USH3, USH3A), wherein the sequence encoding CLRN1 is, for example, SEQ ID NO: 40;

[0058] IFT140 (protein name: intraflagellar transport 140; gene: IFT140; aliases: MZSDS, RP80, SRTD9, WDTC2, c305C8.4, c380F5.1, gs114), wherein the sequence encoding IFT140 is, for example, SEQ ID NO: 41;

[0059] IFT172 (protein name: intraflagellar transport 172; gene: IFT172; aliases: BBS20, NPHP17, RP71, SLB, SRTD10, osm-1, wim), wherein the sequence encoding IFT172 is, for example, SEQ ID NO: 42;

[0060] IQCB1 (protein name: IQ motif containing B1; gene: IQCB1; aliases: NPHP5, PIQ, SLSN5), wherein the sequence encoding IQCB1 is, for example, SEQ ID NO: 43;

[0061] KIZ (protein name: kizuna centrosomal protein; gene: KIZ; aliases: C20orf19, HT013, Kizuna, NCRNA00153, PLK1S1, RP69), wherein the sequence encoding KIZ is, for example, SEQ ID NO: 44;

[0062] LCA5 (protein name: lebercilin LCA5; gene: LCA5; alias C6orf152), wherein the sequence encoding LCA5 is, for example, SEQ ID NO: 45;

[0063] MAK (protein name: male germ cell-associated kinase; gene: MAK; alias RP62), wherein the sequence encoding MAK is, for example, SEQ ID NO: 46;

[0064] NEK2 (protein name: NIMA-associated kinase 2; gene: NEK2; aliases: HsPK21, NEK2A, NLK1, PPP1R111, RP67), wherein the sequence encoding NEK2 is, for example, SEQ ID NO: 47;

[0065] OFD1 (protein name: OFD1 centriole and centriole satellite protein; gene: OFD1; aliases 71-7A, CXorf5, JBTS10, RP23, SGBS2), wherein the sequence encoding OFD1 is, for example, SEQ ID NO: 48;

[0066] OFD1 (protein name: RAB28, member of the RAS oncogene family; gene: RAB28; alias CORD18), wherein the sequence encoding OFD1 is, for example, SEQ ID NO: 49;

[0067] RP1 (protein name: RP1 axonemal microtubule-associated protein; gene: RP1; aliases ORP1, DCDC4A), wherein the sequence encoding RP1 is, for example, SEQ ID NO: 50;

[0068] RP1L1 (protein name: RP1 analog 1; gene: RP1L1; aliases OCMD, RP88, DCDC4B), wherein the sequence encoding RP1L1 is, for example, SEQ ID NO: 51;

[0069] RP2 (protein name: RP2 activator of ARL3 GTPase; gene: RP2; aliases: XRP2, NME10, TBCCD2, NM23-H10, DELXp11.3), wherein the sequence encoding RP2 is, for example, SEQ ID NO: 52;

[0070] RPGR (protein name: retinitis pigmentosa GTPase regulator; gene: RPGR; aliases COD1, CORDX1, CRD, PCDX, RP15, RP3, XLRP3, orf15), wherein the sequence encoding RPGR is, for example, SEQ ID NO: 53 (RPGR isoform C) or SEQ ID NO: 54 (RPGR isoform J);

[0071] RPGRIP1 (protein name: RPGR interacting protein 1, gene: RPGRIP1; aliases CORD13, LCA6, RGI1, RGRIP, RPGRIP, RPGRIP1d), wherein the sequence encoding RPGRIP1 is, for example, SEQ ID NO: 55 (RPGRIP1 isoform 1) or SEQ ID NO: 56 (RPGRIP1 isoform 3);

[0072] SPATA7 (protein name: spermatogenesis-related 7; gene: SPATA7; aliases: HEL-S-296, HSD-3.1, HSD3, LCA3), wherein the sequence encoding SPATA7 is, for example, SEQ ID NO: 57;

[0073] TOPORS (protein name: arginine / serine-rich protein that binds TOP1, E3 ubiquitin ligase; gene: TOPORS; aliases: LUN, RP31, P53BP3, TP53BPL), wherein the sequence encoding TOPORS is, for example, SEQ ID NO: 58;

[0074] TTC8 (protein name: tetratricopeptide repeat domain 8; gene: TTC8; aliases: BBS8, RP51), wherein the sequence encoding TTC8 is, for example, SEQ ID NO: 59;

[0075] USH2A (protein name: usherin; gene: USH2A; aliases RP39, US2, USH2, dJ1111A8.1), wherein the sequence encoding USH2A is, for example, SEQ ID NO: 60;

[0076] WDR19 (protein name: WD repeat domain 19; gene: WDR19; aliases ATD5, CED4, DYF-2, FAP66, IFT144, NPHP13, ORF26, Oseg6, PWDMP, SRTD5), wherein the sequence encoding WDR19 is, for example, SEQ ID NO: 61, or a fimbrin fragment, or a fimbrin variant.

[0077] CFAP410 (protein name: Cilia and Flagella-associated protein 410; gene: CFAP410; alias RDMS; SMDAX; LRRC76; YF5 / A2; C21orf2), wherein the sequence encoding CFAP410 is, for example, SEQ ID NO: 62.

[0078] In one embodiment, the sequence encoding the ciliary protein is selected from SEQ ID NO: 29 to SEQ ID NO: 62, or a fragment or variant thereof.

[0079] In one embodiment, SEQ ID NO:29 to SEQ ID NO:62 are cDNA sequences.

[0080] It will be appreciated that the promoter according to the present invention drives sufficient expression levels to allow expression of the ciliary protein at the correct location without causing the protein to diffuse into the cell body.

[0081] Vectors and plasmids

[0082] In one embodiment, a vector suitable for delivering a nucleic acid sequence encoding a ciliary protein, or a fragment or variant thereof, to a photoreceptor is provided.

[0083] In one embodiment, the vector is a viral vector.

[0084] In one embodiment, the viral vector is a recombinant adeno-associated virus (rAAV).

[0085] In one embodiment, the vector comprises a promoter, which is a FCBR1 promoter (F, CBR, Crx binding region of the FAM161A gene), such as encoded by SEQ ID NO: 7 or SEQ ID NO: 63, or a fragment thereof, or a variant thereof. In another embodiment, the vector comprises a promoter, which comprises a FCBR1 promoter of SEQ ID NO: 7 or SEQ ID NO: 63, or a fragment thereof, or a variant thereof.

[0086] In one embodiment, the vector comprises a promoter, which is a F0.4 promoter (a 460-nucleotide FAM161A core promoter), such as one encoded by SEQ ID NO: 9 or SEQ ID NO: 65, or a fragment thereof, or a variant thereof. In another embodiment, the vector comprises a promoter comprising a core promoter of SEQ ID NO: 9 or SEQ ID NO: 65, or a fragment thereof, or a variant thereof.

[0087] In one embodiment, the vector comprises a promoter, which is a FCBR1-F0.4 promoter (F of the FAM161A gene; CBR, Crx binding region 1; F0.4, FAM161A core promoter), for example, encoded by SEQ ID NO: 10 or SEQ ID NO: 66, or a fragment thereof, or a variant thereof. In another embodiment, the vector has a promoter, which includes a FCBR1-F0.4 promoter of SEQ ID NO: 10 or SEQ ID NO: 66, or a fragment thereof, or a variant thereof.

[0088] In one embodiment, SEQ ID NO: 10 comprises SEQ ID NO: 7 and SEQ ID NO: 9. In one embodiment, SEQ ID NO: 66 comprises SEQ ID NO: 63 and SEQ ID NO: 65.

[0089] In one embodiment, the vector comprises a promoter, which is a FCBR2 promoter (F of the FAM161A gene; CBR, Crx binding region 2), for example, encoded by SEQ ID NO: 8 or SEQ ID NO: 64, or a fragment thereof, or a variant thereof. In another embodiment, the vector has a promoter, which comprises a CBR2 promoter of SEQ ID NO: 8 or SEQ ID NO: 64, or a fragment thereof, or a variant thereof.

[0090] In one embodiment, the vector comprises a promoter, which is a FCBR2-F0.4 promoter (CBR, Crx binding region; F0.4, FAM161A core promoter), for example, encoded by SEQ ID NO: 11 or SEQ ID NO: 67, or a fragment thereof, or a variant thereof. In another embodiment, the vector has a promoter, which includes a FCBR2-Core promoter of SEQ ID NO: 11 or SEQ ID NO: 67, or a fragment thereof, or a variant thereof.

[0091] In one embodiment, SEQ ID NO: 11 includes SEQ ID NO: 8 and SEQ ID NO: 9. In one embodiment, SEQ ID NO: 67 includes SEQ ID NO: 64 and SEQ ID NO: 65.

[0092] FCBR1-F0.4 or FCBR2-F0.4 may be referred to as a promoter. In one embodiment, the promoter is FCBR1-F0.4 or FCBR2-F0.4. In another embodiment, the promoter comprises FCBR1-F0.4 or FCBR2-F0.4.

[0093] In the description, "F0.4" may be labeled as "core", and "FCBR1" and "FCBR2" may be labeled as "CBR1" and "CBR2", so that "FCBR1-F0.4" and "FCBR2-F0.4" may be "CBR1-core" and "CBR2-core".

[0094] In one embodiment, the vector comprises a promoter, which is a GRK1 promoter (G protein coupled receptor kinase 1), for example, encoded by SEQ ID NO: 13, or a fragment thereof, or a variant thereof. In another embodiment, the vector has a promoter, which includes a GRK1 promoter of SEQ ID NO: 13, or a fragment thereof, or a variant thereof. The GRK1 promoter may be referred to herein as GRK1 or hGRK1 or GRK1p.

[0095] In one embodiment, the vector comprises an enhancer, which is an IRBP enhancer (inter-photoreceptor retinoid binding protein), for example, encoded by SEQ ID NO: 12, or a fragment thereof, or a variant thereof. In another embodiment, the vector has an enhancer, which includes an IRBP enhancer of SEQ ID NO: 12, or a fragment thereof, or a variant thereof. The IRBP enhancer may be referred to herein as IRBPen (or IRBP, depending on the context).

[0096] In one embodiment, the vector comprises a promoter, which is an IRBP-GRK1 promoter, for example, encoded by SEQ ID NO: 14, or a fragment thereof, or a variant thereof. In another embodiment, the vector has a promoter, which comprises an IRBP-GRK1 promoter of SEQ ID NO: 14, or a fragment thereof, or a variant thereof. IRBP-GRK1 may be referred to as a promoter. In one embodiment, the promoter is IRBP-GRK1. In another embodiment, the promoter comprises IRBP-GRK1.

[0097] In one embodiment, the vector comprises a nucleic acid sequence encoding the hFAM161A gene or a fragment or variant thereof. The nucleic acid sequence encoding the hFAM161A gene is referred to herein as a transgenic or target gene. In one embodiment, the transgenic is the hFAM161A gene or a fragment or variant thereof. In another embodiment, the transgenic comprises the hFAM161A gene or a fragment or variant thereof.

[0098] In one aspect, the vector comprises a nucleic acid sequence encoding the long isoform of the human FAM161A gene of SEQ ID NO: 1 (hFAM161A-L) or the short isoform of the human FAM161A gene of SEQ ID NO: 2 (hFAM161A-S), or a fragment or variant thereof.

[0099] In one aspect, the vector comprises a nucleic acid sequence encoding the long isoform of the human FAM161A gene (hFAM161A-L) of SEQ ID NO: 1, or a fragment or variant thereof.

[0100] In one aspect, the vector comprises a nucleic acid sequence encoding the short isoform of the human FAM161A gene (hFAM161A-S) of SEQ ID NO: 2, or a fragment or variant thereof.

[0101] In one embodiment, the vector comprises a nucleic acid sequence encoding a pilin gene, or a fragment or variant thereof.

[0102] In one aspect, the vector comprises a nucleic acid sequence encoding a ciliary protein gene or a fragment or variant thereof, wherein the nucleic acid sequence is selected from the group consisting of the POC5 gene of SEQ ID NO: 29, the NPHP4 gene of SEQ ID NO: 30, the ARL6 gene of SEQ ID NO: 31, the BBS1 gene of SEQ ID NO: 32, the BBS2 gene of SEQ ID NO: 33, the BBS9 gene of SEQ ID NO: 34, the PCARE gene of SEQ ID NO: 35, the CFAP418 gene of SEQ ID NO: 36, the CEP164 gene of SEQ ID NO: 37 (CEP164 isoform 1) or SEQ ID NO: 38 (CEP164 isoform 2), the CEP290 gene of SEQ ID NO: 39, the CLRN1 gene of SEQ ID NO: 40, the IFT140 gene of SEQ ID NO: 41, the IFT172 gene of SEQ ID NO: 42, the IQCB1 gene of SEQ ID NO: 43, the KIZ gene of SEQ ID NO: 44, the KIZ gene of SEQ ID NO: 45, the KIZ gene of SEQ ID NO: 46, the KIZ gene of SEQ ID NO: 47, the KIZ gene of SEQ ID NO: 48, the KIZ gene of SEQ ID NO: 49, the KIZ gene of SEQ ID NO: 50, the KIZ gene of SEQ ID NO: 51, the KIZ gene of SEQ ID NO: 52, the KIZ gene of SEQ ID NO: 53, the KIZ gene of SEQ ID NO: 54, the KIZ gene of SEQ ID NO: 55, the KIZ gene of SEQ ID NO: 56, the KIZ gene of SEQ ID NO: 57, the KIZ gene of SEQ ID NO The invention further comprises the following: the LCA5 gene of SEQ ID NO:45, the MAK gene of SEQ ID NO:46, the NEK2 gene of SEQ ID NO:47, the OFD1 gene of SEQ ID NO:48, the OFD1 gene of SEQ ID NO:49, the RP1 gene of SEQ ID NO:50, the RP1L1 gene of SEQ ID NO:51, the RP2 gene of SEQ ID NO:52, the RPGR gene of SEQ ID NO:53 (RPGR isoform C) or SEQ ID NO:54 (RPGR isoform J), the RPGRIP1 gene of SEQ ID NO:55 (RPGRIP1 isoform 1) or SEQ ID NO:56 (RPGRIP1 isoform 3), the SPATA7 gene of SEQ ID NO:57, the TOPORS gene of SEQ ID NO:58, the TTC8 gene of SEQ ID NO:59, the USH2A gene of SEQ ID NO:60, the WDR19 gene of SEQ ID NO:61, and the CFAP410 gene of SEQ ID NO:62.

[0103] In one aspect, the vector comprises the FCBR1-F0.4 promoter and a nucleic acid sequence encoding the hFAM161A gene (eg, the hFAM161A-L gene or the hFAM161A-S gene) or a fragment or variant thereof.

[0104] In one aspect, the vector comprises the FCBR2-F0.4 promoter and a nucleic acid sequence encoding the hFAM161A gene (eg, the hFAM161A-L gene or the hFAM161A-S gene) or a fragment or variant thereof.

[0105] In one aspect, the vector comprises an IRBP-GRK1 promoter and a nucleic acid sequence encoding an hFAM161A gene (eg, an hFAM161A-L gene or an hFAM161A-S gene) or a fragment or variant thereof.

[0106] In one aspect, the vector comprises the FCBR1-F0.4 promoter and a nucleic acid sequence encoding a ciliary protein gene or a fragment or variant thereof, such as gene POC5, NPHP4, ARL6, BBS1, BBS2, BBS9, PCARE, CFAP418, CEP164 isoform 1 or isoform 2, CEP290, CLRN1, IFT140, IFT172, IQCB1, KIZ, LCA5, MAK, NEK2, OFD1, OFD1, RP1, RP1L1, RP2, RPGR isoform C or isoform J, RPGRIP1 isoform 1 or isoform 3, SPATA7, TOPORS, TTC8, USH2A, WDR19, CFAP410.

[0107] In one aspect, the vector comprises the FCBR2-F0.4 promoter and a nucleic acid sequence encoding a ciliary protein gene, or a fragment or variant thereof, such as gene POC5, NPHP4, ARL6, BBS1, BBS2, BBS9, PCARE, CFAP418, CEP164 isoform 1 or isoform 2, CEP290, CLRN1, IFT140, IFT172, IQCB1, KIZ, LCA5, MAK, NEK2, OFD1, OFD1, RP1, RP1L1, RP2, RPGR isoform C or isoform J, RPGRIP1 isoform 1 or isoform 3, SPATA7, TOPORS, TTC8, USH2A, WDR19, CFAP410.

[0108] In one aspect, the vector comprises an IRBP-GRK1 promoter and a nucleic acid sequence encoding a ciliary protein gene, or a fragment or variant thereof, such as gene POC5, NPHP4, ARL6, BBS1, BBS2, BBS9, PCARE, CFAP418, CEP164 isoform 1 or isoform 2, CEP290, CLRN1, IFT140, IFT172, IQCB1, KIZ, LCA5, MAK, NEK2, OFD1, OFD1, RP1, RP1L1, RP2, RPGR isoform C or isoform J, RPGRIP1 isoform 1 or isoform 3, SPATA7, TOPORS, TTC8, USH2A, WDR19, CFAP410.

[0109] In one embodiment, the vector comprises a post-transcriptional regulatory element, such as WPRE (Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element). In one embodiment, the vector comprises a post-transcriptional regulatory element, such as SINEUP non-coding RNA (modular antisense long non-coding RNA).

[0110] In another embodiment, the vector comprises a post-transcriptional regulatory element such as a WPRE of SEQ ID NO: 15, or a fragment thereof, or a variant thereof. In another embodiment, the vector has a post-transcriptional regulatory element comprising a WPRE of SEQ ID NO: 15, or a fragment thereof, or a variant thereof.

[0111] In one aspect, the vector comprises the FCBR1-F0.4 promoter, a nucleic acid sequence encoding the hFAM161A gene (eg, the hFAM161A-L gene or the hFAM161A-S gene) or a fragment or variant thereof, and WPRE.

[0112] In one aspect, the vector comprises the FCBR2-F0.4 promoter, a nucleic acid sequence encoding the hFAM161A gene (eg, the hFAM161A-L gene or the hFAM161A-S gene) or a fragment or variant thereof, and WPRE.

[0113] In one aspect, the vector comprises an IRBP-GRK1 promoter, a nucleic acid sequence encoding an hFAM161A gene (eg, an hFAM161A-L gene or an hFAM161A-S gene) or a fragment or variant thereof, and a WPRE.

[0114] In one aspect, the vector comprises the FCBR1-F0.4 promoter, a nucleic acid sequence encoding a pilin gene, or a fragment or variant thereof, and WPRE, for example, the pilin gene is selected from the group consisting of POC5, NPHP4, ARL6, BBS1, BBS2, BBS9, PCARE, CFAP418, CEP164 isoform 1 or isoform 2, CEP290, CLRN1, IFT140, IFT172, IQCB1, KIZ, LCA5, MAK, NEK2, OFD1, OFD1, RP1, RP1L1, RP2, RPGR isoform C or isoform J, RPGRIP1 isoform 1 or isoform 3, SPATA7, TOPORS, TTC8, USH2A and WDR19, CFAP410.

[0115] In one aspect, the vector comprises the FCBR2-F0.4 promoter, a nucleic acid sequence encoding a pilin gene, or a fragment or variant thereof, and a WPRE, for example, the pilin gene is selected from the group consisting of POC5, NPHP4, ARL6, BBS1, BBS2, BBS9, PCARE, CFAP418, CEP164 isoform 1 or isoform 2, CEP290, CLRN1, IFT140, IFT172, IQCB1, KIZ, LCA5, MAK, NEK2, OFD1, OFD1, RP1, RP1L1, RP2, RPGR isoform C or isoform J, RPGRIP1 isoform 1 or isoform 3, SPATA7, TOPORS, TTC8, USH2A and WDR19, CFAP410.

[0116] In one aspect, the vector comprises an IRBP-GRK1 promoter, a nucleic acid sequence encoding a pilin gene, or a fragment or variant thereof, and a WPRE, for example, the pilin gene is selected from the group consisting of POC5, NPHP4, ARL6, BBS1, BBS2, BBS9, PCARE, CFAP418, CEP164 isoform 1 or isoform 2, CEP290, CLRN1, IFT140, IFT172, IQCB1, KIZ, LCA5, MAK, NEK2, OFD1, OFD1, RP1, RP1L1, RP2, RPGR isoform C or isoform J, RPGRIP1 isoform 1 or isoform 3, SPATA7, TOPORS, TTC8, USH2A and WDR19, CFAP410.

[0117] In one embodiment, the vector comprises a sequence selected from SEQ ID NO: 25 (AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE) or SEQ ID NO: 26 (AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE), or a fragment or variant thereof.

[0118] In one embodiment, the vector comprises a sequence selected from SEQ ID NO: 21 (AAV2 / 8-FCBR2-F0.4-hFAM161A-L-WPRE) or SEQ ID NO: 17, SEQ ID NO: 22 (AAV2 / 8-FCBR2-F0.4-hFAM161A-S-WPRE), or a fragment or variant thereof.

[0119] In one embodiment, the vector comprises a sequence selected from SEQ ID NO: 16 (AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE) or SEQ ID NO: 17 (AAV2 / 8-IRBP-GRK1-hFAM161A-S-WPRE), or a fragment or variant thereof.

[0120] It will be appreciated that SEQ ID NO: 16 to SEQ ID NO: 28 are AAV transgene cassettes (from 5' ITR to 3' ITR, inclusive).

[0121] In one embodiment, the rAAV has a capsid protein selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV2 / 1, AAV2 / 2, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, and AAV2 / 9. In a preferred embodiment, the rAAV has an AAV2 / 8 or AAV2 / 5 capsid protein.

[0122] In one embodiment, a mixture of at least two vectors according to the present invention is provided. In another embodiment, the mixture of at least two vectors comprises a vector comprising SEQ ID NO: 25 (AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE) and SEQ ID NO: 26 (AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE), wherein the two vectors are preferably mixed in a 1:1 ratio. In one embodiment, a plasmid suitable for producing a vector for delivering a nucleic acid sequence encoding a ciliary protein or a fragment thereof or a variant thereof to a photoreceptor is provided, as described herein. The plasmid according to the present invention comprises the elements described for the vector of the present invention.

[0123] In another embodiment, a method of vector production is provided, such as using an engineered cell line, a baculovirus (BV) expression system, or any transient transfection method in HEK293T cells or any other suitable cell line for AAV production (e.g., Sf9 or ExpresSf+ cells). It should be understood that any known vector production method can be used. In another embodiment, a method of vector purification is provided, such as using an iodixanol gradient, ultrafiltration or chromatographic separation or any other suitable method for AAV purification. It should be understood that any known vector purification method can be used.

[0124] In another embodiment, a nucleic acid sequence encoding rAAV according to the present invention is provided.

[0125] In another embodiment, a cell comprising a nucleic acid sequence encoding a rAAV according to the present invention is provided.

[0126] In another embodiment, a vector production method is provided, which comprises transfecting HEK293 cells with two or three plasmids, wherein one plasmid encodes the target gene described herein, one plasmid carries the AAV rep / cap gene, and another plasmid contains auxiliary genes provided by adenovirus or herpes virus.

[0127] Compositions, administration and kits

[0128] In one embodiment, a composition is provided, comprising at least one vector of the present invention and at least one physiologically acceptable carrier or vehicle, wherein the vector comprises a nucleic acid sequence encoding a FCBR1-F0.4 promoter or a FCBR2-F0.4 promoter or an IRBP-GRK1 promoter and a cilia protein (e.g., hFAM161A) or a fragment or variant thereof. Exemplary physiologically acceptable carriers, particularly carriers suitable for administration to the eye, include sterile pyrogen-free phosphate buffered saline, sterile pyrogen-free TSSM-buffer (20 mM tromethamine, 100 mM NaCl, 10 mg / mL sucrose and 10 mg / mL mannitol), and the like.

[0129] In another embodiment, a composition is provided, comprising at least one vector of the present invention and at least one pharmaceutically acceptable carrier or vehicle, the vector carrying a nucleic acid sequence encoding FCBR1-F0.4 promoter or FCBR2-F0.4 promoter or IRBP-GRK1 promoter and a ciliary protein (e.g., hFAM161A) or a fragment or variant thereof. Exemplary pharmaceutically acceptable carriers or vehicles, particularly those suitable for administration to the eye, include buffered saline or other buffers, such as HEPES, to maintain pH at an appropriate physiological level, and optionally, other medicinal agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, and the like.

[0130] The compositions and pharmaceutical compositions of the invention may contain one or more vectors of the invention comprising any of the elements described herein. The compositions and pharmaceutical compositions according to the invention are particularly suitable for administration to the eye, for example by subretinal or intravitreal injection, but also by systemic administration.

[0131] In one embodiment, if the vector is to be stored long term, it can be frozen in the presence of a cryoprotectant (eg, glycerol or Tween 20).

[0132] In one embodiment, there is provided a composition or pharmaceutical composition according to the present invention which is suitable for administration to the eye, preferably by subretinal injection.

[0133] The compositions of the present invention can be delivered in a volume of about 50 μL to about 1 ml (including all numbers within this range), depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume is about 100 μL to about 300 μL.

[0134] The effective concentration range of rAAV according to the present invention is about 10 8 Up to 10 13 The concentration of rAAV infectious units is between 1.5 × 10 9 vg / mL to about 5×10 14 In another embodiment, the effective concentration is about 1.5×10 12 vg / mL to about 1.5×10 14 vg / mL. It is desirable to use the lowest effective concentration of rAAV in order to reduce the risk of undesirable effects (e.g., toxicity, retinal dysplasia, and detachment). Other doses within these ranges may be selected taking into account the physical condition of the subject being treated, the age of the subject, the particular ocular condition, and the extent to which the condition, if advanced, has progressed.

[0135] According to one aspect, the invention relates to a kit for carrying out a method or use according to the invention.

[0136] According to one embodiment, a kit is provided, comprising at least one rAAV or a composition thereof according to the present invention and instructions for use thereof.

[0137] Methods and uses

[0138] Retinal ciliopathies, FAM161A-related retinopathy, such as retinitis pigmentosa-28 (RP28), are associated with a number of retinal changes. These include loss of photoreceptor structure and / or function, thinning or thickening of the outer nuclear layer (ONL), thinning or thickening of the outer plexiform layer (OPL), loss of rod and cone outer segments followed by disarray, shortening of rod and cone inner segments, retraction of bipolar cell dendrites, thinning or thickening of the inner retinal layers including the inner nuclear layer, inner plexiform layer, ganglion cell layer, and nerve fiber layer, mislocalization of opsins, overexpression of neurofilaments, loss of ERG function, loss of visual acuity and contrast sensitivity, and loss of visually guided behavior. In one embodiment, the present invention provides a method of altering, preventing, arresting progression, or ameliorating any retinal changes associated with a retinal ciliopathic disease. Thus, the subject's vision is improved, or vision loss is delayed or inhibited and / or ameliorated.

[0139] In one embodiment, a method of altering, preventing, delaying or arresting progression or ameliorating vision loss associated with a retinal ciliopathy (e.g., a FAM161A-related retinopathy, preferably, e.g., RP28) in a subject in need thereof is provided, the method comprising administering an effective amount of a rAAV according to the present invention or a composition thereof.

[0140] In one embodiment, a method of treating a retinal ciliopathy (eg, a FAM161A-related retinopathy, preferably, eg, RP28) in a subject in need thereof is provided, the method comprising administering an effective amount of a rAAV according to the present invention or a composition thereof.

[0141] In another embodiment, a method of improving photoreceptor structure in a subject in need thereof is provided, the method comprising administering an effective amount of a rAAV according to the present invention or a composition comprising a rAAV according to the present invention.

[0142] In a specific embodiment, a method of improving photoreceptor outer segment structure in a subject in need thereof is provided, the method comprising administering an effective amount of a rAAV according to the present invention or a composition thereof.

[0143] In another embodiment, a method for inducing photoreceptor fimbriae protein stabilization by expressing at least one fimbriae protein in photoreceptor fimbriae is provided, comprising administering an effective amount of rAAV according to the present invention or a composition thereof.

[0144] The method according to the present invention comprises administering to a subject in need thereof an effective amount of a rAAV according to the present invention or a composition comprising the same, preferably by subretinal injection.

[0145] According to one embodiment, a use of the rAAV according to the present invention as a medicament is provided.

[0146] According to one embodiment, a rAAV according to the present invention or a composition comprising the same is provided for use in altering, preventing, delaying, arresting progression or improving vision loss associated with retinal ciliopathy (such as FAM161A-related retinopathy, preferably RP28).

[0147] According to one embodiment, a rAAV according to the present invention or a composition comprising the same is provided for use in treating retinal ciliopathy, such as FAM161A-related retinopathy, preferably RP28.

[0148] According to one embodiment, the present invention provides a use of the rAAV according to the present invention or a composition comprising the same for treating RP28.

[0149] In another embodiment, a use of the rAAV according to the present invention or a composition comprising the same for improving the structure of a photoreceptor is provided.

[0150] In a specific embodiment, a use of the rAAV or a composition thereof according to the present invention for improving the structure of the photoreceptor outer segment is provided.

[0151] In another embodiment, a use of the rAAV or a composition thereof according to the present invention for inducing stabilization of photoreceptor ciliary proteins is provided.

[0152] The use according to the present invention comprises administering an effective amount of the rAAV according to the present invention or a composition comprising the rAAV according to the present invention to a subject in need thereof, preferably by subretinal injection, or by intravitreal injection, or by intravenous injection.

[0153] According to another embodiment, the present invention provides a use of an rAAV according to the present invention for preparing a composition for preventing, modifying, arresting progression or improving vision loss associated with retinal ciliopathy (e.g., FAM161A-related retinopathy, preferably, e.g., RP28), and / or treating retinal ciliopathy (e.g., RP28), and / or improving photoreceptor structure, and / or improving photoreceptor outer segment structure, and / or for inducing photoreceptor ciliary protein stabilization.

[0154] In one embodiment, an effective amount of a rAAV according to the invention or a composition according to the invention is administered only to one or more regions of the eye, for example, regions where photoreceptors are preserved. In another embodiment, the composition is administered to the entire eye.

[0155] It will be appreciated that transduction with rAAV results in expression of the product of the gene encoded in the rAAV, in particular expression of at least one fibrillary protein. It will be appreciated that expression of at least one fibrillary protein results in stabilization of photoreceptor cilia and improved photoreceptor function. Thus, the subject's vision is improved, or vision loss is inhibited and / or improved.

[0156] In yet another embodiment of the invention, any of the above methods is performed in combination with another or secondary therapy. The therapy may be any currently known or still unknown therapy that helps prevent, stop or ameliorate retinal ciliopathy, such as RP28, or any of the effects associated therewith. The secondary therapy may be administered before, simultaneously with or after administration of the above rAAV. In certain embodiments of the methods of the invention, an effective amount of rAAV according to the invention or a composition according to the invention is administered to a subject by subretinal injection.

[0157] In one aspect, photoreceptor function can be assessed using functional studies known to those skilled in the art, such as described in the Examples below, such as ERG, FST or microperimetry or pupil light response or fMRi, which are routine in the art. As used herein, "loss of photoreceptor function" refers to a decrease in photoreceptor function compared to a normal, non-diseased eye or the same eye at an earlier time point. As used herein, "increasing photoreceptor function" refers to improving the function of a photoreceptor or increasing the number or percentage of functional photoreceptors compared to a diseased eye (having the same ocular disease at the same stage), the same eye at an earlier time point, an untreated portion of the same eye, or the contralateral eye of the same subject. As used herein, "maintaining photoreceptor function" refers to controlling the function of a photoreceptor and preventing the degradation of a photoreceptor.

[0158] In another aspect, the present invention provides a method of improving the photoreceptor structure of a subject. As used herein, "improving photoreceptor structure" (in the area of ​​the treated retina) refers to reducing the shortening and loss of outer segments (OS) and / or maintaining the thickness of the outer nuclear layer (ONL) and / or preventing or reducing the progression of ONL thickening (due to edema) or thinning (due to photoreceptor loss) in the entire retina, central retina or periphery (corresponding to the treated area); increasing or decreasing the thickness of the outer plexiform layer (OPL), or preventing the progression of OPL thickening or thinning; reducing the shortening of rod and cone inner segments (IS); reducing bipolar cell dendrite retraction, or increasing bipolar cell dendrite length or amount; and reversing opsin mislocalization.

[0159] For each of the methods or uses described, the treatment can be used to prevent the onset of retinal damage or to rescue eyes with mild or advanced disease. As used herein, the term "rescue" refers to preventing or delaying the progression of the disease to complete blindness, preventing the spread of damage to undamaged photoreceptor cells, or reducing damage to damaged photoreceptor cells. Therefore, in one embodiment, an effective amount of rAAV according to the present invention or a composition comprising rAAV according to the present invention is administered before the onset of the disease. In another embodiment, an effective amount of rAAV according to the present invention or a composition comprising rAAV according to the present invention is administered after the onset of the disease. In another embodiment, an effective amount of rAAV according to the present invention or a composition comprising rAAV according to the present invention is administered before the onset of photoreceptor loss. In another embodiment, an effective amount of rAAV according to the present invention or a composition comprising rAAV according to the present invention is administered after the onset of photoreceptor loss.

[0160] In yet another embodiment, an effective amount of rAAV according to the present invention or a composition comprising rAAV according to the present invention is administered when the subject has 10% or more, or 20% or more, or 30% or more, or 40% or more, or 50% or more, or 60% or more, or 70% or more, or 80%, or 90% or more functional or remaining photoreceptors compared to a non-diseased eye. In one embodiment, an effective amount of rAAV according to the present invention or a composition comprising rAAV according to the present invention is administered only to one or more areas of the eye (e.g., those areas with retained photoreceptors). In another embodiment, the composition is administered to the entire eye. In another embodiment, a method for treating or preventing retinal ciliopathic disease in a subject in need is provided. The method comprises administering to the subject an effective amount of rAAV according to the present invention or a composition comprising rAAV according to the present invention. For example, the method comprises:

[0161] - Identify subjects with or at risk of developing retinal ciliopathy (e.g. RP28);

[0162] - Perform genotyping and identify at least one mutation in a gene associated with retinal ciliopathy;

[0163] - Perform non-invasive retinal imaging and / or functional studies and identify spared photoreceptor areas to be targeted for treatment; and

[0164] - administering to said subject an effective amount of a rAAV according to the invention or a composition comprising a rAAV according to the invention; thereby altering, preventing, arresting or ameliorating retinal ciliopathy. Genotyping is routine in the art.

[0165] patient

[0166] In one embodiment, a subject according to the present invention comprises a mammalian subject, particularly a human suffering from or susceptible to a retinal ciliopathy, such as a FAM161A-related retinopathy, such as retinitis pigmentosa-28 (RP28).

[0167] In one embodiment, the subject according to the present invention suffers from RP28.

[0168] In one embodiment, the subject already displays clinical signs of RP28, such as visual impairment and related retinal degeneration.

[0169] In another embodiment, the subject according to the present invention is susceptible to RP28. In one embodiment, the subject does not show clinical signs of RP28.

[0170] In certain embodiments, the subject has a genetic mutation associated with RP28.

[0171] In another specific embodiment, a subject having or at risk for RP28 has a pathogenic variant in the gene FAM161A. Subjects at risk for RP28 include subjects with a family history of RP28, subjects with one or more confirmed mutations in both alleles of the FAM161A gene.

[0172] In another embodiment, the subject according to the present invention is being treated with RP28. In one embodiment, the retinal ciliopathy is a disease selected from the group consisting of retinitis pigmentosa (RP), retinitis pigmentosa such as retinitis pigmentosa-28 (RP28), FAM161A-related RP, BBS (Bardet-Biedl syndrome), SLS (Senior-Loken syndrome), JBS (Joubert syndrome), MKS (Meckel-Gruber syndrome), USH (Usher syndrome), JATD (Jeune asphyxiating thoracic dysplasia), MZSDS (Mainzer-Saldino syndrome), OFD (oral-facial-digital syndrome), OMD (occult macular dystrophy), CRD (cone-rod dystrophy), LCA (Leber congenital amaurosis), CED (cranial ectodermal dysplasia, also known as Sensenbrenner syndrome).

[0173] In one embodiment, a subject according to the present invention suffers from a retinal ciliopathy, such as those listed herein.

[0174] In another embodiment, the subject according to the present invention is susceptible to retinal ciliopathies, such as those listed herein. In one embodiment, the subject does not show clinical signs of retinal ciliopathies. Subjects at risk of retinal ciliopathies include subjects with a family history of retinal ciliopathies, subjects with mutations in one or more confirmed retinal ciliopathies-associated genes, and offspring of female carriers of retinal ciliopathies-associated gene mutations in both alleles.

[0175] In another embodiment, a subject according to the present invention is being treated for a retinal ciliopathy, such as those listed herein.

[0176] In one embodiment, the subject has 10% or more, or 20% or more, or 30% or more, or 40% or more, or 50% or more, or 60% or more, or 70% or more, or 80%, or 90% or more photoreceptor damage / loss.

[0177] The references cited herein are incorporated herein by reference in their entirety. The scope of the present invention is not limited by the specific embodiments and drawings described herein, which are intended to be separate illustrations of various aspects of the present invention, and functionally equivalent methods and components are within the scope of the present invention. The embodiments illustrating the present invention are not intended to limit the scope of the present invention in any way.

[0178] Example

[0179] List of abbreviations: 661W-mouse photoreceptor cell line; Ac-tub-acetylated tubulin; ARPE19-human RPE; CBR-Crx binding region; F0.4-FAM161A core promoter (or core); ERG-electroretinogram; HEK293T-human embryonic kidney cells; GRK1-human G protein-coupled receptor kinase 1; HL (hFAM161A-L)-human long FAM161A protein isoform; HS (hFAM161A-S)-human short FAM161A protein isoform; hTERT-RPE1- Human immortalized RPE cells; IRBP-interphotoreceptor retinoid binding protein; ML(mFam161a-L)-mouse long Fam161a protein isoform; ML1(mFam161a-L1)-mouse long 1 Fam161a protein isoform; MS(mFam161a-S)-mouse short Fam161a protein isoform; MS1(mFam161a-S1)-mouse short 1 Fam161a protein isoform; ONL-outer nuclear layer; WPRE-woodchuck hepatitis virus (WHP) posttranscriptional regulatory element.

[0180] Example 1: Generation of different plasmids

[0181] Several plasmids were constructed to generate different AAV2 / 8 vectors encoding the long or short isoform of the human FAM161A gene or the mouse Fam161a gene as described below.

[0182] Materials and methods

[0183] Plasmids: Several plasmids were constructed to generate different AAV2 / 8 vectors encoding the long isoform of the human FAM161A gene (hFAM161A-L (CCDS56120.1), SEQ ID NO: 1, referred to herein as HL) or the short isoform of the human FAM161A gene (hFAM161A-S (CCDS42687.2), SEQ ID NO: 2, referred to herein as HS). Promoter sequences were PCR amplified from human or mouse DNA. Human and mouse cDNAs were RT-PCR amplified from human or mouse RNA, respectively. Different AAV transgene cassettes were cloned using recombination (Invitrogen's MultiSite Gateway© system), overlap extension PCR (e.g., In-fusion cloning), or conventional restriction enzyme ligation methods. As a proof of concept, four mouse Fam161a isoforms and two human isoforms were also FLAG-tagged and cloned into plasmids. The four mouse Fam161a isoforms are mFam161a-L (SEQ ID NO:3, referred to herein as ML), mFam161a-L1 (SEQ ID NO:4, referred to herein as ML1), mFam161a-S (SEQ ID NO:5, referred to herein as MS), and mFam161a-S1 (SEQ ID NO:6, referred to herein as MS1). Several different cis-regulatory elements were tested, including the GRK1 (human G protein-coupled receptor kinase 1) promoter in the presence of the IRBP (inter-photoreceptor retinoid binding protein) enhancer, endogenous human FAM161A promoter fragments (FCBR1-F0.4 and FCBR2-F0.4) (CBR, Crx binding region in the FAM161A gene (F), therefore FCBR1 or FCBR2; F0.4, FAM161A core promoter) and the RNA stabilizer WPRE (woodchuck hepatitis virus (WHP) post-transcriptional regulatory element). Figure 1 Possible construct compositions are shown. Note that F0.4 can be labeled "core" and FCBR1 and FCBR2 can be labeled CBR1 and CBR2, such that FCBR1-F0.4 and FCBR2-F0.4 can be CBR1-core and CBR2-core.

[0184] The sequences of the cis-regulatory elements are as follows: FCBR1 is SEQ ID NO:7 or SEQ ID NO:63, FCBR2 is SEQ ID NO:8 or SEQ ID NO:64, F0.4 is SEQ ID NO:9 or SEQ ID NO:65, FCBR1-F0.4 is SEQ ID NO:10 or SEQ ID NO:66, FCBR2-F0.4 is SEQ ID NO:11 or SEQ ID NO:67, IRBP is SEQ ID NO:12, GRK1 is SEQ ID NO:13, IRBP-GRK1 is SEQ ID NO:14, and WPRE is SEQ ID NO:15.

[0185] The difference between SEQ ID NO:7 of FCBR1 and the sequence FCBR1 of SEQ ID NO:63 found in the database is 2608 a>g (wherein “>” means that one nucleotide is replaced by another nucleotide; here a is replaced by g).

[0186] SEQ ID NO:8 of FCBR2 differs from the sequence FCBR2 of SEQ ID NO:64 found in the database at 1862 a>g and 2141 a>g.

[0187] SEQ ID NO:9 of F0.4 differs from the sequence F.04 of SEQ ID NO:65 found in the database at 2881 t>c, 2954 a>g and 3226 t>c.

[0188] The resulting AAV2 / 8 vectors were used in the following experiments and are as follows:

[0189] (1)AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE (SEQ ID NO:16)

[0190] (hFAM161A-L (HL) comprising SEQ ID NO: 1);

[0191] (2)AAV2 / 8-IRBP-GRK1-hFAM161A-S-WPRE (SEQ ID NO: 17)

[0192] (hFAM161A-S (HS) comprising SEQ ID NO: 2);

[0193] (3)AAV2 / 8-IRBP-GRK1-mFam161a-L-WPRE (SEQ ID NO:18)

[0194] (mFam161a-L(ML) comprising SEQ ID NO:3);

[0195] (4)AAV2 / 8-IRBP-GRK1-mFam161a-S-WPRE (SEQ ID NO:19)

[0196] (mFam161a-S (MS) comprising SEQ ID NO: 5);

[0197] (5)AAV2 / 8-IRBP-GRK1-mFam161a-S1-WPRE (SEQ ID NO: 20)

[0198] (mFam161a-S (MS1) comprising SEQ ID NO: 6);

[0199] (6)AAV2 / 8-FCBR2-F0.4-hFAM161A-L-WPRE (SEQ ID NO: 21)

[0200] (hFAM161A-L (HL) comprising SEQ ID NO: 1);

[0201] (7)AAV2 / 8-FCBR2F0.4-hFAM161A-S-WPRE (SEQ ID NO:22)

[0202] (hFAM161A-S (HS) comprising SEQ ID NO: 2);

[0203] (8)AAV2 / 8-FCBR2-F0.4-mFam161a-L-WPRE (SEQ ID NO: 23)

[0204] (mFam161a-L(ML) comprising SEQ ID NO:3);

[0205] (9)AAV2 / 8-FCBR2-F0.4-mFam161a-L1-WPRE (SEQ ID NO: 24)

[0206] (mFam161a-L 1 (ML1) comprising SEQ ID NO: 4);

[0207] (10)AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE (SEQ ID NO: 25)

[0208] (hFAM161A-L (HL) comprising SEQ ID NO: 1);

[0209] (11)AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE (SEQ ID NO: 26)

[0210] (hFAM161A-S (HS) comprising SEQ ID NO: 2);

[0211] (12)AAV2 / 8-FCBR1-F0.4-mFam161a-L-WPRE (SEQ ID NO: 27)

[0212] (mFam161a-L(ML) comprising SEQ ID NO:3);

[0213] (13)AAV2 / 8-FCBR1-F0.4-mFam161a-L1-WPRE (SEQ ID NO: 28)

[0214] (mFam161a-L 1 (ML1) comprising SEQ ID NO: 4).

[0215] SEQ ID NO: 16 to SEQ ID NO: 28 are AAV-transgene cassettes from 5' ITR to 3' ITR (inclusive).

[0216] Example 2: In vitro FAM161A protein expression and function.

[0217] Several plasmids were constructed to generate different plasmids encoding human FAM161A or mouse Fam161a isoforms under the control of the GRK1 promoter in the presence of the IRBP enhancer or the ubiquitous promoter EFs (elongation 1 factor) short promoter and the RNA stabilizer WPRE. Subsequently, FAM161A protein expression was observed in vitro in mouse photoreceptors and human cell lines.

[0218] Materials and methods

[0219] Additional expression plasmids were generated as described above using the EF promoter to express transgenes in vitro in different cell lines. (1)-SEQ ID NO:16, (2)-SEQ ID NO:17, (3)-SEQ ID NO:18, (5)-SEQ ID NO:20, and (6)-SEQ ID NO:21 sequences were used to replace the promoter with EF and to test the protein cellular fate of different FAM161A isoforms.

[0220] Since the anti-FAM161A antibody does not recognize the mouse protein well, the mouse FAM161A isoforms (ML, MS, MS1) were also FLAG-tagged in order to be accurately detected by the anti-FLAG antibody. Transfection of the different plasmids was performed in 661W mouse photoreceptor cell line, HEK293T (human embryonic kidney), ARPE19 (human RPE), and hTERT-RPE1 (human immortalized RPE) cells using lipofectamine 3000. At 3 days post-transfection, cells were extracted in RIPA (radioimmunoprecipitation assay) buffer using sonication for western blot analysis or fixed on coverslips with PAF 4% at 3 days post-transfection for immunocytochemical analysis. Antibodies raised against FAM161A (Sigma HPA032119, 1:2000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Chemicon MAB374, 1:5000), and Flag® tag (Sigma F1804, clone M2, 1:2500), also known as DYKDDDDK-tag (D = aspartic acid; K = lysine; Y = tyrosine), were used for western blotting. Antibodies raised against Flag® tag (Sigma F1804, clone M2, 1:2500) and acetylated tubulin (Sigma T7451, 1:1000) were used for immunohistochemistry.

[0221] FAM161A protein expression: Plasmids were first tested in vitro in 661W and HEK293T to verify isoform expression of EF or IRBP-GFK expression plasmids. After transfection of 661W with EFs constructs ( Figure 2A ) or after transfection of HEK293T cells with the IRBP-GFK1 construct (data not shown), Western blotting was used to detect FAM161A-L (HL) and hFAM161A-S (HS), confirming the different sizes of the two human isoforms. The same method was used to detect mFam161a-L (ML), mFam161a-S (MS), and mFam161a-S1 (MS1) in HEK293T cells after transfection with the IRBP-GFK1 construct (SEQ ID NO:18, 19, 20) ( Figure 2B Then in hTERT-RPE1 cells ( Figure 2C-2G) or ARPE-19 cells, the cellular localization of the different isoforms (i.e., hFAM161A-L (HL), hFAM161A-S (HS), mFam161a-L (ML), mFam161a-S (MS), mFam161a-S1 (MS1)) was examined by immunocytochemistry. Interestingly, while the human isoforms (HL, HS) as well as the mouse long isoform (ML) are expressed along the cytoskeleton (the levels of Ac-tub correspond to those of FAM161A, see Figure 2E and Figure 2H ), but the MS and MS1 isoforms are expressed only in or around the nucleus. High expression of the human FAM161A protein and the mouse L protein (as evidenced by their strong expression in cells and their ubiquitous distribution) leads to the accumulation of acetylated α-tubulin (Ac-tub, Figure 2C , Figure 2D , Figure 2E , Figure 2H ) expression increased, following the pattern of FAM161A distribution ( FIG. 2C to FIG. 2E Quantification of stable acetylated tubulin as a percentage of transfected cells (ARPE19) suggests that FAM161A is involved in microtubule stabilization ( Figure 2H Since the mouse short isoforms (MS and MS1) show specific subcellular localization in the nucleus and fail to induce cytoskeletal stabilization ( Figure 2H ), so an additional mouse isoform, L1, was cloned from mouse retina to match the mouse L isoform (mFam161a-L1 (SEQ ID NO:4)).

[0222] These in vitro experiments confirmed that both EF and IRBP-GRK1 can drive expression of human and mouse isoforms, and that overexpression of hFAM161A-L and hFAM161A-S as well as mFAM161A-L extended and stabilized the microtubule network. However, overexpression of FAM161A in the cell body appeared to reorganize the entire cytoskeleton, altering photoreceptor function (see in vivo data below). These results suggest that low / physiological and localized expression of FAM161A is required in vivo to obtain correct FAM161A protein localization in cilia and to obtain adequate function.

[0223] Example 3: Generation of different AAV2 / 8 vectors with IRBP-GRK1 and mouse Fam161a isoforms and expression in vivo in rod and cone photoreceptors.

[0224] Materials and Methods. Electroretinogram: Full-field ERG (FFERG) was performed using a Ganzfeld dome and a computerized system (Espion E2, Diagnosys LLC, Littleton, MA) after anesthetized animals were dark-adapted overnight. Briefly, the pupil was dilated and a gold wire active electrode was placed on the central cornea. A reference electrode was placed on the tongue and a needle ground electrode was placed in the gluteal muscle. Flicker responses of dark-adapted rods and mixed cones and rods and light-adapted 1 Hz and 16 Hz cones to a series of white flashes of increasing intensity (0.00008–9.6 cd·s / m2) were recorded. All ERG responses were filtered at 0.3–500 Hz and signal averaging was applied.

[0225] Optical coherence tomography (OCT) and fundus autofluorescence (FAF): Retinal structure was studied in vivo by OCT b-scan 30 degree testing, infrared (IR), and fundus autofluorescence (FAF) imaging (SPECTRALIS, Heidelberg). OCT is a non-invasive technique used to obtain cross-sectional images of the retina at very high resolution. Two other methods, infrared (IR) and fundus autofluorescence (FAF), enable the assessment of retinal degeneration status. IR imaging enables the detection of retinal pathology, especially specific features such as intraretinal fluid, chorioretinal atrophy, and retinal pigment epithelial tears. FAF imaging focuses on the fluorescent properties of the pigment in the retina and enables the detection of retinal pathology, especially monitoring debris accumulation in the RPE, which can eventually lead to photoreceptor degeneration.

[0226] Experiments similar to those in Example 4 were performed using vectors encoding mouse Fam161a isoforms (see Vectors). In one set of experiments, Fam161a - / - Mice were treated with AAV2 / 8-IRBP-GRK1-mFam161a-L-WPRE (3) at PN31, and one eye was treated with 0.5E11 VG and 0.001% pluronic acid (single injection, or two injections to cover the maximum retinal area), while the other eye served as an untreated control. Retinal activity was assessed by electroretinographic (ERG) recordings using various stimuli of increasing light intensity. FIG. 6A to FIG. 6D Examples are provided in . ERG recordings were collected at 2, 3.5, 5, and 8 months after injection. In mice, the maximum B-wave ERG response was well maintained at 3 months after injection compared to untreated mice (dark bars), and this trend continued until at least 8 months after injection. The response was slightly reduced due to degeneration of the untreated area (Figures 6 and 7). Visual acuity was significantly improved at 8 months after injection ( Fig. 6FOptical coherence tomography (OCT) images to evaluate the in vivo retinal structure were taken at 3, 6, and 8 months after injection (4, 7, and 9 months of age, respectively) and revealed that the retinal ONL (black band pointed by white arrows) was well preserved in the vector-treated area, while a significant reduction in thickness was observed in the untreated area ( Figure 5 ).

[0227] Similarly, FAF images taken 2, 3.5, 5, and 7 months after injection showed evidence of well-preserved retina in the injected eyes, whereas the uninjected eyes showed fundoscopic findings typical of retinal degeneration ( Figure 4 ).

[0228] In conclusion, after a single injection of AAV2 / 8-IRBP-GRK1-mFam161a-L, Fam161a - / - Gene enhancement therapy in mouse models showed improved retinal function and structure.

[0229] Example 4: Generation of different AAV2 / 8 vectors with IRBP-GRK1 and human FAM161a isoforms and expression in vivo in rod and cone photoreceptors.

[0230] Two different AAV2 / 8 vectors were generated as described, encoding either the long or short isoform of human FAM161A. Subsequently, the expression and function of the FAM161A protein in rod and cone photoreceptors were observed.

[0231] Materials and methods

[0232] Vectors: As described above, two different AAV2 / 8 vectors were produced using plasmids (1)-SEQ ID NO:16, (2)-SEQ ID NO:17, which encode the long or short isoform of the human FAM161A gene (hFAM161A-L (HL) or hFAM161A-S (HS)) and contain the GRK1 promoter in the presence of the IRBP enhancer and the RNA stabilizer WPRE ( Figure 3A , Figure 3B ).

[0233] Subretinal injection: The vector was injected subretinally into the retina of Fam161a-deficient mice at a dose of 1E10 vector genomes (VG) in 1 μL of PBS + 0.001% Pluronic on postnatal day 7 or 14 (PN7 or PN14). - / -(as described in Beryozkin et al., 2021, supra). The therapeutic vector (1) or (2) was injected into one eye, while the contralateral side was treated with a control vector with a GFP transgene (and the same regulatory elements, i.e., AAV-GFP; 1E10 VG) or left untreated.

[0234] In another experiment, therapeutic vectors (1) and (2) were mixed 1:1 in 0.001% pluronic acid solution and injected at a total dose of 1E10 VG at PN 14. Some fellow eyes were injected with AAV-GFP (at a dose of 1E10 VG at PN 14) and used as controls.

[0235] Electroretinogram responses: Electroretinograms (ERGs) were monitored at different time points after vector injection (i.e., 1, 2, or 3 months post-injection (pi)), followed by immunochemical analysis of FAM161A expression in the retina and assessment of retinal integrity. The stimulation protocol was similar to that used in Kircher et al. (2019, Frontiers in Neurol, 10:56). Briefly, animals were dark-adapted overnight and anesthetized with a mixture of ketamine (100 mg / kg) and xylazine (15 mg / kg), and pupils were dilated by topical administration of 0.5% tropicamide, followed by recording retinal activity in response to light flashes of 0.0001 cds / m2 to 30 cds / m2 for scotopic ERGs, and after 5 minutes of light adaptation, photopic ERGs of 1 cds / m2 to 30 cds / m2 were recorded.

[0236] Immunohistological staining: Eyeballs were enucleated, fixed in 4% paraformaldehyde for 30 min at 4°C, rinsed with PBS and incubated in 10%, 20% and 30% sucrose for 2 h to overnight at 4°C. After embedding in egg white albumin (Fluka, Buchs, Switzerland), 14 μm cryosections were collected. Sections were incubated with antibodies raised against FAM161A (Sigma HPA032119, 1:1000) and acetylated tubulin (Sigma T7451, 1:1000) at 4°C overnight and then detected with goat anti-rabbit-Alexa Fluor® 488 and goat anti-mouse-Alexa Fluor® 633 (Invitrogen, 1:2000).

[0237] Statistical analysis: Nonparametric two-tailed paired T tests (Wilcoxon rank test) were performed using Graphpad Prism software to assess significant differences in b-wave amplitude between delivery of AAV-GFP and HL (1) or AAV-GFP and HS (2) at specific time points after injection. For some experiments, data were also obtained at 4 and 8 months after injection.

[0238] Results: Fam161a cells injected with AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE (1), AAV2 / 8-IRBP-GRK1-hFAM161A-S-WPRE (2), or AAV-GFP vector - / - In mice, ERG recordings at 3 months post-injection did not reveal significant improvements in retinal activity in retinas treated with AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE (1) or AAV2 / 8-IRBP-GRK1-hFAM161A-S-WPRE (2). Nevertheless, the HL vector (1) produced a small but significant protection of ERG responses, but with higher changes at 2 and 3 months post-injection compared to control eyes (Figure 8) (maximum b-wave response increased by +4% - +178%, p=0.0039). Histological examination of FAM161A expression in treated retinas at 3 months post-injection revealed strong expression of the protein throughout the cilia and cell bodies, whereas this pattern was not observed in WT retinas, suggesting that overexpression of the protein is not controlled ( Fig.10 ). For combined injections, the distribution and expression of FAM161A were the same as Fig.10 These results indicate that, despite an imperfect expression pattern, short-term rescue can be observed with AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE (1).

[0239] Still in Fam161a - / - Combinations of AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE (1) and AAV2 / 8-IRBP-GRK1-hFAM161A-S-WPRE (2) vectors were tested in the retina. The control group received AAV-GFP. At 2 months post-injection, ERG responses were significantly preserved in treated animals, corresponding to +23.9% (p=0.0039) of controls that received 10 cd*s / m2 of the GFP transgene ( Fig. 9B ), but 1 month after injection ( Fig.9A ) and 3 months ( Fig. 9C ). It is speculated that the transient rescue observed with the mixture of AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE (1) and AAV2 / 8-IRBP-GRK1-hFAM161A-S-WPRE (2) vectors is due to the use of human sequences in mice (see Example 4).

[0240] Subsequent histological analysis showed that compared with the uninjected areas of the same eyes ( Fig.10 D- Fig.10 F) Injected areas with the human short or long isoforms showed thicker retina, i.e., outer nuclear layer (ONL) thickness was 38.69 ± 3.82 μm versus 17.16 ± 1.18 μm, respectively. FAM161A expression was detected in segments and cell bodies of photoreceptors and was not restricted to the CC as expected ( Fig.10 C). Thus, the treatment partially reestablished the normal structure of photoreceptor cells, effectively protecting them from degeneration and temporarily preserving their function. However, ectopic expression of FAM161A led to uncontrolled growth of tubulin, which could affect cell function.

[0241] These vectors do not appear to be optimal for the recovery of Fam161a - / - Intact structure and function in mice. Nevertheless, since there is only 58% sequence similarity between mouse and human FAM161A protein sequences, it cannot be excluded that human FAM161A will not be effective in restoring mouse retinal function. Indeed, experiments performed with a vector encoding the long isoform of mouse Fam161a provided better rescue and support the fact that the use of the FAM161A human sequence in humans will be effective.

[0242] Example 5: Generation of plasmids with FCBR1-F0.4 or FCBR2-F0.4.

[0243] Endogenous regulatory sequences of the hFAM161A gene were used to engineer a specific photoreceptor promoter.

[0244] Materials and Methods: Two Crx binding regions (FCBR1 and FCBR2) and the core promoter region (F0.4) from the human gene were amplified by PCR and fused to construct AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE ((10) SEQ ID NO:25), AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE ((11) SEQ ID NO:26), AAV2 / 8-FCBR2-F0.4-hFAM161A-L-WPRE ((6) SEQ ID NO:21), AAV2 / 8-FCBR2-F0.4-hFAM161A-S-WPRE ((7) SEQ ID NO:22) and their equivalents of mouse L and L1 isoforms (8, 9, 12, 13 - SEQ ID NOs:23, 24, 27 and 28, respectively).

[0245] Results: The graphs of AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE (10) and AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE (11) are shown in Figures 1 and 2. Figure 3C and 3D shown.

[0246] Example 6: Generation of vectors with FCBR1-F0.4 or FCBR2-F0.4 and FAM161A isoforms and in vivo testing.

[0247] Several different AAV2 / 8 vectors encoding the human FAM161A gene or the mouse Fam161a gene, including the FCBR1-F0.4 or FCBR2-F0.4 promoter and the RNA stabilizer WPRE were compared. Subsequently, the expression and function of the FAM161A protein in rod and cone photoreceptors were observed and compared.

[0248] Materials and methods: The vector was obtained as described above.

[0249] The vector was injected subretinal into the retina of Fam161a-deficient mice on postnatal day 14 to 24. - / - (As described in Beryozkin et al., 2021, supra). Animals received the vector (6) AAV2 / 8-FCBR2-F0.4-hFAM161A-L-WPRE, (7) AAV2 / 8-FCBR2-F0.4-hFAM161A-S-WPRE, (10) AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE, or (11) AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE in one eye and the AAV2 / 8-GFP vector in the contralateral eye.

[0250] Electroretinogram responses: Obtained as described above.

[0251] FAM161A immunohistological staining: obtained as described above.

[0252] Results: Two months after injection, eyes injected with vectors containing FCBR2 regulatory elements (6 or 7) showed a significant increase in Fam161a compared with control eyes. - / - Mice showed a significant preservation of ERG responses in 3 of 11 animals (maximal amplitudes in this group were +25% to +156% higher compared with control eyes (P = 0.0078)), whereas responses were reduced in the other eyes (Table 1). In several eyes, histological examination revealed that FAM161A expression was abundant in photoreceptor cells, from cilia to bipolar synapses. In addition, several interneurons and retinal ganglion cells expressed long FAM161A filaments, including in the optic nerve. This abnormal pattern of FAM161A expression led to the failure to restore photoreceptor function in most cases, despite preservation of photoreceptor cells as measured by ONL thickness.

[0253] Compared with eyes injected with AAV-GFP, retinas of mice injected with the vector AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE (10) showed smaller but better b-wave amplitudes in response to several stimuli at 3 months post-injection (improvements of +31% to +87%, p = 0.0039, Table 1, Fig.11A Improved ERG responses were also observed in mice treated with AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE (11) compared to control eyes of the same animals (increase of +14% to +100%, p=0.0039, Table 1, Fig. 11B Histological examination revealed discrete expression of FAM161A in the CC of the HL group similar to healthy retinas ( Fig. 12A ), some cells have a small number of long thin filaments extending along the entire photoreceptor. The HS group showed more ectopic expression of FAM161a. It has been previously shown that at PN7, cilia in Fami161aKO photoreceptors are already altered and open like leakiness. Cilia can be reconstructed using FCRB1-F0.4-vector when injected at PN14.

[0254] Thus, the FCBR1-F0.4 vector was shown to restore physiological FAM161A expression, retinal structure, and prolong the physiological function of photoreceptors. In general, for the same dose, the FCBR2-F0.4 vector caused high expression of FAM161A protein, which exceeded the connecting ciliary structure and extended to the ectopic cell area, forming an abnormal network. This phenotype was associated with reduced vector efficacy compared with the FCRB1-F0.4 vector. FCBR1-F0.4-hFAM161A-L-WPRE and FCBR1-F0.4-hFAM161A-S-WPRE seemed most promising to change the course of FAM161A to delay photoreceptor loss and prolong retinal activity.

[0255] Co-injection of AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE and AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE resulted in a significant rescue of ERG responses at 3 months post-injection (3 mpi) compared to control eyes injected with AAV2 / 8-FCBR1-F0.4-GFP-WPRE vector ( Fig. 11C Indeed, an improvement in retinal activity to low-light stimulation was observed, indicating preservation of rod function, and maximal b-wave amplitudes (indicative of rod and cone responses) were 57% to 340% higher compared to control eyes (p=0.002).

[0256] Histological analysis showed that the treated area was preferentially positive for FAM161a in the connecting cilia, and some photoreceptors close to the injected area were also positive in the inner segments and cell bodies.

[0257] This series of experiments demonstrated the value of AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE and AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE vectors and mixtures of AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE (1) and AAV2 / 8-IRBP-GRK1-hFAM161A-S-WPRE (2) for maintaining retinal viability and prolonging retinal cell survival ( Fig. 12C and Fig.12F ).

[0258] Given the efficacy of the FCBR1-F04 vector expressing the hFAM161A isoform in restoring retinal function, targeting Fam161a - / - Mice were tested with AAV2 / 8-FCBR1-F0.4-mouse-L-Fam161a-WPRE. Analysis was performed at 8 mpi. At this age, GFP control treated eyes showed only very weak responses to light stimulation ( Fig.13A ), while eyes treated with a single or double injection showed a significant rescue of retinal activity (+200% to +300%). Histological analysis showed that the treated area still contained several rows of photoreceptors, while the ONL contained zero to one row of photoreceptors in the untreated area ( Fig. 13B ). Targeting Fam161a - / - Mice were tested with the vectors detailed in Table 1 , summarizing protein expression and localization as well as ERG recordings.

[0259] Targeting Fam161a - / - The AAV2 / 8-FCBR2-F0.4 vector was tested in mice (i.e., (6), (7), (8)), and strong expression was observed, with localization of the FAM161A protein beyond the connecting cilium and overexpression into the photoreceptor cell bodies (Table 1). In contrast, the use of the AAV2 / 8-FCBR1-F0.4 vector (i.e., (10), (11), (12), (13)) resulted in good and specific localization of the protein in the connecting cilium (Table 1, Fig. 12B -C).

[0260] Table 1

[0261]

[0262]

[0263] Table 1: Summary of vector efficacy. Note that the vector with the FCBR1-F0.4 sequence enabled FAM161a-specific localization and improved retinal viability after 3 m.pi. The AAV-IRBP-GRK1 vector encoding the long mouse isoform also rescued Fam161a - / - retinal activity, and FAM161A protein was mainly localized in cilia and inner segments.

[0264] The literature (Langmann et al., Am J Hum Genet 2010) suggested that the CBR2 sequence and the short FAM161A isoform would be the most promising combination to induce adequate expression of FAM161A due to the activity of Crx box-2 (CBR2) and the abundance of the short isoform in the mouse retina, but the above results contradict this hypothesis. Indeed, surprisingly, in vivo vector efficacy testing showed that the FCBR1-F0.4 promoter drove more adequate expression of the FAM161A isoform in the retina than FCBR2-F0.4, which mimics the endogenous expression of this gene in photoreceptors. Indeed, the FAM161A protein was correctly localized in the connecting cilium and did not extend its expression throughout the cell, as observed with the FCBR2-F0.4 promoter.

[0265] The human FAM161A gene produces two different mRNA isoforms in the retina, and the current literature does not provide any guidance as to whether one or both isoforms are essential for retinal structural preservation and functional rescue. Surprisingly, in vivo vector efficacy testing showed that the FCBR1-F0.4 promoter drives sufficient expression of both FAM161A isoforms in the retina. Therefore, both isoforms can be used alone or in combination to ensure retinal activity and survival of photoreceptors in patients affected by FAM161A deficiency.

[0266] It has been described that fine-tuned expression of microtubule-associated proteins such as FAM161A is required in order not to interfere with the cytoskeleton which could affect general cellular trafficking. As photoreceptor function is highly dependent on intracellular trafficking of proteins to the outer segments, insufficient FAM161A expression could negatively affect vision. Surprisingly, in vivo vector efficacy testing showed that the FCBR1-F0.4 promoter drives sufficient expression of both FAM161A isoforms in photoreceptor connecting cilia. Therefore, the use of either isoform could be used to maintain or remodel cilia in RP28 patients. The efficacy of gene therapy is highly dependent on the accurate expression of the therapeutic gene. Therefore, mimicking the endogenous expression pattern is essential to correctly address cellular function. This is even more important for ciliary proteins, which when overexpressed can lead to adverse cellular effects such as "paralysis" of the cytoskeleton. It is shown that it is crucial to correctly target FAM161A expression to the connecting cilium using the FCBR1-F0.4FAM161A promoter and its properties. Therefore, use of the FCBR1-F0.4 FAM161A promoter can be used for any other gene transfer aimed at restoring the normal structure of photoreceptor cilia.

[0267] The Fam161a sequence was used to validate the described gene therapy approach in a Fam161a-deficient mouse model. Given the limited homology between mouse and human protein sequences (58%), examining the mouse sequence in a mouse model is more relevant for assessing gene therapy success than using human sequence genes in a mouse model. However, despite this limitation, administration of AAV-IRBP-GKR1-HL-W (1), AAV-FCBR1-F0.4-HL-W (10), AAV-FCBR1-F0.4-HS-W (11), AAV-FCBR2-F0.4-HS-W (7), and a mixture of AAV-FCBR1-F0.4-HL-W (10) and AAV-FCBR1-F0.4-HS-W (11) showed improved photoreceptor survival and varying levels of increases in retinal activity in the mouse model. Additionally, the success of cell survival and functional maintenance of mouse Fam161a encoding AAV vectors allows anticipation of the success of treating RP28 patients with human FAM161a encoding AAV vectors.

[0268] By combining appropriate promoters and FAM161A isoform sequences, vectors with different potentials are provided herein to maintain retinal integrity and / or function. In particular, AAV2 / 8-IRBP-GRK1-hFAM161A-L-WPRE and AAV2 / 8-FCBR1-F0.4-hFAM161A-L-WPRE and AAV2 / 8-FCBR1-F0.4-hFAM161A-S-WPRE have been shown to be effective vectors.

Claims

1. A vector comprising a FCBR1-F0.4 promoter and a nucleic acid sequence encoding a cilia protein or a fragment or variant thereof.

2. A vector comprising an IRBP-GRK1 promoter and a nucleic acid sequence encoding a ciliary protein or a fragment or variant thereof.

3. The carrier according to claim 1, wherein The FCBR1-F0.4 promoter is SEQ ID NO: 10 or SEQ ID NO:

66.

4. The carrier according to claim 2, wherein The IRBP-GRK1 promoter is SEQ ID NO:

14.

5. The carrier according to any one of claims 1 to 4, wherein The nucleic acid sequence encoding the ciliary protein is selected from the long isoform of the human FAM161A gene of SEQ ID NO: 1 and the short isoform of the human FAM161A gene of SEQ ID NO: 2, or a fragment or variant thereof.

6. The carrier according to any one of claims 1 to 4, wherein The nucleic acid sequence encoding the ciliary protein is selected from the group consisting of the POC5 gene of SEQ ID NO: 29, the NPHP4 gene of SEQ ID NO: 30, the ARL6 gene of SEQ ID NO: 31, the BBS1 gene of SEQ ID NO: 32, the BBS2 gene of SEQ ID NO: 33, the BBS9 gene of SEQ ID NO: 34, the PCARE gene of SEQ ID NO: 35, the CFAP418 gene of SEQ ID NO: 36, the CEP164 gene of SEQ ID NO: 37 (CEP164 isoform 1) or SEQ ID NO: 38 (CEP164 isoform 2), the CEP290 gene of SEQ ID NO: 39, the CLRN1 gene of SEQ ID NO: 40, the IFT140 gene of SEQ ID NO: 41, the IFT172 gene of SEQ ID NO: 42, the IQCB1 gene of SEQ ID NO: 43, the KIZ gene of SEQ ID NO: 44, the LCA5 gene of SEQ ID NO: 45, the CLRN1 gene of SEQ ID NO: 46, the IFT140 gene of SEQ ID NO: 47, the IFT172 gene of SEQ ID NO: 48, the IQCB1 gene of SEQ ID NO: 49, the KIZ gene of SEQ ID NO: 50, the LCA5 gene of SEQ ID NO: 51, the CLRN1 gene of SEQ ID NO: 52 NO:46, the MAK gene, the NEK2 gene, the OFD1 gene, the OFD1 gene, the RP1 gene, the RP1L1 gene, the RP2 gene, the RPGR gene, the RPGR gene, the RPGR gene, the RPGRIP1 gene, the RPGRIP1 gene, the SPATA7 gene, the TOPORS gene, the TTC8 gene, the USH2A gene, the WDR19 gene, the CFAP410 gene, or a fragment or variant thereof.

7. The vector according to any one of claims 1 to 6, further comprising a WPRE (Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element) post-transcriptional regulatory element of SEQ ID NO: 15, or a fragment or variant thereof.

8. The vector according to any one of claims 1 to 7, which is a recombinant AAV vector having an AAV2 / 8 capsid protein or an AAV2 / 5 capsid protein.

9. A pharmaceutical composition comprising at least one carrier according to any one of claims 1 to 8 or a combination thereof and at least one pharmaceutically acceptable carrier.

10. Use of the carrier according to any one of claims 1 to 8 as a medicament.

11. Use of a vector according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 for modifying, preventing, delaying, arresting the progression or ameliorating vision loss associated with retinal ciliopathy.

12. Use of a vector according to any one of claims 1 to 8 in the treatment of retinal ciliopathy.

13. Use of the carrier according to claim 11 or 12, wherein, The retinal ciliopathy is a FAM161A-related retinopathy.

14. Use of the carrier according to claim 11 or 13, wherein The retinal ciliary disease is retinitis pigmentosa28.

15. Use of the carrier according to any one of claims 1 to 8 for improving or maintaining the structure of a photoreceptor.

16. Use of the vector according to any one of claims 1 to 8 for inducing stabilization of photoreceptor ciliary proteins.

Images