阅读说明：本技术 用于眼部病症的基因疗法 (Gene therapy for ocular disorders ) 是由 琼·贝内特 珍妮特·班尼切利 孙俊伟 宋知伦 塞奇·尼科诺夫 于 2018-03-01 设计创作，主要内容包括：本发明提供了用于治疗受试者的莱伯氏先天性黑蒙症(LCA)的组合物和方法。一方面,提供了重组腺相关病毒载体,其包括包含编码Lebercilin的序列的核酸分子。另一方面,Lebercilin具有氨基酸序列SEQ ID NO:1。又一方面,所述核酸分子具有序列SEQ ID NO:3或其变体。在所需实施方案中,所述受试者为人、猫、狗、绵羊或非人灵长类动物。(The present invention provides compositions and methods for treating Leber Congenital Amaurosis (LCA) in a subject. In one aspect, a recombinant adeno-associated viral vector is provided, which includes a nucleic acid molecule comprising a sequence encoding lebrcilin. In another aspect, Lebercilin has the amino acid sequence SEQ ID NO 1. In yet another aspect, the nucleic acid molecule has the sequence SEQ ID NO 3 or a variant thereof. In a desired embodiment, the subject is a human, cat, dog, sheep, or a non-human primate.)

1. A codon optimized, engineered nucleic acid sequence of SEQ ID NO 3 encoding human Lebercilin.

2. An expression cassette comprising a codon optimized nucleic acid sequence of SEQ ID NO 3 encoding human Lebercilin.

3. A recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising:

(a) AAV 5' Inverted Terminal Repeat (ITR) sequences;

(b) A promoter;

(c) A coding sequence encoding human Lebercilin;

(d)AAV 3'ITR。

4. The rAAV of claim 3, wherein the coding sequence of (c) is codon-optimized human LCA5 that is at least 70% identical to native human Lebercilin coding sequence SEQ ID NO 2.

5. The rAAV of claim 3 or 4, wherein the coding sequence of (c) is SEQ ID NO 3.

6. The rAAV of any one of claims 3 to 5, wherein the rAAV capsid is an AAV7m8 or variant thereof, an AAV8 capsid, an AAV6 capsid or variant thereof, an AAV9 capsid or variant thereof, an AAV7 capsid or variant thereof, an AAV5 capsid or variant thereof, an AAV2 capsid or variant thereof, an AAV1 capsid or variant thereof, an AAV3 capsid or variant thereof, or an AAV4 capsid or variant thereof.

7. The rAAV of any one of claims 3 to 6, wherein the promoter is a Cytomegalovirus (CMV) promoter or a hybrid promoter comprising a CMV promoter sequence and a Chicken Beta Actin (CBA) promoter sequence.

8. The rAAV of any one of claims 3 to 7, wherein the AAV5 'ITRs and/or AAV3' ITRs are from AAV 2.

9. The rAAV of any one of claims 3 to 8, wherein the vector genome further comprises polyA.

10. The rAAV according to any one of claims 3 to 9, further comprising an intron.

11. The rAAV according to any one of claims 3 to 10, further comprising an enhancer.

12. A composition comprising the rAAV of any one of claims 3-11 and a pharmaceutically acceptable carrier or excipient suitable for delivery to the eye.

13. An aqueous suspension suitable for administration to a LCA patient, said suspension comprising an aqueous suspending liquid and about 1x10 per eye¹⁰Virions of recombinant adeno-associated virus (rAAV) to about 1X10¹²A GC or virion, a rAAV suitable for use as a therapeutic agent for an LCA, the rAAV having an AAV capsid and having packaged therein a vector genome comprising:

(a) AAV 5' Inverted Terminal Repeat (ITR) sequences;

(b) A promoter;

(c) A coding sequence encoding human Lebercilin; and

(d)AAV 3'ITR。

14. The suspension of claim 13, wherein the suspension is suitable for subretinal or intravitreal injection.

15. The suspension of claim 13 or 14, wherein the promoter is SEQ ID No. 10 and the coding sequence encoded is SED ID No. 3.

16. The suspension according to any one of claims 13-15, wherein the rAAV capsid is an AAV7m8 capsid.

17. The suspension according to any of claims 13 to 16, further comprising CBA exon 1 and intron from nt 824 to nt1795 of SEQ ID No. 8.

18. Use of the rAAV according to any one of claims 3 to 11, the composition according to claim 12, or the suspension according to any one of claims 13 to 18 for treating a subject with LCA.

19. The use of claim 18, wherein the rAAV is in an aqueous suspension at about 1x10⁹To about 1X10¹³Individual vector genomes per eye (vg per eye) delivery.

20. The use of claim 18 or 19, wherein the rAAV is administered subretinally or intravitreally.

21. The use of any one of claims 18 to 20, wherein the rAAV is in a volume comprising about or at least 150 microliters of 1x10⁹To 1X10¹³Dose administration of individual rAAV, thereby restoring visual function to the subject.

22. A method of treating a subject having LCA with the rAAV according to any one of claims 3 to 11, the composition according to claim 12 or the suspension according to any one of claims 13 to 18.

23. The method of claim 22, wherein the rAAV is in aqueous suspension at about 1x10⁹To about 1X10¹³Individual vector genomes per eye (vg per eye) delivery.

24. The method of claim 22 or 23, wherein the rAAV is administered subretinally or intravitreally.

25. The method of any one of claims 22-24, wherein the rAAV is in a volume comprising about or at least 150 microliters of 1x10⁹To 1X10¹³Dose administration of individual rAAV, thereby restoring visual function to the subject.

Background

Leber's congenital amaurosis (LCA; OMIM 204000) is one of the most serious inherited blinding diseases. LCA is rare, occurring in 1:50,000 individuals, is usually inherited in an autosomal recessive manner, and can be caused by mutations in any of at least 22 different genes (sph. Clinical features include severe abnormalities of vision (decreased vision, diminished visual field) in infancy or early childhood, nystagmus, and progressive loss of poor vision occurring early in life. Clinical trials revealed disappearing dark and bright adaptation to Electroretinogram (ERG) responses, dark pupils, decreased light sensitivity, and pigment changes in the retina. There is currently no approved treatment for LCA.

The form of LCA caused by mutations in the retinal pigment epithelium 65kDa protein-encoding gene RPE65 (Redmond TM, Yu S, Lee E, Bok D, Hamasaki D, Chen N et al Rpe65is processing for production of11-cis-vitamin A in the recombinant visual cycle Nat Genet (1998)20(4): 344-51; Redmond T, Hamel C.genetic analysis of RPE65: from human disease to patients in Enzymol (2000)317:705-24, all of which are incorporated herein by reference) has gained much interest in recent years as it has become a target for clinical trials for enhanced therapies. By using adeno-associated virus (AAV) serotype 2, several groups have demonstrated that it is safe to deliver wild-type copies of the RPE65cDNA to the Retinal Pigment Epithelium (RPE), and this can reverse many of the drawbacks including night blindness. (3-9) random, multicenter phase 3 studies (or voretigene neuropravec, sponsored by Spark Therapeutics, Philadelphia, PA) testing AAV2-hRPE65v2 have demonstrated that subretinal injection of this agent results in an improvement in photosensitivity, visual field, and ability to navigate accurately and rapidly using visual cues even under certain luminance conditions. (10,11) the U.S. Food and Drug Administration (FDA) approved Voretigene neuropivvec-rzyl drugs on 19.12.2017, making it one of the first approved gene therapy drugs in the united states. Progress in developing treatments for LCA caused by RPE65 mutant LCA2 paved the way to develop treatments for other forms of early onset retinal degeneration, most of which are caused by mutations in photoreceptor-specific genes and not only RPE-specific genes.

One of the most severe forms of this already severe pathology (LCA) is caused by a mutation in the photoreceptor-specific gene LCA5 encoding Lebercilin (12-20). It is estimated that the LCA5 mutation accounts for approximately 2% of LCA cases, although it may be more prevalent in genomically isolated populations. (16) LCA5 mutations are also thought to be responsible for other early onset forms of retinal degeneration including cone dystrophy and autosomal cryptopigmented retinitis (ARRP). (15, 16, 21)

Thus, there is a need for compositions suitable for expressing lebrcilin in a subject in need thereof.

Disclosure of Invention

Embodiments described herein relate to compositions and methods relating to AAV gene therapy vectors for delivering human LCA5 to a subject in need thereof that produce long-term, potentially 10 years or more, clinically meaningful correction of Leber Congenital Amaurosis (LCA) following intravitreal or subretinal administration of the vector.

In one aspect, a codon optimized, engineered nucleic acid sequence encoding human Lebercilin is provided. In one embodiment, the codon-optimized nucleic acid sequence is a variant of SEQ ID NO. 3 or SEQ ID NO. 2. In another embodiment, the codon optimized nucleic acid sequence is SEQ ID NO 3. In another embodiment, the nucleic acid sequence is codon optimized for expression in humans.

In another aspect, an expression cassette is provided comprising a codon-optimized nucleic acid sequence encoding Lebercilin. In one embodiment, the expression cassette comprises the nucleic acid sequence SEQ ID NO 3 encoding human Lebercilin. In other embodiments, the lebrcilin coding sequence is located between the 5 'and 3' AAV ITR sequences.

In another aspect, a recombinant adeno-associated virus (rAAV) vector is provided. The rAAV comprises an AAV capsid and a vector genome packaged therein. In one embodiment, the vector genome comprises: (a) AAV 5' Inverted Terminal Repeat (ITR) sequences; (b) a promoter; (c) a coding sequence encoding human Lebercilin; and (d) AAV3' ITRs. In one embodiment, the rAAV vector further comprises expression control sequences that direct expression of lebrcilin in a host cell. In other embodiments, the Lebercilin sequence is the protein sequence SEQ ID NO 1. In one embodiment, the vector genome is the sequence of nt 1-4379 of SEQ ID NO. 8. In another embodiment, the vector genome is the sequence of nt 1-4368 of SEQ ID NO 9. In yet another embodiment, the LCA5 coding sequence in any one of the identified vector genomes is exchanged with another LCA5 coding sequence as described herein.

In another aspect, an aqueous solution suitable for administration to an LCA patient is providedAnd (4) suspending. In one embodiment, the suspension comprises an aqueous suspending liquid and about 1 × 10 per eye¹⁰GC or virions of a recombinant adeno-associated virus (rAAV) suitable for use as an LCA therapeutic as described herein to about 1x10¹³GC or virus particles.

In another aspect, a pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, excipient, and/or adjuvant and a nucleic acid sequence, plasmid, vector, or viral vector, such as a rAAV as specifically described herein.

In another aspect, a method for treating Lebercilin congenital amaurosis and/or restoring visual function caused by a defect in the Lebercilin gene (LCA5) in a mammalian subject suffering from LCA comprises delivering a recombinant AAV vector encoding Lebercilin as described herein to a subject in need thereof via intravitreal, subretinal, or intravascular injection.

In another aspect, there is provided use of an AAV vector as described herein in the treatment of lebercilin congenital amaurosis and/or restoration of visual function caused by a defect in the lebercilin gene (LCA5) in a mammalian subject having LCA. The use comprises delivering a recombinant AAV vector encoding lebrcilin as described herein via intravitreal, subretinal, or intravascular injection to a subject in need thereof.

Other aspects and advantages of the invention will become readily apparent from the following detailed description of the invention.

Drawings

Figure 1A shows the transgene cassettes for the production of aav7m8.cba.hopt.lca5 and aav7m8.cba.egfp as described herein.

FIGS. 1B-1D provide an alignment of the human nucleic acid sequence SEQ ID NO 2 of LCA5 (native _ LCA5) with the codon optimized LCA5 (codon optimized _ LCA5) sequence SEQ ID NO 3.

Figures 1E-1F provide plasmid maps and feature lists of paav.cmv.cba. human codon optimized lebrcilin vector. The nucleic acid sequence is reproduced in SEQ ID NO. 8.

FIGS. 1G-1H provide an immunofluorescence analysis of eyes injected with AAV7m 8-hop-LCA 5 at P5 and analyzed at P15, showing the co-localization of Lebercilin with the base of the tubulin positive outer segment as described in examples 1,2 and 4. After intravitreal injection (IVT, fig. 1G) or subretinal injection (SR, fig. 1L), Lebercilin was distributed throughout the retina, which was barely photoreceptor at P95. In contrast, lebercilin was not present in untreated P15 and P95Lca 5-/-retinas. SR: subretinal; IVT: in the vitreous body; (-) untreated Lca 5-/-. A sketch showing an intravitreal injection protocol (fig. 1G) and a subretinal injection protocol (fig. 1H) is provided.

Figure 1I shows Lebercilin expression in Lca 5-/-mice subretinally injected with aav.lca5 vector in wild type mice and P20.

Figures 2A-2I show normalized pupillary reflex amplitudes measured at 3 months of age in aav7m8. hop-Lca 5 injected mice compared to control (sham injected) eyes in Lca 5-/-mice treated at PN5 and PN 15. Results are shown in (a) intravitreal and (B) after subretinal injection or (C) in untreated (-) control mice. (D) The relative pupillary reflex amplitude (% of baseline pupil diameter) of the right eye of the animals in each subgroup shown in (a-C) plus wild type (C57B16) positive (+) control mice is shown graphically (E). Fig. 2F and 2G are representations of test protocols used to produce the results shown in fig. 2A-2D. Figures 2H and 2I show a comparison of normalized pupillary reflex amplitude in the experimental and control mice shown in figures 2A-2D treated via subretinal (SR, I) or intravitreal (IV, H) injection at PN5 versus PN 15. *. p < 0.1; p < 0.05; p < 0.01.

figures 3A-3F show the water maze test results in the eyes of comparative control (sham-injected) injected with aav7m8. hop-Lca 5 of Lca 5-/-mice treated at PN5 and PN15 and measured at 3 months of age as described in example 3. Fig. 3A is a table showing the results of statistical analysis.

Fig. 3B is a graphical representation of representative results of the water maze test. Figure 3D is a bar graph showing the number of days before Lca 5-/-mice were first successful when trained on intravitreal or subretinal treatment of PN5 or PN15 with aav7m8.Lca5 vector at birth. Wild type mice were provided as controls. Figure 3D is a line graph of the success rate of Lca 5-/-mice at PN5 treated intravitreally with aav7m8.Lca5 vector at birth at various light intensities (x-axis). Figure 3E is a line graph of Lca 5-/-mice success rate at various light intensities (x-axis) at PN5 treated subretinally with aav7m8.Lca5 vector at birth. Figure 3F is a line graph of the success rate of Lca 5-/-mice at PN15 treated intravitreally with aav7m8.Lca5 vector at birth at various light intensities (x-axis).

Figure 4 is a graph providing representative histological results after delivery of aav7m8. hop-LCA 5 to the LCA5gt/gt retina at Postnatal (PN)5 days. The figure shows the number of rows of Outer Nuclear Layer (ONL) versus sham injections after IVT or SR treatment with aav.

Figure 5A provides immunofluorescence results showing the continued presence of rhodopsin (red) in treated (but not control untreated) photoreceptors by the 3 month time point. Occasional photoreceptor cells were eGFP positive (injected area identified by co-injection with aav7m8. eGFP).

Fig. 5B-5D provide multi-electrode array (MEA) responses of rods and cones from Lca5gt/gt aav7m8. hop-Lca 5 treatment similar to those of Wild Type (WT) retina. (B) The reaction amplitude (difference in discharge rate before and after flash occurrence) measured from each flash mean trace (not shown) is compared to the flash intensity data. Responses from treated retinas were up to 70% of responses from WT retinas; untreated retinas present minimal to abrogated responses. (C) The magnitude of the response of group a retinas from the first and second round intensity series (stimulation series intensities increased from dark to brightest adaptation values in increments of approximately 0.5log during each round of intensity series before/during and after the brightest exposure at the end of the first round). (D) The amplitude of the instantaneous ON- (difference in discharge rate before and after flash occurrence), the sustained ON- (difference before flash occurrence and shift) and the OFF-response (difference before and after flash shift) varies with the flash intensity of the first and second series of intensity rounds. The light blue shaded area indicates the range of WT responses (4 retinas, MEAN + -STD), with only the wired traces giving the average WT response amplitude. The horizontal arrow indicates a rightward shift in sensitivity due to exposure to the brightest flash at the end of the first round of intensity series. Treated Lca5gt/gt and WT retinas showed similar responses/intensity dependence before and after bleaching, whereas untreated Lca5gt/gt retinal responses were flattened. The circles connected by lines represent the LCA5gt/gt treated. The triangles connected by lines represent the control LCA5 gt/gt.

Fig. 6A-6E show the presence of massive cell death during photoreceptor degeneration early in life (and the delay in this degeneration after treatment with aav.hop.lca5) in untreated Lca 5-/-retinas as evidenced by (a) TUNEL assay (fig. 6A; fig. 6B-E, third row) and (B) rhodopsin immunofluorescence analysis (fig. 6B-E, lines 1, 3 and 4).

Figures 7A-7D show that the outer segment is present in aav7m8.hop. Lca5 treated Lca 5-/-retina but not in the control Lca 5-/-retina. Transmission electron microscopy evaluation of retinas injected with aav7m8.hop. Lca5 revealed rod and cone photoreceptor outer segments with stacked discs and connecting cilia in 3-month-old Lca 5-/-. This structure is not present in untreated Lca 5-/-retinas. (a, B) PN80 after intravitreal injection with AAV7m8p643 (codon optimized Lebercilin) at PN5, representative panels; (A1-A3): resolution 12K, collage pictures showing rows of photoreceptor cells, rod cells (arrowhead), cone cells (arrowhead), many mitochondria of photoreceptor cells (asterisks); (B) resolution 20K; (B1) the cilia cross-section shows the 9+0 structure of the microtubules, the membrane disc from the rod cell's OS (arrows); (C) resolution 30K, substrate (arrow); (C1) a sagittal section connecting the cilia; (D) resolution 12K, photoreceptor cells were not left on the ONL; (D1) pycnotic nuclei of dead cells (arrowhead); (D2) floating debris of dead cell organelles, including rough ER (arrows).

Figure 8 shows the results of pupillary light responses of a retina of an adult male with LCA5 (red trace-top line) with similar temporal characteristics as an age-matched normal-vision male (blue trace-bottom line). The magnitude of the reduction in LCA5 patients was reduced compared to normal individuals.

Fig. 9 is a table of various retinal layer thicknesses showing that the Outer Nuclear Layer (ONL) thickness in the treated (.) retina remained thicker for at least 3 months compared to the control retina. There is no such clear trend in the other retinal layers (outer plexiform layer: OPL; inner nuclear layer: INL; inner plexiform layer: IPL). For each time point, the bar graphs from left to right represent the thicknesses of the ONL, OPL, INL and IPL, respectively.

Fig. 10 provides the results of light-mediated changes in the location of light transduction specific molecules following injection with aav7m8.hop.

FIGS. 11A-11B provide plasmid maps and feature lists of the pAAV.CMV.CBA. human native Lebercilin vector. The nucleic acid sequence is reproduced in SEQ ID NO 9.

Figures 12A-12F show cilium phenotypes rescued in homozygous human LCA5p. (Q279) iPSC-RPE following treatment with aav7m8. hop-LCA 5. (A) Confocal images show the hexagonal morphology of mature RPE cells and the immunofluorescent detectable RPE markers ZO-1 and MITF. Phase Contrast (PC) images show the construction of iPSC-RPE cultures derived from RPE of normal-sighted and LCA5 patients; (B) quantitative real-time PCR (qRT-PCR) of LCA5mRNA expression in normal vision control RPEs and RPEs from LCA5 patients. GAPDH was used to normalize expression levels. (C) Western blot analysis showed endogenous (×) lebrcilin proteins in untreated ("-") normal vision control cells or cells treated with aav7m8.egfp ("G"). There were no endogenous lebercilins in untreated or aav7m8. gfp-treated LCA5 affected cells. Robust levels of lebercilin were present after cell infection from normal vision and LCA5 individuals ("L") treated with aav7m8.LCA 5. Immunofluorescence analysis showed the presence of Arl13b positive primary cilia in normal vision (D) and LCA 5-derived (E) iPSC-RPE. Lebercilin was present in normal vision controls and LCA5 affected cells treated with aav7m8.LCA5 (but not aav7m8. egfp). (F) Quantitative analysis of the number of cilia per cell in normal vision versus LCA5-iPSC-RPE showed a rescue effect of cilia formation after treatment of LCA5-iPSC-RPE cells with aav7m8.LCA5 (instead of with aav7m8. egfp).

Detailed Description

The methods and compositions described herein relate to compositions and methods for delivering an LCA5 nucleic acid sequence encoding a Lebercilin protein to a subject in need thereof for treatment of Leber Congenital Amaurosis (LCA). In one embodiment, such compositions involve codon optimization of the Lebercilin coding sequence. It is desirable to improve the efficacy of the product and thus safety, since lower doses of the agent can be used. Also encompassed herein are compositions comprising the native Lebercilin coding sequence as shown in SEQ ID NO: 2.

Technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and refer to publications that provide those skilled in the art with a general guide to many of the terms used in this application. The definitions contained in this specification are provided for clarity in describing the components and compositions herein and are not intended to limit the claimed invention.

"Lebercilin" is encoded by the LCA5 gene on chromosome 6q14 and is a cilial protein that localizes to the connective cilia of photoreceptors as well as microtubules, centromeres and promilli of cultured mammalian cells.

Lebercilin is widely expressed during development and is found in cilia of cultured cells as well as the connective cilia of mature photoreceptor cells. The connecting cilia are narrow structures between the inner segment of the photoreceptor carrying the biosynthetic machinery of the cell and the outer segment containing the opsin-driven visual cascade. The attached cilia serve as a conduit supporting the bidirectional transport of proteins and vesicles along the ciliary microtubule track in a process called intraflagellar transport (IFT). By applying quantitative affinity proteomics to a genetically engineered Lca5 mouse model, Boldt et al demonstrated that loss of Lca5 function disrupts IFT, thereby causing defects in photoreceptor ectologic development and failure to inhibit protein and opsin trafficking. Lca5 null (Lca5gt/gt) mice lack cone and rod ERG responses and undergo early and progressive retinal degeneration in which only one row of scattered nuclei (in contrast to 8-10 rows of adjacent cells in the retina of wild type mice) is present in the Outer Nuclear Layer (ONL) by 2 months of age 19.

Mutations in LCA5 result in a genetic form of retinal degeneration known as Leber Congenital Amaurosis (LCA). The phenotype in the affected individual is confined to the eye and causes blindness. Of the 6 families studied by den Hollander, 5 had homozygous nonsense and frameshift mutations present and in one family the LCA5 transcript was completely absent. The nucleic acid encoding the Lebercilin cDNA or codon-optimized form thereof is of appropriate size to accommodate adeno-associated virus (AAV) vectors. See, e.g., the sequences in FIGS. 1A and 1E through 1F and FIGS. 11A-11B. As described in the examples below, using rAAV-mediated gene enhancement strategies, retinal degeneration caused by the LCA5 mutation was shown to be correctable. Such a therapy is particularly advantageous if the wild-type or optimized copy of the gene is delivered early in life (e.g., early in childhood or postnatal). Furthermore, in one embodiment, this intravitreal or subretinal administration used effectively provides the gene to the target cell (e.g., photoreceptor).

The Lebercilin gene LCA5, which encodes Lebercilin (i.e., a 697 amino acid protein), is thought to be involved in centrosome or ciliary function and negative terminal-directed microtubule transport. As used herein, the terms "LCA 5" and "Lebercilin" are used interchangeably when referring to a coding sequence. The natural nucleic acid sequence encoding human Lebercilin is reported in the NCBI reference sequences NM-181714.3 (transcript 1), NM-001122769.2 (transcript 2), XM-011535504.1 (transcript X1) and XM-005248665.4 (transcript X2) and is reproduced here in SEQ ID NO:4, 5,6 and 7, respectively. The natural human amino acid sequence of Lebercilin is reproduced here in SEQ ID NO:1(NCBI reference sequences: NP-001116241.1 or NP-859065.2 and UniProtKB/Swiss-Prot ID: Q86VQ 0-1). The LCA5 gene mutation is associated with Leber's Congenital Amaurosis (LCA). In certain embodiments, the terms "LCA 5" and "Lebercilin" are used interchangeably.

Leber's Congenital Amaurosis (LCA) is an eye disorder that primarily involves the retina, a special tissue that detects light and color at the back of the eye. People with this disorder often have severe visual impairment beginning in infancy. Visual impairment tends to stabilize, although it may deteriorate very slowly over time. Leber's congenital amaurosis is also associated with other vision problems, including increased sensitivity to light (photophobia), involuntary movements of the eye (nystagmus), and extreme hyperopia (hyperopia). Pupils, which typically dilate and constrict with the amount of light entering the eye, do not respond normally to light. Instead, they expand and contract more slowly than normal, or they do not react at all to light. Furthermore, the transparent covering (cornea) in front of the eye can be conical and extremely thin, a condition known as keratoconus. A particular behavior known as freund's finger-eye sign is characteristic of leber's congenital amaurosis. This sign consists of poking, pressing and rubbing the eyes with the knuckles or fingers. Researchers suspect that this behavior may lead to deep eye puffiness and keratoconus in the affected children. In a very few cases, developmental delays and intellectual impairment have been reported in people with characteristics of leber's congenital amaurosis. However, researchers are unable to determine whether these individuals are actually having leber's congenital amaurosis or another syndrome with similar signs and symptoms. At least 13 types of leber's congenital amaurosis have been described. These types differ in their genetic cause, pattern of vision loss, and associated ocular abnormalities.

The terms "percent (%) identity", "sequence identity", "percent sequence identity", or "percent identity" in the context of nucleic acid sequences refer to the residues at which two sequences that are identical align for identity. The length of the sequence identity comparison can be over the full-length genome, the full-length gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, as desired. However, identity in smaller fragments, e.g., at least about nine nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.

The percent identity of the amino acid sequence, polypeptide, about 32 amino acids, about 330 amino acids, or peptide fragments thereof, or corresponding nucleic acid sequence encoding sequences of a full-length protein can be readily determined. Suitable amino acid fragments may be at least about 8 amino acids in length, and may be up to about 700 amino acids in length. In general, when referring to "identity", "homology" or "similarity" between two different sequences, reference is made to "aligning" the sequences to determine "identity", "homology" or "similarity". "aligned" sequences or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences, which typically contain deletions or corrections for additional bases or amino acids as compared to a reference sequence.

Identity can be determined by preparing sequence alignments and by using various algorithms and/or computer programs known in the art or commercially available [ e.g., BLAST, ExPASy; ClustalO; FASTA; determined using, for example, Needleman-Wunsch algorithm, Smith-Waterman algorithm ]. The alignment is performed using any of a variety of publicly or commercially available multiple sequence alignment programs. For amino acid sequences, sequence alignment programs can be used, for example, the "Clustal Omega", "Clustal X", "MAP", "PIMA", "MSA", "BLOCKAKER", "MEME" and "Match-Box" programs. Typically, any of these programs are used under default settings, but those skilled in the art can change these settings as needed. Alternatively, one skilled in the art may utilize another algorithm or computer program that provides at least the level of identity or alignment provided by the reference algorithm and program. See, e.g., J.D.Thomson et al, Nucl.acids.Res., "Acomprehensive compliance of multiple sequence alignments", 27(13): 2682-.

multiple sequence alignment programs can also be used for nucleic acid sequences. Examples of such programs include "Clustal Omega", "Clustal W", "CAP Sequence Assembly", "BLAST", "MAP" and "MEME", which are available through Web servers on the Internet. Other sources of such procedures are known to those skilled in the art. Alternatively, the carrier NTI effect is also used. Many algorithms are also known in the art for measuring nucleotide sequence identity, including those contained in the programs described above. As another example, the polynucleotide sequence may use Fasta^TM(i.e., the program GCG version 6.1). Fasta^TMAlignments and percentage of sequence identity for the best overlapping regions between query and search sequences are provided. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta with its default parameters (word length of 6 and scoring matrix of NOPAM factor) as provided in GCG version 6.1^TMTo be determined, which is incorporated by reference.

In one aspect, a codon optimized, engineered nucleic acid sequence encoding human Lebercilin is provided. Preferably, the codon optimized Lebercilin coding sequence has less than about 80% identity, preferably about 75% identity or less, to the full length native Lebercilin coding sequence (FIGS. 1B-1D, SEQ ID NO: 2). In one embodiment, the codon optimized Lebercilin coding sequence has about 74% identity to the native Lebercilin coding sequence SEQ ID NO. 2. In one embodiment, the codon optimized lebrcilin coding sequence is characterized by an improved translation rate following AAV-mediated delivery (e.g., rAAV) as compared to native lebrcilin. In one embodiment, the codon optimized Lebercilin coding sequence shares less than about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or less identity with the full length native Lebercilin coding sequence, SEQ ID NO. 2. In one embodiment, the codon optimized nucleic acid sequence is a variant of SEQ ID NO. 3. In another embodiment, the codon optimized nucleic acid sequence is a sequence sharing about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or greater identity with SEQ ID NO. 3. In one embodiment, the codon optimized nucleic acid sequence is SEQ ID NO 3. In another embodiment, the nucleic acid sequence is codon optimized for expression in humans. In another embodiment, the lebercilin coding sequence is nt 1883 to nt 3976 of SEQ ID NO. 8. In other embodiments, different lebrcilin coding sequences are selected.

Codon-optimized coding regions can be designed by a variety of different methods. This optimization can be performed using methods available on-line (e.g., GeneArt), published methods, or companies that provide codon optimization services such as DNA2.0(Menlo Park, CA). For example, one codon optimization method is described in U.S. international patent publication No. WO 2015/012924, which is incorporated herein by reference in its entirety. See also, for example, U.S. patent publication No. 2014/0032186 and U.S. patent publication No. 2006/0136184. Suitably, the entire length of the Open Reading Frame (ORF) of the product is modified. However, in some embodiments, only a fragment of the ORF may be altered. By using one of these methods, frequency can be applied to any given polypeptide sequence and a nucleic acid fragment encoding a codon optimized coding region for the polypeptide is generated.

Many options are available for performing the actual changes of codons or for synthesizing codon optimized coding regions designed as described herein. Such modifications or syntheses may be performed using standard and routine molecular biology procedures well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs each 80 to 90 nucleotides in length and spanning the length of the desired sequence are synthesized by standard methods. These oligonucleotide pairs are synthesized such that they, when annealed, form a double-stranded fragment containing a sticky end of 80 to 90 base pairs, e.g., each oligonucleotide of the pair is synthesized to extend 3, 4, 5,6, 7,8, 9, 10 or more bases beyond the region complementary to the other oligonucleotide of the pair. The single stranded ends of each pair of oligonucleotides are designed to anneal together with the single stranded ends of the other pair of oligonucleotides. Annealing the oligonucleotide pairs, then annealing together about five to six of these double-stranded fragments via the sticky single-stranded ends, followed by ligating them together and cloning into a standard bacterial cloning vector, such as that available from Invitrogen Corporation, Carlsbad, CalifAnd (3) a carrier. The constructs were then sequenced by standard methods. Several of these constructs were made from 5 to 6 fragments of 80 to 90 base pair fragments (i.e., fragments of about 500 base pairs) joined togetherComposition such that the entire desired sequence is presented in a series of plasmid constructs. The inserts of these plasmids are then cleaved with appropriate restriction enzymes and joined together to form the final construct. The final construct was then cloned into a standard bacterial cloning vector and sequenced. Other methods will be apparent to the skilled artisan. Furthermore, gene synthesis is readily available commercially.

By "engineered" is meant that the nucleic acid sequence encoding the lebrcilin protein described herein is assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., that transfers the lebrcilin sequence carried thereon into a host cell, e.g., for use in the production of a non-viral delivery system (e.g., an RNA-based system, naked DNA, etc.) or for use in the production of a viral vector in a packaging host cell and/or for delivery to a host cell of a subject. In one embodiment, the genetic element is a plasmid. Methods for making such engineered constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

As used herein, the term "host cell" can refer to a packaging cell line in which the recombinant AAV is produced from a production plasmid. In the alternative, the term "host cell" may refer to any target cell in which expression of a coding sequence is desired. Thus, "host cell" refers to a prokaryotic or eukaryotic cell containing exogenous or heterologous DNA that has been introduced into the cell by any means such as electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA coating precipitation, viral infection, and protoplast fusion. In certain embodiments herein, the term "host cell" refers to a cell used to produce and package a viral vector or recombinant virus. In other embodiments herein, the term "host cell" refers to a culture of ocular cells of various mammalian species used for in vitro evaluation of the compositions described herein. Also, in other embodiments, the term "host cell" is intended to refer to an ocular cell of a subject treated in vivo for LCA.

As used herein, the term "ocular cell" refers to any cell in or associated with the function of the eye. The term may refer to any one of the photoreceptors, including rod photoreceptors, cone photoreceptors, and light-sensitive ganglion cells, Retinal Pigment Epithelium (RPE) cells, muller cells, choroidal cells, bipolar cells, horizontal cells, and amacrine cells. In one embodiment, the ocular cell is a photoreceptor. In another embodiment, the ocular cell is a cone photoreceptor. In another embodiment, the ocular cell is a rod photoreceptor.

In one embodiment, the nucleic acid sequence encoding lebrcilin further comprises a nucleic acid encoding a tag polypeptide covalently linked thereto. The tag polypeptide may be selected from known "epitope tags," including, but not limited to, myc tag polypeptide, glutathione-S-transferase tag polypeptide, green fluorescent protein tag polypeptide, myc-pyruvate kinase tag polypeptide, His6 tag polypeptide, influenza hemagglutinin tag polypeptide, flag tag polypeptide, and maltose binding protein tag polypeptide.

In another aspect, an expression cassette is provided comprising a nucleic acid sequence encoding Lebercilin. In one embodiment, the sequence is a codon optimized sequence. In another embodiment, the codon optimized nucleic acid sequence is SEQ ID NO 3 encoding human Lebercilin.

As used herein, an "expression cassette" refers to a nucleic acid molecule comprising the coding sequence of the lebrcilin protein, a promoter, and may include other regulatory sequences, which cassette may be packaged into the capsid of a viral vector (e.g., a viral particle). Typically, such expression cassettes used to generate viral vectors comprise the LCA5 sequences described herein flanked by packaging signals and other expression control sequences of the viral genome, such as those described herein. For example, for AAV viral vectors, the packaging signals are the 5 'Inverted Terminal Repeats (ITRs) and the 3' ITRs. When packaged into an AAV capsid, the ITRs, along with the expression cassette, may be referred to herein as a "recombinant AAV (raav) genome" or a "vector genome. In one embodiment, the expression cassette comprises a codon-optimized nucleic acid sequence encoding a Lebercilin protein. In one embodiment, the cassette provides codon optimized LCA5 in operable combination with an expression control sequence that directs the expression of a codon optimized nucleic acid sequence encoding Lebercilin in a host cell. In one embodiment, the vector genome is the sequence of nt 1-4379 of SEQ ID NO 8. In another embodiment, the vector genome is the sequence of nt 1-4368 of SEQ ID NO 9. In yet another embodiment, the LCA5 coding sequence in any one of the identified vector genomes is exchanged with another LCA5 coding sequence as described herein.

In another embodiment, an expression cassette for use in an AAV vector is provided. In that embodiment, the AAV expression cassette comprises at least one AAV Inverted Terminal Repeat (ITR) sequence. In another embodiment, the expression cassette comprises a 5'ITR sequence and a 3' ITR sequence. In one embodiment, the 5 'and 3' ITRs are flanked by codon-optimized nucleic acid sequences encoding lebrcilin, optionally with additional sequences that direct expression of the codon-optimized nucleic acid sequence encoding lebrcilin in a host cell. Thus, as described herein, an AAV expression cassette is intended to describe an expression cassette as described above flanked at its 5 'end by a 5' AAV Inverted Terminal Repeat (ITR) and at its 3 'end by a 3' AAV ITR. Thus, this rAAV genome contains the minimal sequences required to package the expression cassette into an AAV virion (i.e., AAV5 'and 3' ITRs). AAV ITRs can be obtained from the ITR sequences of any AAV as described herein. These ITRs may be of the same AAV origin as the capsid used in the resulting recombinant AAV or of a different AAV origin (to generate an AAV pseudotype). In one embodiment, the ITR sequence from AAV2 or a deleted form thereof (Δ ITR) is used for convenience and to expedite regulatory approval. However, ITRs from other AAV sources may be selected. When the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped. Typically, the AAV vector genome comprises AAV5 'ITRs, Lebercilin coding sequences and any regulatory sequences, and AAV3' ITRs. However, other configurations of these elements may be suitable. A shortened form of the 5' ITR (termed. DELTA. ITR) has been described in which the D-sequence and terminal melting site (trs) are deleted. In other embodiments, full length AAV5 'and 3' ITRs are used. Each rAAV genome can then be introduced into a production plasmid.

as used herein, the term "regulatory sequence", "transcription control sequence" or "expression control sequence" refers to DNA sequences, such as promoter sequences, enhancer sequences, and promoter sequences, that induce, repress, or otherwise control the transcription of an operably linked protein-encoding nucleic acid sequence.

As used herein, the term "operably linked" or "operably associated" refers to an expression control sequence that is contiguous with a nucleic acid sequence encoding lebrcilin and/or an expression control sequence that functions in trans or at a distance to control its transcription and expression.

In one aspect, a vector is provided comprising any of the expression cassettes described herein. As described herein, such vectors may be plasmids of various origins and, in certain embodiments, suitable for use in the generation of recombinant replication-defective viruses as further described herein.

As used herein, a "vector" is a nucleic acid molecule into which an exogenous or heterologous or engineered nucleic acid transgene can be inserted, which can then be introduced into an appropriate host cell. The vector preferably has one or more origins of replication and one or more sites into which recombinant DNA may be inserted. Vectors often have means to select vector cells from vector-free cells, e.g., they encode drug resistance genes. Common vectors include plasmids, viral genomes, and (mainly in yeast and bacteria) "artificial chromosomes". Certain plasmids are described herein.

In one embodiment, the vector is a non-viral plasmid comprising the expression cassette, e.g., "naked DNA," "naked plasmid DNA," RNA, and mRNA; coupled to various compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, polysaccharide compositions and other polymers, lipid and/or cholesterol based nucleic acid conjugates, and other constructs, as described herein. See, e.g., x.su et al, mol. pharmaceuticals, 2011,8(3), pages 774-; network release: year 2011, 3, 21; WO2013/182683, WO 2010/053572 and WO 2012/170930, all incorporated herein by reference. Such non-viral Lebercilin vectors may be administered by the routes described herein. Viral or non-viral vectors can be formulated with physiologically acceptable carriers for use in gene transfer and gene therapy applications.

In another embodiment, the vector is a viral vector comprising the expression cassette described herein. "viral vector" is defined as a replication-defective virus containing an exogenous or heterologous LCA5 nucleic acid transgene. In one embodiment, an expression cassette as described herein can be engineered onto a plasmid for drug delivery or the production of a viral vector. Suitable viral vectors are preferably replication-defective and selected from those that target ocular cells. The viral vector may include any virus suitable for gene therapy, including but not limited to adenovirus; herpes virus; a lentivirus; a retrovirus; parvovirus, and the like. However, for ease of understanding, adeno-associated viruses are referred to herein as exemplary viral vectors.

"replication-defective virus" or "viral vector" refers to a synthetic or recombinant virion in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, wherein any viral genomic sequence that is also packaged in the viral capsid or envelope is replication-defective; that is, they are unable to produce progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding enzymes required for replication (the genome may be engineered to be "gut-free" -containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Thus, it is considered safe for gene therapy because replication and infection by progeny virions will not occur except for the presence of viral enzymes required for replication.

In another embodiment, a recombinant adeno-associated virus (rAAV) vector is provided. The rAAV comprises an AAV capsid and a vector genome packaged therein. In one embodiment, the vector genome comprises: (a) AAV 5' Inverted Terminal Repeat (ITR) sequences; (b) a promoter; (c) a coding sequence encoding human Lebercilin; and (d) AAV3' ITRs. In another embodiment, the vector genome is an expression cassette as described herein. In one embodiment, the LCA5 sequence encodes the full-length lebrcilin protein. In one embodiment, the Lebercilin sequence is the protein sequence SEQ ID NO 1. In another embodiment, the coding sequence is SEQ ID NO 3 or a variant thereof.

Adeno-associated viruses (AAV), members of the parvovirus family, are small non-enveloped icosahedral viruses with a single-stranded linear DNA genome of 4.7 kilobases (kb) to 6 kb. Known AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and the like. ITRs or other AAV components can be readily isolated or engineered from AAV using techniques available to those skilled in the art. Such AAV may be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., american type culture collection, Manassas, VA). Alternatively, AAV sequences can be engineered by synthesis or other suitable means, with reference to published sequences (e.g., available in the literature or databases such as GenBank, PubMed, etc.). AAV viruses can be engineered by conventional molecular biology techniques, making it possible to optimize these particles for cell-specific delivery of nucleic acid sequences, minimization of immunogenicity, tailoring stability and particle lifetime, efficient degradation, precise delivery to the nucleus, and the like.

AAV fragments can be readily used in a variety of vector systems and host cells. The ideal AAV fragment is a cap protein, including vp1, vp2, vp3, and the hypervariable region; rep proteins, including rep 78, rep 68, rep 52, and rep 40; and sequences encoding such proteins. Such fragments may be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements from other AAV or non-AAV viral sequences. As used herein, an artificial AAV serotype includes, but is not limited to, an AAV having a non-naturally occurring capsid protein. Such an artificial capsid may be produced by any suitable technique using the novel AAV sequences of the invention (e.g., a fragment of the vp1 capsid protein) in combination with a heterologous sequence obtainable from another AAV serotype (known or novel), a non-contiguous portion of the same AAV serotype, a non-AAV viral source, or a non-viral source. The artificial AAV serotype can be, but is not limited to, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. In one embodiment, the vector contains the AAV8cap and/or rep sequences of the invention. See, for example, U.S. patent application publication No. US2009/02270030, which is incorporated herein by reference.

As used herein, the term "AAV" or "AAV serotype" refers to a number of naturally occurring and available adeno-associated viruses as well as artificial AAV. Among the AAVs isolated or engineered and well characterized from human or non-human primates (NHPs), human AAV2 is the first AAV developed as a gene transfer vector; it has been widely used for effective gene transfer experiments in different target tissues and animal models. Unless otherwise specified, the AAV capsid, ITRs and other selected AAV components described herein can be readily selected from any AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVAnc80, variants of any of the known or mentioned AAVs or AAVs to be found, or variants or mixtures thereof. See, for example, WO 2005/033321, which is incorporated herein by reference. In another embodiment, the AAV capsid is an AAV8bp capsid that preferentially targets bipolar cells. See, WO 2014/024282, which is incorporated herein by reference. In another embodiment, the AAV capsid is an AAV7m8 capsid, which is shown to be preferentially delivered to the outer retina. The AAV7m8 capsid nucleic acid sequence is reproduced in SEQ ID NO. 11 and the amino acid sequence is reproduced in SEQ ID NO. 12. See, Dalkara et al, In Vivo-Directed Evolution of a New Adeno-Associated Virus for Therapeutic OuterRetinal Gene Delivery from the vitamins, Sci Transl Med 5,189ra76(2013), which is incorporated herein by reference.

As used herein, an "AAV 7m8 capsid" is a self-assembled AAV capsid composed of multiple AAV7m8vp (variable protein) proteins. AAV7m8vp protein is typically represented as an alternative splice variant encoded by the nucleic acid sequence SEQ ID No. 11 or a sequence thereof encoding at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% of the vp1 amino acid sequence SEQ ID No. 12. These splice variants produce proteins of different lengths of SEQ ID NO 12. In certain embodiments, the "AAV 7m8 capsid" comprises an AAV having an amino acid sequence with up to 99% identity to SEQ ID NO 12.

In another embodiment, the rAAV capsid is selected from an AAV8 capsid or variant thereof, an AAV6 capsid or variant thereof, an AAV9 capsid or variant thereof, an AAV7 capsid or variant thereof, an AAV5 capsid or variant thereof, an AAV2 capsid or variant thereof, an AAV1 capsid or variant thereof, an AAV3 capsid or variant thereof, and an AAV4 capsid or variant thereof. In one embodiment, a recombinant adeno-associated virus (rAAV) vector is provided comprising an AAV7m8 capsid and an expression cassette as described herein, wherein the expression cassette comprises a nucleic acid sequence encoding lebrcilin, an inverted terminal repeat, and an expression control sequence that directs expression of lebrcilin in a host cell.

In yet another embodiment, a recombinant adeno-associated virus (AAV) vector is provided for delivering the LCA5 construct and the optimized sequences described herein. Adeno-associated virus (AAV) viral vectors are AAV DNase resistant particles having an AAV protein capsid and nucleic acid sequences packaged therein for delivery to a target cell. The AAV capsid is composed of 60 capsid (cap) protein subunits VP1, VP2, and VP3 arranged in icosahedral symmetry at a ratio of about 1:1:10 to 1:1:20 depending on the AAV selected. AAV may be selected as the source of the AAV viral vector capsid identified above. See, e.g., U.S. published patent application No. 2007-0036760-A1; U.S. published patent application No. 2009-0197338-a 1; EP 1310571. See also WO 2003/042397(AAV7 and other monkey AAV), US 7790449 and US 7282199(AAV8), WO 2005/033321 and US 7,906,111(A AV9), and WO2006/110689, and WO 2003/042397(rh.10) and (Dalkar a D, Byrne LC, Klimczak RR, Visel M, Yin L, Merigan WH et al In vivo-directed evolution of a new adono-associated virus for thermal output reliable gene delivery Med (2013)5(189) (AAV 3976. doi: 10.1126/scientific measured Med.3005708.) (AAV7M 8). Each of these documents is incorporated herein by reference. These documents also describe other AAV capsids that can be selected for production of AAV, and are incorporated by reference. In some embodiments, AAV caps for use in viral vectors can be generated by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the AAV capsids described above or a nucleic acid encoding therefor. In some embodiments, the AAV capsid is chimeric, comprising domains from two or three or four or more of the AAV capsid proteins described above. In some embodiments, the AAV capsid is a chimera of Vp1, Vp2, and Vp3 monomers from two or three different AAV or recombinant AAV. In some embodiments, the rAAV composition comprises more than one Cap described above.

As used herein, with respect to AAV, the term variant means any AAV sequence derived from a known AAV sequence, including those sequences that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or more sequence identity with an amino acid or nucleic acid sequence. In another embodiment, the AAV capsid comprises a variant that may comprise up to about 10% variation from any of the described or known AAV capsid sequences. That is, the AAV capsid shares from about 90% identity to about 99.9% identity, from about 95% to about 99% identity, or from about 97% to about 98% identity with an AAV capsid as provided herein and/or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with the AAV capsid. When determining the percent identity of AAV capsids, any variable proteins (e.g., vp1, vp2, or vp3) can be compared. In one embodiment, the AA V capsid shares at least 95% identity with AAV7m8 on vp1, vp2, or vp 3. In another embodiment, the Capsid is an AAV8 Capsid having Y447F, Y733F, and T494V mutations (also referred to as "AAV 8(C & G + T494V) and rep2-cap8(Y447F +733F + T494V)"), as exemplified by Kay et al, targeted photoreactors via intraviral delivery Using Novel, Capsid-Mutated AAV Vectors, PLoS one.2013; 8(4) e62097. published online on 2013 on 26.4.2013, which is incorporated herein by reference.

In one embodiment, it is desirable to utilize AAV capsids that exhibit tropism for desired target cells such as photoreceptors (e.g., rods and/or cones), RPE, or other ocular cells. In one embodiment, the AAV capsid is a tyrosine capsid-mutant in which certain surface exposed tyrosine residues are substituted with phenylalanine (F). Such AAV variants are described, for example, in Mowat et al, Tyrosine capsule-mutant AAV vectors for Gene delivery to the canoenteretina from a subentinal or intraviral approach, Gene Therapy 21,96-105 (1 month 2014), which is incorporated herein by reference.

As used herein, "artificial AAV" means, but is not limited to, an AAV having a non-naturally occurring capsid protein. Such an artificial capsid may be produced by any suitable technique using selected AAV sequences (e.g., a fragment of the vp1 capsid protein) in combination with heterologous sequences that may be obtained from a different selected AAV, a non-contiguous portion of the same AAV, a non-AAV viral source, or a non-viral source. The artificial AAV may be, but is not limited to, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Pseudotyped vectors are suitable for use in the present invention in which the capsid of one AAV is replaced by a heterologous capsid protein. In one embodiment, AAV2/5 and AAV2/8 are exemplary pseudotyped vectors.

In another embodiment, a self-complementary AAV is used. "self-complementary AAV" refers to a plasmid or vector having an expression cassette in which the coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. Upon infection, the two complementary halves of the scAAV will combine to form a double stranded dna (dsdna) unit ready for immediate replication and transcription, rather than waiting for cell-mediated synthesis of the second strand. See, for example, D M McCarty et al, "Self-minor additional catalytic amplification of DNA synthesis (scAAV) vectors promoter expression of DNA synthesis", Gene Therapy (8.2001), Vol.8, No. 16, p.1248 and 1254. Self-complementary AAV is described, for example, in U.S. patent nos. 6,596,535; 7,125,717, respectively; and 7,456,683, each of which is incorporated by reference herein in its entirety.

The term "exogenous" is used to describe a nucleic acid sequence or protein, meaning that the nucleic acid or protein does not naturally occur in its place in the chromosome or in the host cell. An exogenous nucleic acid sequence also refers to a sequence that is derived from the same host cell or subject and inserted into it, but which sequence is not found in nature, e.g., in different copy numbers, or under the control of different regulatory elements.

The term "heterologous" is used to describe a nucleic acid sequence or protein, meaning that the nucleic acid or protein originates from a different organism or a different species of the same organism than the host cell or subject in which it is expressed. The term "heterologous" when used in reference to a protein or nucleic acid in a plasmid, expression cassette or vector means that the protein or nucleic acid is present with another sequence or subsequence, with which the protein or nucleic acid in question is not present in the same relationship in nature.

In yet another embodiment, the expression cassette (including any of those described herein) is used to produce a recombinant AAV genome.

In one embodiment, the expression cassettes described herein are engineered into suitable genetic elements (vectors) suitable for the generation of viral vectors and/or delivery to host cells, e.g., naked DNA, phage, translocator, cosmid, episome, etc., which transfer the LCA5 sequences carried thereon. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated precipitation, viral infection, and protoplast fusion. Methods for making such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

For packaging of an expression cassette or rAAV genome or production plasmid into a virion, the ITR is the only AAV component required in cis in the same construct as the expression cassette. In one embodiment, the coding sequence for replication (rep) and/or capsid (cap) is removed from the AAV genome and supplied in trans or by a packaging cell line to produce an AAV vector.

Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, for example, U.S. patent 7790449; us patent 7282199; WO 2003/042397; WO 2005/033321; WO 2006/110689; and US 7588772B 2 ]. In one system, a producer cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and constructs encoding rep and cap. In the second system, packaging cell lines stably supplying rep and cap are transiently transfected with constructs encoding transgenes flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with helper adenovirus herpes virus, thereby requiring isolation of rAAV from contaminating viruses. More recently, systems have been developed that do not require infection with helper viruses to recover AAV-which systems also provide the required helper functions in transit (i.e., adenovirus E1, E2a, VA and E4 or herpesviruses UL5, UL8, UL52 and UL29, and herpesvirus polymerase). In these more recent systems, helper functions can be provided by transiently transfecting the cell with a construct encoding the desired helper function, or the cell can be engineered to stably contain the gene encoding the helper function, the expression of which can be controlled at the transcriptional or post-transcriptional level.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system is isolated, even if later reintroduced into the natural system. Such polynucleotides may be part of a vector and/or such polynucleotides or polypeptides may be part of a composition, and they are still isolated in that such vector or composition is not part of its natural environment.

In yet another system, expression cassettes flanked by the ITR and rep/cap genes are introduced into insect cells by infection with baculovirus-based vectors. For an overview of these production systems, see, for example, Zhang et al, 2009, "Adenoviral-assisted viral hybrid for large-scale viral production," Human Gene Therapy 20: 922-. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which are incorporated herein by reference in their entirety: 5,139,941; 5,741,683, respectively; 6,057,152, respectively; 6,204,059, respectively; 6,268,213, respectively; 6,491,907, respectively; 6,660,514, respectively; 6,951,753, respectively; 7,094,604, respectively; 7,172,893, respectively; 7,201,898; 7,229,823, respectively; and 7,439,065. See, for example, Grieger & Samulski,2005, "Adeno-assisted virus a gene therapy Vector: Vector reduction, production and clinical applications," adv. biochem. Engin/Biotechnol.99: 119-145; buring et al, 2008, "recent details in adono-associated virus vector technology," J.Gene Med.10:717 733; and the references cited below, each of which is incorporated herein by reference in its entirety.

Methods for constructing any of the embodiments of the invention are known to those having skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook et al, Molecular Cloning, analytical Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Likewise, methods of producing rAAV virions are well known, and selection of an appropriate method is not limiting of the invention. See, e.g., K.Fisher et al, (1993) J.Virol.,70:520-532 and U.S. Pat. No. 5,478,745.

"plasmids" are generally referred to herein by the preceding lower case p and/or followed by a capital letter and/or number according to standard nomenclature rules well known to those skilled in the art. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those skilled in the art. Furthermore, the skilled artisan can readily construct any number of other plasmids suitable for use in the present invention. The nature, construction and use of such plasmids, as well as other vectors of the invention, will be readily apparent to those skilled in the art from this disclosure.

In one embodiment, the production plasmid is as described herein or as described in WO2012/158757, which is incorporated herein by reference. Various plasmids for producing rAAV vectors are known in the art and are suitable for use herein. The production plasmid is cultured in a host cell that expresses the AAV cap and/or rep proteins. In the host cell, each rAAV genome is rescued and packaged into capsid or envelope proteins to form infectious virions.

In one aspect, a production plasmid is provided comprising an expression cassette as described above. In one embodiment, the production plasmid is as shown in SEQ ID NO 8 and FIGS. 1E-1F, which is designated p 643. This plasmid is an example used to generate rAAV-human codon optimized Lebercilin vectors. Such plasmids contain 5' AAV ITR sequences; a selected promoter; a polyA sequence; and 3' ITRs; in addition, it also contains a padding sequence, such as λ. In yet another embodiment, the stuffer sequence maintains the rAAV vector genome at the following dimensions: between about 3 kilobases (kb) to about 6kb, about 4.7kb to about 6kb, about 3kb to about 5.5kb, or about 4.7kb to 5.5 kb. In one embodiment, the non-coding λ filler region is included in the vector backbone. An example of p643, which contains the Lebercilin coding sequence, can be found in SEQ ID NO 8. In another embodiment, the production plasmid is as shown in FIGS. 11A-11B and SEQ ID NO 9. In another embodiment, the production plasmid is modified to optimize vector plasmid production efficiency. Such modifications include the addition of additional neutral sequences, or the deletion of one or more portions of the entire lambda fill sequence to modulate the level of supercoiling of the vector plasmid. Such modifications are contemplated herein. In other embodiments, terminators and other sequences are included in the plasmid.

In certain embodiments, the rAAV expression cassette, vector (e.g., rAAV vector), virus (e.g., rAAV), production plasmid comprise an AAV inverted terminal repeat sequence, a codon-optimized nucleic acid sequence encoding lebrcilin, and an expression control sequence that directs expression of the encoded protein in a host cell. In other embodiments, the rAAV expression cassette, virus, vector (e.g., rAAV vector), production plasmid, further comprises one or more of the following: introns, Kozak sequences, polyA, post-transcriptional regulatory elements, and the like. In one embodiment, the post-transcriptional regulatory element is a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE).

Expression cassettes, vectors, and plasmids contain other components that can be optimized for a particular species using techniques known in the art, including, for example, codon optimization as described herein. The components of the cassettes, vectors, plasmids and viruses or other compositions described herein comprise a promoter sequence as part of the expression control sequence. In another embodiment, the promoter is cell-specific. The term "cell-specific" means that a particular promoter selected for a recombinant vector can direct expression of an optimized Lebercilin coding sequence in a particular ocular cell type. In one embodiment, the promoter is specific for expression of the transgene in photoreceptor cells. In another embodiment, the promoter is specific for expression in rods and cones. In another embodiment, the promoter is specific for expression in the rods. In another embodiment, the promoter is specific for expression in the cones. In one embodiment, the photoreceptor-specific promoter is a human rhodopsin kinase promoter. The rhodopsin kinase promoter has been shown to be active in both rods and cones. See, e.g., Sun et al, Gene Therapy with a Promoter Targeting Board roads and Conses research recovery used by AIPL1 muscles, Gene ther, month 1 2010; 17(1) 117 and 131, which are incorporated herein by reference in their entirety. In one embodiment, the promoter is modified to add one or more restriction sites, thereby facilitating cloning.

In another embodiment, the promoter is a human rhodopsin promoter. In one embodiment, the promoter is modified to include a restriction for the ends of cloning. See, for example, Nathans and Hogness, Isolation and nucleotide sequence of the gene encoding human rhodopsin, PNAS,81:4851-5 (8 months 1984), which is incorporated herein by reference in its entirety. In another embodiment, the promoter is a portion or fragment of the human rhodopsin promoter. In another embodiment, the promoter is a variant of the human rhodopsin promoter.

Other exemplary promoters include the human G protein-coupled receptor protein kinase 1(GRK1) promoter (Genbank accession No. AY 327580). In another embodiment, the promoter is the 292nt fragment of the GRK1 promoter (position 1793-2087) (see, Beltran et al, Gene Therapy 201017: 1162-74, which is incorporated herein by reference in its entirety). In another preferred embodiment, the promoter is the human inter-photoreceptor retinoid binding protein proximal (IRBP) promoter. In one embodiment, the promoter is a 235nt fragment of the hIRBP promoter. In one embodiment, the promoter is the RPGR proximal promoter (Shu et al, IOVS,2102 5 months, which is incorporated herein by reference in its entirety). Other promoters suitable for use in the present invention include, but are not limited to, rod opsin promoter, red green opsin promoter, blue opsin promoter, cGMP- β -phosphodiesterase promoter (Qgueta et al, IOVS, Invest Ophthalmol Vis Sci, 12 months 2000; 41(13):4059-63), mouse opsin promoter (Beltran et al 2010 cited above), rhodopsin promoter (Mussolino et al, Gene Ther, 7 months 2011, 18(7): 637-45); the alpha subunit of cone transducin (Morrissey et al, BMCDev, Biol, 1 month 2011, 11: 3); a beta Phosphodiesterase (PDE) promoter; retinitis pigmentosa (RP1) promoter (Nicord et al, J.Gene Med, month 12 2007, 9(12): 1015-23); NXNL2/NXNL1 promoter (Lambard et al, PLoS One, 10.2010, 5(10): el3025), RPE65 promoter; the chronic retinal degeneration/peripherin 2(Rds/perph2) promoter (Cai et al, Exp Eye Res.2010, 8 months; 91(2): 186-94); and the VMD2 promoter (Kachi et al, Human Gene Therapy,2009(20: 31-9)). Each of these documents is incorporated by reference herein in its entirety. In another embodiment, the promoter is selected from the group consisting of human EFl α promoter, rhodopsin kinase, interphotoreceptor binding protein (IRBP), cone opsin promoter (red green, blue), cone opsin upstream sequences containing the control region of the red green cone locus, cone transduction and transcription factor promoter (neuroretinal leucine zipper (Nrl) and photoreceptor specific nuclear receptor Nr2e3, bZIP).

In another embodiment, the promoter is a ubiquitous or constitutive promoter. An example of a suitable promoter is a hybrid chicken β -actin (CBA) promoter with a Cytomegalovirus (CMV) enhancer element, such as the sequences shown in fig. 1E-1F-in another embodiment, the promoter is the CB7 promoter. Other suitable promoters include the human β -actin promoter, the human elongation factor-1 α promoter, the Cytomegalovirus (CMV) promoter, the simian virus 40 promoter, and the herpes simplex virus thymidine kinase promoter. See, e.g., Damdindorj et al, (8 months 2014) A Comparative Analysis of connective reactants Located in Adeno-Associated Viral vectors PLoS ONE 9(8) e 106472. Still other suitable promoters include viral promoters, constitutive promoters, regulatable promoters [ see, e.g., WO 2011/126808 and WO 2013/04943 ]. Alternatively, promoters responsive to physiological elicitors can be used in the expression cassettes, rAAV genomes, vectors, plasmids, and viruses described herein. In one embodiment, the promoter has a small size, at 1000bp, due to size limitations of AAV vectors. In another embodiment, the promoter is at 400 bp. Other promoters may be selected by those skilled in the art.

In yet another embodiment, the promoter is selected from the group consisting of the SV40 promoter, the dihydrofolate reductase promoter, and the phosphoglycerate kinase (PGK) promoter, rhodopsin kinase promoter, rod opsin promoter, red and green opsin promoter, blue opsin promoter, inter-photoreceptor binding protein (IRBP) promoter and cGMP-beta-phosphodiesterase promoter, bacteriophage lambda (PL) promoter, Herpes Simplex Virus (HSV) promoter, tetracycline-controlled transactivator-responsive promoter (tet) system, Long Terminal Repeat (LTR) promoters such as RSV LTR, MoMLV LTR, BIV LTR or HIV LTR, Moloney murine sarcoma virus U3 region promoter, granzyme A promoter, regulatory sequences of the metallothionein gene, CD34 promoter, CD8 promoter, Thymidine Kinase (TK) promoter, B19 parvovirus promoter, and combinations thereof, PGK promoter, glucocorticoid promoter, Heat Shock Protein (HSP) promoter such as HSP65 and HSP70 promoter, immunoglobulin promoter, MMTV promoter, Rous Sarcoma Virus (RSV) promoter, lac promoter, CaMV 35S promoter, nopaline synthase promoter, MND promoter or MNC promoter. The promoter sequences thereof are known to the person skilled in the art or are publicly available, as in the literature or in databases, for example GenBank, PubMed, etc.

In another embodiment, the promoter is an inducible promoter. Inducible promoters may be selected from known promoters, including rapamycin/rapamycin analogue promoters, ecdysone promoters, estrogen responsive promoters, and tetracycline responsive promoters, or heterodimeric repression switches. See Sochor et al, An automated regulated expression System for Gene Therapeutic applications, scientific reports, 11/24/2015; 17105 and Daber R, Lewis M., A novel molecular switch.JMol biol.2009, 8 months and 28 days; 391(4) 661-70, Epub 2009, 21/6, both of which are incorporated herein by reference in their entirety.

In yet another embodiment, the promoter is a chicken β -actin promoter having the nucleic acid sequence nt 546 to nt 283 of SEQ ID No. 8.

In other embodiments, expression cassettes, vectors, plasmids, and viruses described herein contain other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; a TATA sequence; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequence); an intron; sequences that improve protein stability; and, if desired, increasing the secretion of the encoded product. The expression cassette or vector may be free of any one or more of the elements described herein.

Examples of suitable polyA sequences include, for example, synthetic polyA or from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β -globin (RGB) or modified RGB (mrgb). In yet another embodiment, poly A has the nucleic acid sequence nt 3993 to nt 4200 of SEQ ID NO 8.

Examples of suitable enhancers include, for example, the CMV enhancer, the RSV enhancer, the alpha-fetoprotein enhancer, the TTR minimal promoter/enhancer, the LSP (TH-binding globulin promoter/alpha 1-microglobulin/bikunin enhancer), the APB enhancer, the ABPS enhancer, the alpha mic/bik enhancer, the TTR enhancer, en34, ApoE, and the like. In one embodiment, the enhancer has the nucleic acid sequence nt 241 to nt 544 of SEQ ID NO 8.

In one embodiment, a Kozak sequence is included upstream of the lebrcilin coding sequence to enhance translation from the correct start codon. In another embodiment, CBA exon 1 and intron are included in the expression cassette. In one embodiment, the Lebercilin coding sequence is placed under the control of a hybrid Chicken Beta Actin (CBA) promoter. This promoter consists of the Cytomegalovirus (CMV) immediate early enhancer, the proximal chicken beta actin promoter, and CBA exon 1 flanked by intron 1 sequences.

In another embodiment, the intron is selected from CBA, human beta globin, IVS2, SV40, bGH, alpha globin, beta globin, collagen, ovalbumin, p53, or a fragment thereof.

In one embodiment, the expression cassettes, vectors, plasmids and viruses contain a 5'ITR, the chicken β -actin (CBA) promoter, CMV enhancer, CBA exon 1 and intron, the human codon optimized Lebercilin sequence, bGH poly A and a 3' ITR. In yet another embodiment, the expression cassette comprises nt1 to 4379 of SEQ ID NO 8. In yet another embodiment, the 5'ITR has the nucleic acid sequence of nt1 to nt 130 of SEQ ID NO.8 and the 3' ITR has the nucleic acid sequence of nt 4250 to nt4379 of SEQ ID NO. 8. In yet another embodiment, CBA exon 1 and intron have the nucleic acid sequence nt 824 to nt1795 of SEQ ID NO 8. In yet another embodiment, the production plasmid has the sequence SEQ ID NO 8, also shown in FIGS. 1E-1F. In yet another embodiment, the production plasmid has the sequence SEQ ID NO 9, also shown in FIGS. 1A-11B.

In another aspect, a method of treating Lebercilin congenital amaurosis and/or restoring visual function in a subject suffering from LCA comprising delivering to a subject in need thereof a vector (e.g., rAAV) encoding Lebercilin as described herein. In one embodiment, a method of treating a subject having LCA with a rAAV described herein is provided.

"administering" as used in the methods means delivering the composition to a selected target cell characterized by LCA. In one embodiment, the method involves delivering the composition to the RPE, photoreceptor cells, or other ocular cells by sub-retinal injection. In another embodiment, intravitreal injection into the subject is employed. In another embodiment, subretinal injection is administered to the subject. In yet another approach, an intravascular injection may be used, such as via the palpebral vein. Other methods of administration may be selected by those skilled in the art in view of this disclosure.

"administration" or "route of administration" is the delivery of a composition described herein to a subject, with or without a pharmaceutical carrier or excipient. The routes of administration can be combined, if desired. In some embodiments, the administering is repeated periodically. The pharmaceutical compositions described herein are designed to be delivered to a subject in need thereof by any suitable route or combination of different routes. In some embodiments, direct delivery to the eye (optionally via ocular delivery, subretinal injection, intraretinal injection, intravitreal, topical) or via systemic routes, such as intravascular, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration, is employed. The nucleic acid molecules, expression cassettes, and/or vectors described herein can be delivered in a single composition or in multiple compositions. Optionally, two or more different AAV, or multiple viruses, can be delivered [ see, e.g., WO 202011/126808 and WO2013/049493], in another embodiment, the multiple viruses can contain different replication-defective viruses (e.g., AAV and adenovirus), alone or in combination with proteins.

Also provided herein are pharmaceutical compositions. The pharmaceutical compositions described herein are designed to be delivered to a subject in need thereof by any suitable route or combination of different routes. These delivery means are designed to avoid direct systemic delivery of suspensions containing the AAV compositions described herein. Suitably, this may have the following benefits: reduced dose, reduced toxicity and/or reduced unwanted immune response to AAV and/or transgene product as compared to systemic administration.

In still other aspects, these nucleic acid sequences, vectors, expression cassettes, and rAAV viral vectors are suitable for use in pharmaceutical compositions that further comprise a pharmaceutically acceptable carrier, excipient, buffer, diluent, surfactant, preservative, and/or adjuvant, and the like. Such pharmaceutical compositions are useful for expressing optimized lebrcilin in host cells by delivery via such recombinantly engineered AAV or artificial AAV.

To prepare these pharmaceutical compositions containing the nucleic acid sequences, vectors, expression cassettes and rAAV viral vectors, the sequences or vectors or viral vectors are preferably assessed for contamination by conventional methods and then formulated into pharmaceutical compositions suitable for ocular administration. Such formulations involve the use of a pharmaceutically and/or physiologically acceptable vehicle or carrier, particularly one suitable for administration to the eye, such as buffered saline or other buffers, e.g., HEPES, to maintain the pH at an appropriate physiological level, and optionally other pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, surfactants or excipients, and the like. For injection, the carrier is typically a liquid. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free phosphate buffered saline. A variety of such known carriers are provided in U.S. patent publication No. 7,629,322, which is incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is a balanced salt solution. In one embodiment, the carrier comprises tween. If the virus is to be stored for long periods, it may be frozen in the presence of glycerol or Tween 20.

In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution containing saline, a surfactant, and a physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for example in the range of pH6 to 9, or pH6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. Since the pH of cerebrospinal fluid is from about 7.28 to about 7.32, a pH in this range may be required for intrathecal delivery; whereas for intravitreal or subretinal delivery a pH of 6.8 to about 7.2 may be required. However, for other routes of delivery, other pH's within this broadest range or these subranges can be selected.

Suitable surfactants or combinations of surfactants may be selected from non-toxic non-ionic surfactants. In one embodiment, a difunctional block copolymer with primary hydroxyl end groups is selectedPhysical surface-active agents, e.g. likeF68[BASF]Also known as poloxamer 188, which has a neutral pH, has an average molecular weight of 8400. Other surfactants and other poloxamers may be selected, i.e. nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly (propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly (ethylene oxide)); SOLUTOL HS 15(Macrogol-15 hydroxystearic acid); LABRASOL (polyoxyglyceryl caprylate); polyoxy 10 oleyl ether; TWEEN (polyoxyethylene sorbitan fatty acid ester); ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are usually named with the letter "P" (for poloxamers) followed by three numbers: the first two numbers x 100 give the approximate molecular weight of the polyoxypropylene core and the last number x10 gives the percent polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension.

In one embodiment, the formulation may contain, for example, a buffered saline solution comprising one or more of the following in water: sodium chloride, sodium bicarbonate, glucose, magnesium sulfate (e.g., magnesium sulfate-7H 2O), potassium chloride, calcium chloride (e.g., calcium chloride-2H 2O), disodium phosphate, and mixtures thereof. Suitably, for intrathecal delivery, the osmotic pressure is in a range compatible with cerebrospinal fluid (e.g., about 275 to 290); see, e.g., emericine, medscape, com/article/2093316-overview. Optionally, for intrathecal delivery, commercially available diluents can be used as suspending agents or in combination with another suspending agent and other optional excipients. See, e.g., Elliots solution[Lukare Medical]. In other embodiments, the formulation may contain one or more penetration enhancers. Examples of suitable penetration enhancers may include, for exampleSuch as mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether or EDTA.

In another embodiment, the composition comprises a carrier, diluent, excipient and/or adjuvant. One skilled in the art can readily select an appropriate carrier in view of the indication for which the transfer virus is intended. For example, one suitable carrier includes saline, which may be formulated with a variety of buffer solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The buffer/carrier should include components that prevent the rAAV from sticking to the infusion line but do not interfere with the in vivo binding activity of the rAAV.

Optionally, the compositions of the invention may contain, in addition to the rAAV and carrier, other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The composition according to the invention may comprise a pharmaceutically acceptable carrier as defined above. Suitably, the compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and/or mixed with a suitable excipient designed for delivery to a subject via injection, osmotic pump, intrathecal catheter or by another device or route. In one embodiment, the composition is formulated for intravitreal delivery. In one embodiment, the composition is formulated for subretinal delivery.

In an exemplary specific embodiment, the composition of the carrier or excipient contains 180mM NaCl, 10mM NaPi, ph7.3, with 0.0001% -0.01% Pluronic F68(PF 68). The exact composition of the saline component of the buffer is in the range of 160mM to 180mM NaCl. Optionally, a different pH buffer (possibly HEPES, sodium bicarbonate, TRIS) is used instead of the buffer specifically described. Alternatively, a buffer containing 0.9% NaCl is useful.

In the case of AAV viral vectors, quantification of genomic copies ("GC"), vector genomes ("VG"), or virions can be used as a measure of the dose contained in the formulation or suspension. Any method known in the art can be used to determine the number of Genomic Copies (GCs) of the replication defective virus compositions of the invention. One method of AAV GC quantitative titration is performed as follows: purified AAV vector samples were first treated with DNase to remove unencapsulated AAV genomic DNA or contaminating plasmid DNA from the production process. The DNase resistant particles are then subjected to a heat treatment in order to release the genome from the capsid. The released genome is then quantified by real-time PCR using a primer/probe set that targets a specific region of the viral genome (usually the poly a signal). In another method, the effective dose of recombinant adeno-associated virus carrying a nucleic acid sequence encoding an optimized Lebercilin coding sequence is measured as described in S.K. McLaughlin et al, 1988J.Virol, 62:1963, which is incorporated herein by reference in its entirety.

As used herein, the term "dose" may refer to the total dose delivered to a subject during a course of treatment or the amount delivered in a single unit (or multiple units or divided doses) administration. The pharmaceutical viral compositions may be formulated in dosage units so as to contain, per dose, the following amounts of replication defective virus carrying a codon-optimized nucleic acid sequence encoding Lebercilin as described herein: at about 1.0x 10⁹GC to about 1.0x 10¹⁵Within the range of GC, all whole or fractional amounts within the range are included. In one embodiment, the composition is formulated so as to contain at least 1x10 per dose⁹、2x10⁹、3x10⁹、4x10⁹、5x10⁹、6x10⁹、7x10⁹、8x10⁹Or 9x10⁹And GC, including all whole or fractional amounts within the range. In another embodiment, the composition is formulated so as to contain at least 1x10 per dose¹⁰、2x10¹⁰、3x10¹⁰、4x10¹⁰、5x10¹⁰、6x10¹⁰、7x10¹⁰、8x10¹⁰Or 9x10¹⁰A GC included inAll whole or fractional amounts within this range. In another embodiment, the composition is formulated so as to contain at least 1x10 per dose¹¹、2x10¹¹、3x10¹¹、4x10¹¹、5x10¹¹、6x10¹¹、7x10¹¹、8x10¹¹Or 9x10¹¹And GC, including all whole or fractional amounts within the range. In another embodiment, the composition is formulated so as to contain at least 1x10 per dose¹²、2x10¹²、3x10¹²、4x10¹²、5x10¹²、6x10¹²、7x10¹²、8x10¹²Or 9x10¹²And GC, including all whole or fractional amounts within the range. In another embodiment, the composition is formulated so as to contain at least 1x10 per dose¹³、2x10¹³、3x10¹³、4x10¹³、5x10¹³、6x10¹³、7x10¹³、8x10¹³Or 9x10¹³And GC, including all whole or fractional amounts within the range. In another embodiment, the composition is formulated so as to contain at least 1x10 per dose¹⁴、2x10¹⁴、3x10¹⁴、4x10¹⁴、5x10¹⁴、6x10¹⁴、7x10¹⁴、8x10¹⁴Or 9x10¹⁴And GC, including all whole or fractional amounts within the range. In another embodiment, the composition is formulated so as to contain at least 1x10 per dose¹⁵、2x10¹⁵、3x10¹⁵、4x10¹⁵、5x10¹⁵、6x10¹⁵、7x10¹⁵、8x10¹⁵Or 9x10¹⁵And GC, including all whole or fractional amounts within the range. In one embodiment, for human use, the dose may be at 1x10 per dose¹⁰To about 1x10¹²Within the range of GC, all whole or fractional amounts within the range are included. All doses can be measured by any known method, including by oqPCR or digital micro-drop PCR (ddPCR), as described, for example, in M.Lock et al, HumGene Ther methods.2014, 4 months; 25(2) 115-25.doi:10.1089/hgtb.2013.131, which is incorporated herein by reference.

In one embodiment, an aqueous suspension suitable for administration to an LCA patient is provided. The suspension comprises an aqueous suspending liquid and about 1x10 per eye¹⁰GC or virion of a recombinant adeno-associated virus (rAAV) described herein to about 1x10¹²A GC or a virion, the recombinant adeno-associated virus being suitable for use as a therapeutic agent for LCA.

It may also be desirable to administer multiple "boost" doses of the pharmaceutical composition of the present invention. For example, depending on the duration of the transgene within the target cells of the eye, booster doses may be delivered at 6 month intervals, or annually after the first administration. The fact that AAV neutralizing antibodies cannot be generated by administration of rAAV vectors should allow for additional enhanced administration.

Such booster doses, and thus the need, may be monitored by the attending physician using, for example, retinal and visual function tests and visual behavior tests as described in the examples below. Other similar tests may be used to determine the status of the treated subject over time. The appropriate test may be selected by the attending physician. Still alternatively, the methods of the invention may also involve injecting larger volumes of virus-containing solution in a single or multiple infection to allow levels of visual function to approach those seen in wild-type retinas.

In another embodiment, the amount of vectors, viruses and replication defective viruses described herein that carry a codon optimized nucleic acid sequence encoding Lebercilin is about 1.0x 10 per eye⁷VG to about 1.0x 10 per eye¹⁵Within the range of VGs, all whole or fractional amounts within the range are included. In one embodiment, the amount is at least 1x10 per eye⁷、2x10⁷、3x10⁷、4x10⁷、5x10⁷、6x10⁷、7x10⁷、8x10⁷Or 9x10⁷VG, including all whole or fractional amounts within the range. In one embodiment, the amount is at least 1x10 per eye⁸、2x10⁸、3x10⁸、4x10⁸、5x10⁸、6x10⁸、7x10⁸、8x10⁸Or 9x10⁸VG, including all rangesIn whole or in part. In one embodiment, the amount is at least 1x10 per eye⁹、2x10⁹、3x10⁹、4x10⁹、5x10⁹、6x10⁹、7x10⁹、8x10⁹Or 9x10⁹VG, including all whole or fractional amounts within the range. In one embodiment, the amount is at least 1x10 per eye¹⁰、2x10¹⁰、3x10¹⁰、4x10¹⁰、5x10¹⁰、6x10¹⁰、7x10¹⁰、8x10¹⁰Or 9x10¹⁰VG, including all whole or fractional amounts within the range. In one embodiment, the amount is at least 1x10 per eye¹¹、2x10¹¹、3x10¹¹、4x10¹¹、5x10¹¹、6x10¹¹、7x10¹¹、8x10¹¹Or 9x10¹¹VG, including all whole or fractional amounts within the range. In one embodiment, the amount is at least 1x10 per eye¹²、2x10¹²、3x10¹²、4x10¹²、5x10¹²、6x10¹²、7x10¹²、8x10¹²Or 9x10¹²VG, including all whole or fractional amounts within the range. In one embodiment, the amount is at least 1x10 per eye¹³、2x10¹³、3x10¹³、4x10¹³、5x10¹³、6x10¹³、7x10¹³、8x10¹³Or 9x10¹³VG, including all whole or fractional amounts within the range. In one embodiment, the amount is at least 1x10 per eye¹⁴、2x10¹⁴、3x10¹⁴、4x10¹⁴、5x10¹⁴、6x10¹⁴、7x10¹⁴、8x10¹⁴Or 9x10¹⁴VG, including all whole or fractional amounts within the range. In one embodiment, the amount is at least 1x10 per dose¹⁵、2x10¹⁵、3x10¹⁵、4x10¹⁵、5x10¹⁵、6x10¹⁵、7x10¹⁵、8x10¹⁵Or 9x10¹⁵And GC, including all whole or fractional amounts within the range. In one embodimentIn the case, the method comprises 1x10 per eye per dose⁹To about 1x10¹³Doses within the VG range, including all whole or fractional amounts within the range. In another embodiment, the method comprises delivering the carrier in an aqueous suspension. In another embodiment, the method comprises administering a composition in a volume of about or at least 150 microliters in a volume of 1x10⁹To 1x10¹³A dose of GC is administered with a rAAV as described herein, thereby restoring visual function to the subject. All doses can be measured by any known method, including by oqPCR or digital micro-drop PCR (ddPCR), as described, for example, in M.Lock et al, Hum Gene Ther methods.2014, 4 months; 25(2) 115-25.doi:10.1089/hgtb.2013.131, which is incorporated herein by reference.

These doses may be administered in various volumes of carrier, excipient or buffer formulation, ranging from about 25 to about 1000 microliters, including all values 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 of carrier, excipient, or buffer is at least about 25 μ L. In one embodiment, the volume is about 50 μ L. In another embodiment, the volume is about 75 μ L. In another embodiment, the volume is about 100 μ L. In another embodiment, the volume is about 125 μ L. In another embodiment, the volume is about 150 μ L. In another embodiment, the volume is about 175 μ L. In yet another embodiment, the volume is about 200 μ L. In another embodiment, the volume is about 225 μ L. In yet another embodiment, the volume is about 250 μ L. In yet another embodiment, the volume is about 275 μ L. In yet another embodiment, the volume is about 300 μ L. In yet another embodiment, the volume is about 325 μ L. In another embodiment, the volume is about 350 μ L. In another embodiment, the volume is about 375 μ L. In another embodiment, the volume is about 400 μ L. In another embodiment, the volume is about 450 μ L. In another embodiment, the volume is about 500. mu.L. In another embodiment, the volume is about 550 pL. In another embodiment, the volume is about 600 pL. In another embodiment, the volume is about 650 pL. In another embodiment, the volume is about 700 μ L. In another embodiment, the volume is about 800 μ L. In another embodiment, the volume is between about 150 and 800 μ L. In another embodiment, the volume is between about 700 and 1000 μ L. In another embodiment, the volume is between about 250 and 500 μ L.

In one embodiment, the viral construct may be at least 1x10⁹To about at least 1x10¹¹Doses of each GC are delivered to a small animal subject, such as a mouse, in a volume of about 1 μ L to about 3 μ L. For larger veterinary subjects with eyes of about the same size as the human eye, larger human doses and volumes as described above are useful. For a discussion of good practice in administering substances to various veterinary animals, see, e.g., Diehl et al, j.applied Toxicology,21:15-23 (2001). This document is incorporated herein by reference.

It is desirable to use the lowest effective concentration of virus or other delivery vehicle to reduce the risk of undesirable effects such as toxicity, retinal dysplasia and exfoliation. Still other dosages within these ranges may be selected by the attending physician after considering the following factors: the physical condition of the subject (preferably a human being) being treated, the age of the subject, the LCA and the extent to which the disorder, if progressive, develops.

Yet another aspect described herein is a method of treating, delaying or stopping the progression of LCA in a mammalian subject. In one embodiment, rAAV carrying the lebrcilin native, modified or codon optimized sequence may be administered to a desired subject, including a human subject, preferably suspended in a physiologically compatible carrier, diluent, excipient and/or adjuvant. The method comprises administering to a subject in need thereof any one of: a nucleic acid sequence, an expression cassette, a rAAV genome, a plasmid, a vector, or a rAAV vector, or a composition comprising the same. In one embodiment, the composition is delivered subretinally. In another embodiment, the composition is delivered intravitreally. In yet another embodiment, the composition is delivered using a combination of administration routes suitable for treating LCA, and may also involve administration via the palpebral vein or other intravenous or conventional administration routes.

For use in these methods, the volume and viral titer of each dose is determined individually, as further described herein, and can be the same or different from other treatments performed in the ipsilateral or contralateral eye. Dosages, administrations, and regimens can be determined by the attending physician in view of the teachings of the present specification. In one embodiment, the composition is administered in a single dose selected from those listed above in the affected eye. In another embodiment, the composition is administered simultaneously or sequentially as a single dose selected from those listed above in both affected eyes. Sequential administration may mean that the time interval between administration from one eye to the other is minutes, hours, days, weeks or months. In another embodiment, the method involves administering two or more doses (e.g., divided doses) of the composition to the eye. In another embodiment, multiple injections are made in different parts of the same eye. In another embodiment, the second administration of the rAAV comprising the selected expression cassette (e.g., a cassette comprising LCA5) is performed at a later point in time. Such time points may be weeks, months or years after the first administration. In one embodiment, this second administration is performed with a rAAV having a different capsid than the rAAV administered first. In another embodiment, the first and second administered rAAV have the same capsid.

In still other embodiments, the compositions described herein can be delivered in a single composition or multiple compositions. Optionally, two or more different AAVs, or multiple viruses, may be delivered [ see, e.g., WO 2011/126808 and WO2013/049493], in another embodiment, the multiple viruses may contain different replication-defective viruses (e.g., AAV and adenovirus).

In certain embodiments of the invention, noninvasive retinal imaging and functional studies are required to identify the areas of rod and cone photoreceptors to which therapy will be targeted and to test treatment efficacy. In these embodiments, clinical diagnostic tests are used to determine the precise location of one or more subretinal injections. These tests may include Electroretinograms (ERG), visual field examinations, topographic mapping of the layers of the retina and measurement of the thickness of its layers by means of confocal laser tomography ophthalmoscopy (cSLO) and Optical Coherence Tomography (OCT), topographic mapping of cone density via Adaptive Optics (AO), functional ophthalmic examinations, multi-electrode arrays (MEA), pupillary light response, etc., depending on the kind of subject treated, its physical condition and health and dose. In view of imaging and functional studies, in some embodiments of the invention, one or more injections are made in the same eye to target different areas of the affected eye. The volume and viral titer of each injection is determined separately, as further described herein, and may be the same or different from other injections made in the ipsilateral or contralateral eye. In another embodiment, a single, larger volume injection is performed in order to treat the entire eye. In one embodiment, the volume and concentration of the rAAV composition are selected such that only a region of the damaged ocular cells is affected. In another embodiment, the volume and/or concentration of the rAAV composition is a greater amount in order to reach more of the eye, including non-damaged photoreceptors.

In another embodiment, the method comprises conducting additional studies, e.g., functional and imaging studies, in order to determine treatment efficacy. For examination in animals, such tests include assessment of retinal and visual function via Electroretinograms (ERGs) investigating rod and cone photoreceptor function, tremor of the eye, pupillometry, water maze test, light-dark preference, optical coherence tomography (to measure the thickness of each layer of the retina), histology (thickness of the retina, lines of nuclei in the outer nuclear layer, immunofluorescence to record transgene expression, cone photoreceptor counting, staining of retinal sections with peanut agglutinin-which identifies the cone photoreceptor sheath).

In particular, for human subjects, following administration of a dose of the composition described in the specification, the subjects were tested for therapeutic efficacy using the following means: electroretinograms (ERGs) to examine rod and cone photoreceptor function, pupillometry visual acuity, contrast-sensitive color vision tests, visual field tests (hanflei/goldmann visual field), visual field examination performance tests (beyond handicap training), and reading speed tests. Other useful post-treatment efficacy tests to which a subject is exposed following treatment with a pharmaceutical composition described herein are functional magnetic resonance imaging (fMRI), full-field photosensitivity tests, retinal structure studies including optical coherence tomography, fundus photography, fundus autofluorescence, adaptive optical laser scanning ophthalmoscopy, motility tests, tests for reading speed and accuracy, microfield examination, and/or ophthalmoscopy. These and other efficacy tests are described in U.S. patent nos. 8,147,823; in co-pending international patent application publication WO2014/011210 or WO 2014/124282, which are incorporated by reference.

In one embodiment of the methods described herein, a single intraocular delivery of a composition as described herein (e.g., AAV delivery of optimized LCA5 cassette) is suitable for treating an LCA in a subject. In another embodiment of the methods described herein, a single intraocular delivery of a composition as described herein (e.g., AAV delivery of optimized LCA5 cassette) is suitable for treating an LCA in a subject at risk.

Thus, in one embodiment, the composition is administered prior to the onset of the disease. In another embodiment, the composition is administered prior to the onset of vision impairment or loss. In another embodiment, the composition is administered after the onset of vision impairment or loss. In yet another embodiment, the composition is administered when less than 90% of the rods and/or cones or photoreceptors function normally or remain as compared to a non-diseased eye. In one embodiment, neonatal treatment is defined as administration of the lebrcilin coding sequence, expression cassette or vector as described herein within 8 hours, the first 12 hours, the first 24 hours or the first 48 hours of delivery. In another embodiment, particularly for primates (human or non-human), neonatal delivery is over a period of about 12 hours to about 1 week, 2 weeks, 3 weeks, or about 1 month or after about 24 hours to about 48 hours. In another embodiment, the composition is delivered after the symptoms appear. In one embodiment, treatment (e.g., first injection) of a patient is initiated prior to the first year of life. In another embodiment, treatment is initiated after 1 year of age, or after 2 to 3 years of age, after 5 years of age, after 11 years of age, or at a later age. In one embodiment, treatment begins at about 4 to about 12 years of age. In one embodiment, treatment begins at or after about 4 years of age. In one embodiment, treatment begins at or after about 5 years of age. In one embodiment, treatment begins at or after about 6 years of age. In one embodiment, treatment begins at or after about the age of 7 years. In one embodiment, treatment begins at or after about 8 years of age. In one embodiment, treatment begins at or after about the age of 9 years. In one embodiment, treatment begins at or after about 10 years of age. In one embodiment, treatment begins at or after about 11 years of age. In one embodiment, treatment begins at or after about 12 years of age. However, treatment may begin at or after about 15 years of age, about 20 years of age, about 25 years of age, about 30 years of age, about 35 years of age, or about 40 years of age. In one embodiment, intrauterine treatment is defined as administration of a composition described herein in a fetus. See, e.g., David et al, Recombinant adheno-associated virus-mediated in Gene transfer viral transfer therapeutic transfer expression in the sheet, Hum Gene ther, 4 months 2011; 22(4) 419-26.doi:10.1089/hum.2010.007.epub 2011, 2/month 2, which is incorporated herein by reference.

In another embodiment, the composition is administered at a later date. Optionally, more than one reapplication is allowed. Such re-administration may be with the same type of vector, a different viral vector, or via non-viral delivery as described herein. In one embodiment, the vector is reapplied to a different portion of the retina from the initial injection to the patient. In one embodiment, the carrier is reapplied to the same portion of the retina that the patient was originally injected with.

In yet another embodiment, any of the above methods can be performed in combination with another or second therapy. The second therapy may be any now known or as yet unknown therapy that helps prevent, prevent or ameliorate these mutations or defects or any effects associated therewith. The second therapy may be administered prior to, concurrently with, or after administration of the composition as described above. In one embodiment, the second therapy involves a non-specific method for maintaining retinal cell health, such as administration of neurotrophic factors, antioxidants, anti-apoptotic agents. Non-specific methods are achieved by injection of proteins, recombinant DNA, recombinant viral vectors, stem cells, fetal tissue or genetically modified cells. The latter may comprise encapsulated genetically modified cells.

In one embodiment, a method of producing a recombinant rAAV comprises obtaining a plasmid containing an AAV expression cassette as described above and culturing a packaging cell carrying the plasmid in the presence of viral sequences sufficient to allow packaging of the AAV viral genome into an infectious AAV envelope or capsid. Particular methods of rAAV vector production are described above and can be used to produce rAAV vectors that can deliver codon-optimized LCA5 in the expression cassettes and genomes described above and in the examples below.

In certain embodiments of the invention, the subject suffers from Leber Congenital Amaurosis (LCA) and the components, compositions and methods of the invention are designed to treat such conditions. As used herein, the term "subject" means a mammal, including humans, veterinary or farm animals, livestock or pets, and animals commonly used in clinical studies. In one embodiment, the subject of these methods and compositions is a human. Still other suitable subjects include, but are not limited to, murine, rat, canine, feline, porcine, bovine, ovine, non-human primates, and the like. As used herein, the terms "subject" and "patient" are used interchangeably.

As used herein, the term "treatment" or "treating" is defined to encompass the administration of one or more compounds or compositions described herein to a subject for the purpose of improving one or more symptoms of LCA. "treating" may thus include one or more of the following in a given subject: reducing the onset or progression of LCA; preventing diseases; reducing the severity of disease symptoms; or delay its progression, including the progression of blindness; eliminating disease symptoms; delay the onset of disease or monitor disease progression or efficacy of therapy.

It should be noted that the term "a" or "an" refers to one or more than one. Thus, the terms "a/an", "one or more" and "at least one" are used interchangeably herein.

The word "comprising" is intended to be inclusive and not exclusive. The word "consisting of" and variations thereof is to be interpreted exclusively, rather than exclusively. Although various embodiments in the specification have been presented using the language "comprising," related embodiments are also intended to be interpreted and described using the language "consisting of or" consisting essentially of.

As used herein, "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal state in a subject.

As used herein, unless otherwise noted, the terms "about" or "to" mean 10% difference from the given reference value.

As used herein, the term "modulate" or variants thereof refers to the ability of a composition to inhibit one or more components of a biological pathway.

Unless defined otherwise herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and with reference to the disclosed text, which provides those skilled in the art with a general guidance for many of the terms used in this application.