Methods and compositions related to TRNA therapeutics for treatment of visual loss

Abstract

Provided herein are compositions and methods related to tRNA therapeutic agents for the treatment of visual loss and blindness.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 63 / 333,442, filed on April 21, 2022, the entire contents of which are incorporated herein by reference.

[0003] Statement Regarding Federally Sponsored Research

[0004] This invention was made with government support under Grant EY032434 awarded by the National Institutes of Health. The government has certain rights in this invention. Field of the Invention

[0005] Provided herein are compositions and methods relating to tRNA therapeutics for treating vision loss and blindness. Summary of the invention

[0006] Premature stop codons (PTCs) are caused by a single nucleotide mutation that converts a typical triplet nucleotide codon to one of three stop codons, such as TAG, TGA, or TAA. One such example is Leber congenital amaurosis (LCA), a rare type of inherited eye disorder that causes severe vision loss at birth, affects approximately 200,000 people and results from a variety of genetic defects. Of these, approximately 22,000 people have PTC-related eye disease (an incidence of approximately 11%), and this number is likely to continue to grow as sequencing analysis becomes more common. The FDA has approved AAV-mediated gene therapy It is only indicated for patients with biallelic RPE65 (mutation-associated retinal dystrophy), but patients who benefit from Luxturna account for a subset of LCA mutations (47).

[0007] Specifically, the eye and retina are highly attractive tissues for drug targets due to their anatomical and immunological advantages, as demonstrated by anti-VEGF delivery via the intravitreal route (49) and RPE65 gene therapy via the subretinal route (50). Systemic delivery of drugs to the retina is challenging because the blood-retinal barrier limits the full utilization of drugs in the retina. Intravitreal administration is considered to be the most advantageous because, in addition to targeting the posterior retina (which can cause diffusion restrictions), multiple dosing intervals can be tolerated (51). Drugs can be delivered to the posterior retina via subretinal and suprachoroidal routes (52, 53). Importantly, there are a variety of methods for viral and non-viral delivery of nucleic acid-based therapies for the eye; however, until recently, the choice of scalable in vivo "delivery" methods has remained limited. A method for repairing one or more PTCs in one or more genes is provided herein. The provided method can be used to treat vision loss and blindness. Therefore, methods and compositions for achieving this purpose are provided herein.

[0008] In one aspect, a method of reading through one or more premature stop codons (PTCs) in an eye cell is provided, comprising delivering one or more tRNAs, one or more oligonucleotides encoding one or more tRNAs, one or more vectors encoding one or more tRNAs, one or more expression cassettes encoding one or more tRNAs, or one or more compositions comprising any of the foregoing provided herein, to an eye cell in an amount effective to read through the one or more PTCs.

[0009] In another aspect, a method for restoring protein function in an eye cell is provided, comprising delivering one or more tRNAs, one or more oligonucleotides encoding one or more tRNAs, one or more vectors encoding one or more tRNAs, one or more expression cassettes encoding one or more tRNAs, or one or more compositions comprising any of the foregoing provided herein to an eye cell in an amount effective to restore protein function in the cell.

[0010] In another aspect, a method of restoring vision in a subject is provided, comprising delivering one or more tRNAs, one or more oligonucleotides encoding one or more tRNAs, one or more vectors encoding one or more tRNAs, one or more expression cassettes encoding one or more tRNAs, or one or more compositions comprising any of the foregoing provided herein, to cells of the eye in an amount effective to restore vision in the subject.

[0011] In one embodiment of any of the methods provided herein, the composition used is any of the compositions provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The folded tRNA structure is shown (left), with the anticodon region shown as a loop at the bottom of the structure. In this example, UCG is the wild-type tRNA anticodon for decoding the CGA arginine codon. G To UC A The (underlined) mutation converts this tRNA to a UGA(RNA) stop codon suppressor.

[0013] Figure 2 The tRNA suppressors for each human pathogenic PTC identified are shown.

[0014] Figure 3 It is shown that the engineered tRNA does not suppress normal protein stop codons, also known as "true stop codons". The average log2-fold change in the normalized ribosome footprint occupancy of each nucleotide from -50 to +50nt around the stop codon of the transcriptome (18,101 sequences) is plotted. The cartoon illustrates the offset of about 15nt from the 5' end of the ribosome footprint to the first base position of the stop codon in the ribosome. The illustration on the right shows that the ribosome footprint density on the 3'UTR is plotted as the log2-fold change of the readings of the treated cells compared to the control cells (puc57GG empty vector). Transcripts are grouped according to their endogenous UAA, UAG and UGA stop codons. Each point represents the average of two replicates of the transcript. The error bar represents the mean ± SD of the log2-fold change.

[0015] Figures 4A to 4D show high fidelity post-transcriptional PTC repair using ace-tRNA. Figure 4A shows the PTC repair peptides for glycine in HEK cells as determined by mass spectrometry. UGA and tryptophan UGA Figure 4B shows ace-tRNA (for Gly TGA ) showed stop codon specificity and was superior to the read-through compounds gentamicin (100 mM) and G418 (10 mM). FIG. 4C shows missense mutations W53Q and W53E potassium channels. W53Q and W53E channels showed reduced expression and altered gating. FIG. 4D shows an average graph of membrane potential, showing depolarization of W53E and W53Q channels.

[0016] Figure 5Shown is the PTC rescue that is independent on position.Center, the topology of 24 transmembrane, 250kD, Nav1.2 sodium channel encoded by SNC2A gene, the topology of this channel is similar to CACNA1F, and wherein PTC causes night blindness.The upper left corner shows the representative family of wild-type voltage-gated sodium current caused by -140mV holding potential expressed in HEK cells by 20 milliseconds of depolarization.Shown are five PTC sites relevant to nervous system disease (developmental delay): R607X, S700X, G1013X, R1626X and W1716X from (ClinVar).Each mutant does not show measurable activity when expressed separately.For each site, the exemplary rescue sodium current under each condition of mutant SCN2A cDNA cotransfection is shown.

[0017] Fig. 6A 6C show schematic representations of SNPs designed for intravitreal injection. Fig. 6A Shown is the design of intravitreal injection of SNPs for PR / RPE targeted delivery. Figure 6B Flow chart showing the microemulsion process for preparing SNPs. Figure 6C shows a schematic diagram of the cellular uptake of SNPs and subcellular release of the payload into the cytosol.

[0018] 7A to 7D Shows the delivery efficiency of SNP to nucleic acids (pDNA and mRNA) and CRISPRRNP. Fig. 7A ) SNP loaded with pDNA and ( Figure 7B ) Transfection efficiency of SNP loaded with mRNA in HEK 293T cells. Figure 7C ) Genome editing efficiency of RNP-loaded SNPs in HEK293T cells expressing GFP. Fig.7D ) Viability of HEK 293T cells treated with Lipo2000 and different concentrations of SNPs.

[0019] FIG. 8A to FIG. 8E SNPs loaded with Cre mRNA or SNPs loaded with Cas9 RNP are shown to induce genome editing in vivo in Ai14 mice by subretinal injection. ( Fig. 8A )TdTomato locus in Ail4 reporter mice. A LoxP-flanked stop cassette containing three Ai14 sgRNA target sites prevents downstream tdTomato expression. Cre-Lox recombination or Cas9RNP-guided excision of the stop cassette results in tdTomato expression. FIG. 8B to FIG. 8D ) Representative images of tdTomato cells 4 days after injection: ( Figure 8B ) SNP-11cRal loaded with Cre mRNA edits PR, ( Figure 8C) SNP-ATRA loaded with Cre mRNA edits RPE, ( Fig.8D )PBS was used as control. Fig. 8E ) Representative images of tdTomato+ cells 12 days after injection of SNP-ATRA loaded with Cas9 RNP. Scale bar: 500 μm in RPE / retinal floret images; 100 μm in magnified images.

[0020] FIG. 9A to FIG. 9B Shown is the induction of transfection of SNP loaded with 5X tRNA in Ai14 mice by subretinal injection. ( FIG. 9A to FIG. 9B ) Representative images of tRNA+ cells (GFP+) 4 days after injection: ( Fig. 9A ) PBS as a control, and ( Fig. 9B ) SNP-ATRA loaded with 5XtRNA targets RPE cells.

[0021] Figures 10A to 10D show the absorption of SNP-CPP loaded with RNP+ssODN by iPSC-RPE after 4 days of treatment. Control cells in 20X and 50X (Figure 10A) bright field images. (Figure 10B) ATTO-488 fluorescence after delivery of 3ug RNP in superimposed images and reconstructed z-stack fluorescence images (lower sub-figures). (Figure 10C) Same as Figure 10B, but for ATTO-488 positive cells after delivery of 6ug RNP. (Figure 10D) Same as Figure 10B and Figure 10C, for ATTO-488 positive cells after delivery of 12ug RNP.

[0022] FIG. 11A to FIG. 11I We show that putative Kir7.1 loss of function can be cured by genetic enhancement. Fig.11A ) LCA16 patient-derived iPSC-RPE and TAG stop codon ( Fig. 11B ). Fig. 11C ) Graph of average current-voltage (I / V) curves of Kir7.1 current from patient iPSC-RPE and control iPSC-RPE. ( Fig.11D ) control iPSC-RPE and TGG genotype ( Fig.11E ). Fig.11F )like Fig.11F Diagram IV in Figure 4 shows the depolarization of the membrane potential. Fig.11G ) Diagram IV of K+ or Rb+ use in LCA16hiPSC-RPE cells after gene therapy. Fig.11H ) Graph of mean membrane potentials for control, LCA16, and after gene therapy. ( Fig.11I ) Distribution of wild-type Kir7.1 channels in LCA16 iPSC-RPE after gene therapy.

[0023] FIG. 12A to FIG. 12F Displays the use of tRNA Trp Functional restoration of Kir7.1LCA16 mutants. Fig. 12A )W53X cells, showing the cytoplasmic distribution of GFP. Fig. 12B ) via tRNA Trp Membrane localization of processed Kir7.1 channels ( Fig. 12C ) membrane potential, ( Fig.12D )Normal Kir7.1 current, ( Fig.12E , Fig.12F ). Rb permeability increased after treatment.

[0024] FIG. 13A to FIG. 13B (From Capowski et al., 2019) shows light microscopy (LM) identification of different morphological stages in live cultures of differentiated hPSC-derived 3D retinal organoids. Fig.13A ) Schematic representation of the hPSC retinal differentiation protocols that can be used. ( Fig. 13B ) Timing and overlap of morphological stages observed in all 16 cell lines tested. Representative in vivo LM stage images of stage 1, stage 2, and stage 3 organoids. Scale bar = 100 microns. Stage 3 right panel is a magnification of the box in the stage 3 light panel showing the outer segments, the far right panel is a multi-lobed stage 3 organoid. Scale bar = 50 microns.

[0025] FIG. 14A to FIG. 14L Stage 3 organoids are shown to have a well-defined outer nuclear-like layer (ONL) with mature rods and cones. Fig.14A ) ICC image of stage 3 organoid (d200) shows that the ONL region contains an outermost layer of M / L-OPSIN+ / CRX+ cones with M / L-OPSIN+ surface processes (arrows) and a 3-5 nuclei thick bottom layer of NRL+ / CRX+ rods. Mislocalized CRX+PRs, some co-expressing M / L OPSIN or NRL, are also present beneath the ONL (asterisks) (Scale bar = 100 μm). ( FIG. 14B to FIG. 14C ) Higher magnification ICC image of stage 3 organoid (d160) showing M / L-OPSIN+( Fig. 14B ) and S-OPSIN+( Fig. 14C ) Location and morphology of cones and NR2E3+ rods (B′ and C′, fused in B″ and C″) (scale bar = 25 μm). Fig.14D and Fig.14E ) Percentage of NRL+ rods in the ONL from three different WT hiPSC cell lines ( Fig.14D ) and percentage of ARR3+ cones ( Fig.14E ). Fig.14F , Figure 14G ) M / L-OPSIN+( Fig.14F ) and S-OPSIN+( Figure 14G )Cones percentage. Fig.14H ) 3D multiphoton rendering of a 259-day-old organoid showing the surface distribution of RHO+ rods and M / L-OPSIN+ cones (scale bar = 100 μm). ( FIG. 14I to FIG. 14K )For M / L OPSIN( Fig.14I ) and RHO( Fig.14J ; integrated into Figure 14K ) Confocal image of a single z-plane through the immunostained organoid shown in panel H. ( Figure 14L ) Light microscopy image of an organoid taken before fixation, showing the lobulated regions of the pigmented RPE ( FIG. 14I to FIG. 14L ) and hair-like protrusions covering the rest of the organoid surface (arrows). FIG. 14I to FIG. 14L Scale bar = 500 μm.

[0026] Figures 15A to 15G Transduction of photoreceptors in hiPSC-derived retinal organoids using two different AAV capsids is shown. The CAG-GFP expression construct was packaged in AAV5 or AAV7m8 and used to transduce retinal organoids at day 90 of differentiation. GFP expression was present throughout the organoids two weeks after transduction (no infection control: Fig.15A (left panel), Fig.15A (right panel); AAV5: Fig. 15B (left panel), Fig. 15B (right panel); AAV7m8: Fig. 15C (left panel), Fig. 15C (right panel) and in sections (AAV5: FIG. 15D to FIG. 15F ; AAV7m8: Figure 15G ). Overall, AAV5 is more potent than AAV7m8; however, both capsids may be improved after further titer optimization. Scale bar: FIG. 15A to FIG. 15C =200mm; FIG. 15D to FIG. 15F =50mm; Figure 15G =25mm.

[0027] FIG. 16A to FIG. 1 6I shows AAV2.7m8-mediated transduction of iPSC-RPE and mouse RPE in vivo. ( Fig.16A The small tRNA size enables multiplexing of multiple tRNA copies of payload and reporter genes within 4 kb. Fig. 16B) Nanoluciferase-terminated and eGFP-terminated reporter genes will be used with the corresponding stop codons (TGA, TAA, TAG) in the reporter construct. Fig. 16C ) Delivery and inhibitory activity of AAV (2 / 2) of Nluc-TGA + 3X Arg-UGA inhibitor induced differentiation of iPSCs into excitatory neurons in day 14 lentiviral-mediated NGN2 expression. X-axis, 1 μl or 0.1 μl per well (titer vg / ml 2.2E13) (96-well plate). ( Fig.16D , left panel) Bright field images of hiPSC-RPE and ( Fig.16D , right panel) GFP expression after transduction. The same rAAV2.7m8 vector was delivered into the mouse eye by intravitreal injection ( Fig.16E ). Fig.16F )Fundus autofluorescence images of mouse RPE after transduction. Figure 16G ) OCT image of the complete retinal layers. Fig.16H , left panel) Flat mounts of RPE cells were imaged for GFP expression. ( Fig.16H , right panel) Higher magnification of GFP expressing RPE as in (16H, left panel).

[0028] FIG. 17A to FIG. 17C The design of SNPs for subretinal injection is shown. ( Fig.17A ) Schematic representation of SNP, injection method, and SNP penetration through the retinal barrier. Fig. 17B ) Schematic flow chart for fabrication of SNPs and surface modification with HA coating. ( Fig. 17C ) Synthesis scheme of HA conjugated with ligands (11cRal and ATRA) for SNP surface coating.

[0029] 18A to 18C The design of subretinal injection of SNPs is shown as a potential alternative approach. Fig.18A ) Schematic representation of SNP and injection method. Fig.18B ) Schematic flow chart for SNP fabrication and surface modification. ( Fig. 18C ) Synthesis scheme of SNPs conjugated to ligands (e.g., 11cRal, ATRA, and CPP (e.g., TAT or POD)).

[0030] Fig.19 Schematic representation showing AAV / SNP / MC-mediated RPE or PR targeting for delivery of therapeutic tRNA.

[0031] FIG. 20A to FIG. 20F Ace-tRNA delivery via minicircle and viral approaches is shown.

[0032] Fig.21 A schematic representation of HA-SNP fabrication is provided.

[0033] Fig. 22 Figure 2 shows the efficiency of nucleic acid and RNP delivery of SNP in Ai14 mice by subretinal injection. Efficient delivery of Cre-mRNA by SNP-PEG-ATRA in mouse RPE. D1, RPE florets of eyes injected subretina with Cre-mRNA encapsulated SNP; D2, 20x magnification image of tdTomato+RPE tissue; D3, RPE florets of PBS control.

[0034] FIG. 23A to FIG. 23E Shows that Kv8.2E148* modulates Kv2.1 currents. FIG. 23A to FIG. 23C )Current trace to voltage pulse. ( Fig.23D ) Maximum current amplitude diagram. ( Fig.23E )like Fig.23D Color coding of activation curves in .

[0035] Fig.24 Nuclear localization of ATTO 550 in 3D organoid PR is shown.

[0036] Fig.25 PCR products and Sanger sequencing results are shown. DETAILED DESCRIPTION

[0037] There are limited therapeutic options for repairing premature stop codons (PTCs). Nonsense mutations can result from the insertion of inherited or spontaneous nucleotide changes within a single codon, thereby introducing an in-frame PTC into the mRNA sequence (1). Ten amino acid codons are susceptible to PTC conversion by single nucleotide substitutions: arginine, tryptophan, tyrosine, cysteine, glutamate, lysine, glutamine, serine, leucine, and glycine (2, 3). This is because one of their triplets is within one base of one of the three stop codons (TGA, TAA, TAG). Because these mutations terminate protein synthesis, they are phenotypically severe and more pathogenic, and they account for 10-15% of all human genetic diseases (4). Collectively, “PTC diseases” represent a group of hundreds of genes containing PTCs, resulting in a large number of individual rare diseases with a common molecular basis: the introduction of a stop codon. Each diseased gene itself may have hundreds of unique PTC genetic lesions, which together pose a huge challenge to treatment. Recent estimates suggest that as many as 2.5 to 3 million patients in the United States alone suffer from PTC caused by either spontaneous or inherited mutations. Clinical or therapeutic options for PTC repair are limited. Small molecules such as aminoglycosides (5), dipeptides (6), and oxadiazoles (7) promote “readthrough” of PTC (8) but result in encoding a near-homologous amino acid (9), effectively creating a missense mutation in place of the PTC. Ion channels critical for visual transduction, such as burcortin, Kir7.1, Kv8.2, TRPM1, and Ca2+ channels, are poorly tolerated to missense mutations, which can result in gain-of-function or loss-of-function neurological disorders (10, 11). Furthermore, aminoglycosides, the first-line read-through drugs, are ototoxic and nephrotoxic when used chronically (12), and first-in-class read-through compounds such as oxadiazoles and ataluren show unexpectedly low efficacy in this patient population (ACT DMD Phase 3 clinical trial, NCT01826487; ACTCF, NCT02139306).

[0038] Recent and ongoing advances in CRISPR / Cas9-mediated genome editing offer potential permanent solutions for diseases caused by nonsense mutations, and such approaches have recently been successful in repairing retinal genes (13). However, certain aspects of this technology present obstacles to its rapid therapeutic application (14, 15), including cell type-specific delivery, efficiency of homologous recombination, and frequency of off-target editing. It is also a complex process of CRISPR / Cas9 construct validation and off-target assessment for each of the hundreds of unique PTCs in the context of blindness. AAV delivery is promising, but at 5-9 kb per gene transcript, ion channel genes greatly exceed AAV payload capacity limitations, hindering the practicality of rapidly advancing AAV therapies for tissue-specific gene replacement. In addition, when gene delivery is successful, the potential complications caused by AAV-mediated gene enhancement are not trivial and include unexpected gene over- or under-expression, as well as evasion of regulatory pathways that fine-tune ion channel expression in delicate detection systems (such as those in vision). As a result, clinical options for ion channel PTCs are limited and the therapeutic pipeline is weak.

[0039] A universal therapeutic approach based on anticodon-edited short transfer RNA (ace-tRNA) to repair retinal PTCs in vivo has been developed (16). Data suggest that this approach exhibits the versatility of small molecule therapeutics and the precision of gene editing, with essentially no interaction with “true” stop codons. This approach is noteworthy for its agnostic nature, that is, an ace-tRNA formulation used to repair the Arg->TGA mutation can be used to treat any such gene carrying the Arg->TGA mutation at any position within the diseased gene. Furthermore, novel ace-tRNA formulations have been identified for repairing each known PTC in retinal disease, thereby providing a single comprehensive approach for treating all nonsense-mediated inherited retinal disorders. Once ace-tRNA technology achieves stable delivery to retinal tissue, it could provide a novel, long-acting gene therapy for a variety of inherited blinding disorders. This article provides an ace-tRNA technology as a retinal therapy for the treatment of inherited eye diseases caused by premature stop codons.

[0040] Provided herein are compositions and methods for repairing one or more PTCs in retinal genes using tRNA therapeutic agents. A variety of gene mutations lead to inherited retinal diseases, including retinitis pigmentosa, cone-rod dystrophy, Leber congenital amaurosis (LCA), macular dystrophy and degeneration (17, 18). Subjects who may benefit from the provided compositions and methods may suffer from any disease or condition that causes vision loss or blindness, such as any disease or condition provided herein. Nonsense mutations lead to about 15% of inherited human diseases, such as retinopathy. However, for a given gene, the percentage may be much higher, even up to about 40% in some cases (HGMD 2019.2). The first step mechanism of the behavior of "seeing" involves phototransduction of PR and its maintenance by RPE (19). Nonsense mutations in the following genes in RPE and PR can lead to retinopathy, all of which are targets of tRNA. Therefore, in one embodiment, any one of the methods or compositions provided herein can be directed to one or more PTCs in one or more of these genes. Subject can be any subject that needs vision restoration, and can be any subject with PTC mutation in one or more retinal genes, such as those subjects provided herein, for example, the subject below.Show genes and PTC that can be repaired in vitro and in vivo, and it is known in the art that there are other genes and PTC.The leftmost is the mutation of disease type, OMIM list, gene name and DNA and RNA level.Identify the candidate tRNA type (table 1) of each amino acid and mutation, and have shown the limited contact with the real stop codon of each major tRNA family type.In any one of compositions or methods provided herein, the tRNA used for us is any one of these tRNA types for the amino acid and mutation identified.In any one of compositions or methods provided herein, such tRNA is used for related diseases or illness as provided herein or as otherwise known in the art.

[0041] Table 1

[0042] PTC Blindness OMIM Gene DNA RNA References VMD 607854 BEST1, TYR29TER 87C-G UAC-UAG (20) VMD 611809 BEST1, ARG200TER 598C-T CGA-UGA (21) VMD 611809 BEST1, SER517TER 1550C-G UCA-UGA (22) Cone dystrophy 610356 KCNV2, GLU143TER 427G-T GAA-UAA (23) Cone dystrophy 610356 KCNV2, GLU306TER 916G-T GAG-UAG (23) Cone dystrophy 610356 KCNV2, GLN76TER 226C-T CAA-UAA (24) Cone dystrophy 610356 KCNV2, GLU148TER 442G-T GAA-UAA (24) Cone dystrophy 610478 CACNA2D4, TYR802TER 2406C-A UAC-UAA (25) Cone dystrophy 610478 CACNA2D4,ARG628TER 1882C-T CGA-UGA (26) CSNB2A 300071 CACNA1F, ARG958TER 2172C-T CGA-UGA (27) CSNB2A 300071 CACNA1F, ARG830TER 2488C-T CGA-UGA (28) LCA16 614186 KCNJ13, TRP53TER 158G-A UGG-UAG (29) LCA16 614186 KCNJ13,ARG166TER 496C-T CGA-UGA (30) CSNB1C 613216 TRPM1, GLN11, TER 31C-T CAA-UAA (31) CSNB 613216 TRPM1, LYS294TER 880A-T AAG-UAG (32) CSNB 613216 TRPM1,ARG877TER 2629C-T CGA-UGA (32) CSNB 613216 TRPM1, SER882, TER 2645C-G UCA-UGA (33)

[0043] For example, KCNJ13 encodes Kir7.1, a homotetrameric inwardly rectifying potassium channel localized to the apical membrane of the RPE (34). This channel protein is essential for maintaining potassium homeostasis in the subretinal space, and its function is critical for vision (35). Sergouniotis et al. identified the R166X mutation in a Middle Eastern family, in which a C to T mutation results in a premature stop codon (TGA) instead of an arginine (CGA) (30). The R166X mutant lacks the C-terminal tail that is required for homotetramer formation and localization to the membrane, resulting in greatly reduced channel activity. A 12-year-old proband of Middle Eastern descent was studied who was homozygous for the nonsense mutation 158G>A, resulting in W53X PTC. Clinical manifestations were characteristic of the LCA phenotype, including abnormal ERGs, nystagmus, RPE pigmentation, and loss of outer retinal architecture. At age six, the ERG a- and b-wave amplitudes were reduced. At nine years of age, the pattern electroretinogram is undetectable, indicating loss of PR function (29, 30, 36-38).

[0044] As another example, the KCNV2 gene is located on chromosome 9p24.2 (39). A rare form of cone dystrophy with enhanced rod electroretinogram is caused by a homozygous nonsense mutation in the KCNV2 gene encoding the Kv8.2 protein (40). Kv8.2 forms a heterotetrameric complex with Kv2.1 to form a unique photoreceptor outwardly rectifying potassium channel responsible for the circulating dark current. Wu et al. first demonstrated a nonsense mutation 427G-T within exon 1 of the KCNV2 gene, which results in the mutation E143X. Within exon 1, Wissinger et al. discovered several nonsense mutations (E73X, Q76X, E148X, K260X, and Q287X) by sequencing the entire coding sequence, and found a 442G-T transition resulting in E148X in several patients tested (24). Another homozygous 916G-T mutation results in the E306X stop codon (23).

[0045] As a further example, burocortin 1 (BEST1 or VMD2) is located on chromosome 11 (11q 12.3) and encodes a transmembrane 585 amino acid chloride channel that is localized to the basement membrane of the RPE (41, 42). In the RPE, burocortin is essential for regulating fluid and ion homeostasis and ion transport (42). BEST1 mutations can cause autosomal dominant yolk lesions (Best yolk macular dystrophy, BVMD) and autosomal recessive yolk lesions (ARB). Burgess et al. identified a nonsense mutation in BEST1 that can cause BVMD (R200X mutation caused by 598C>T substitution (21)), while Schatz et al. described a Y29X nonsense mutation (20). Recently, Chibani et al. found a nonsense mutation C1550G (p.S517X) in a Tunisian BVMD family (22). However, VMD is a rare disease with unknown incidence.

[0046] The TRPM1 gene is mapped to chromosome 15q 13-q 14 and encodes a cation-permeable transient receptor ion channel. TRPM1 channels are present in the ON-bipolar cells of the retina, where they control the mGluR6 transduction cascade. In a recent cohort study, an autosomal recessive inheritance rate of 1:6210 was found in the Jewish community (32). Several nonsense mutations are present in a compound heterozygous state, including Q11X, S882X, and Y1035X (31, 33). CACNA2D4 is present on chromosome 12p13.3 and encodes the voltage-dependent calcium channel α-2 / δ subunit 4 (43). At the PR terminus, these specialized calcium channels mediate calcium influx, thereby initiating glutamate release (25). By whole genome sequencing, two probands carrying the homozygous nonsense mutation R628X have presented with non-progressive cone dysfunction (26). Similarly, two siblings with slowly progressive cone dysfunction were diagnosed with the homozygous nonsense mutation Y802X (26).

[0047] As a further example, CACNA1F is present on Xp11.23 and encodes a 1,985 amino acid L-type calcium channel protein present in the outer nuclear layer, inner nuclear layer, and ganglion cell layer (44-46). Two nonsense mutations identified in patients with congenital stationary night blindness are R830X and R958X (27, 46).

[0048] Still other nonsense mutations are obvious to those of ordinary skill in the art. Increased genetic testing has enabled the discovery of pathogenic gene mutations that cause blindness in the outer retinal channel disease. Although these mutations have definite physiological relevance, their diverse clinical manifestations present challenges to disease management. Currently, there are no FDA-approved treatment options, and the complexity of these membrane proteins exacerbates this problem. In addition, although specific diseases rarely have genetic heterogeneity, they have common genetic damage (early termination codons). Therefore, the methods and compositions provided herein and associated with tRNA therapeutics can provide treatments that have been difficult to achieve so far.

[0049] There are multiple tRNA genes for each amino acid type, each with a unique sequence (isodecoder) for a given cognate amino acid (isoacceptor), resulting in >400 tRNAs annotated in the human genome (56, 57). Screening for tRNAs with single (human) anticodon mutations that retain PTC inhibitory efficacy in mammalian cells has been performed (16). This approach has identified a large number of inhibitory tRNAs. Figure 1 The left figure shows the folded tRNA structure, where the anticodon region is shown as a loop at the bottom of the structure. In this example, UCG is the wild-type tRNA anticodon for decoding the CGA arginine codon. G To UC A The (underlined) mutation converts this tRNA to a UGA(RNA) stop codon suppressor.

[0050] tRNA suppressors have been identified for each known human disease-causing PTC ( Figure 2 ). Data are shown relative to normalized folded background NLuc luminescence on a Log10 scale (below), with each bar representing a single engineered tRNA sequence. There are multiple unique tRNA sequences (isodecoders) within each tRNA family, with the anticodon editing type indicated in superscript in each case. The dashed line represents baseline tRNA inhibitory activity. Each tRNA dataset was obtained in triplicate and is shown as mean ± SEM.

[0051] Nucleotide sequences encoding hundreds of human tRNAs are known and generally available to those skilled in the art through sources such as Genbank. The structure of tRNA is highly conserved, and tRNA can function across species. Therefore, bacterial or other eukaryotic tRNA sequences are also potential sources of tRNAs disclosed herein. Determining whether a specific tRNA has the desired function (such as in a desired mammalian cell) can be determined as described herein or by other experiments that will be apparent to those of ordinary skill in the art who benefit from the teachings provided herein. In some embodiments, the tRNA sequence can be any sequence provided in PCT / US2018 / 059065, WO2019 / 090 154, WO2019 / 090169, and Lueck et al. Nature Communications 10,822,2019, and the disclosure of tRNA and its sequence is incorporated herein by reference. Therefore, in one embodiment of any one of the compositions or methods provided herein, the one or more tRNAs are selected from any of the foregoing.

[0052] tRNA and nucleotide sequences encoding the tRNA can be produced synthetically. The tRNA can be delivered as DNA or RNA (e.g., viral vectors (e.g., AAV) / minicircles / nanoparticles). Data are provided for viral and non-viral delivery methods of cDNA and RNA to deliver nucleic acids to retinal cells, providing proof of principle. tRNA is generally small (~100 bp). It should be noted that:

[0053] 1. The rescue protein level of PTC-containing genes is based on the activity of the endogenous gene promoter, and the mRNA present in the targeted cells will therefore be affected by normal cellular regulatory events. This is important for ion channel genes that require a tightly regulated expression pattern.

[0054] 2. The tRNA suppression approach is "position agnostic," that is, it can be repaired regardless of where the in-frame stop codon is located or the context within the gene of interest or any gene. In addition, preliminary data suggest that the size of the gene containing the PTC does not affect the rescue efficacy, allowing the targeting of large genes that exceed the viral payload, such as cytoskeletal proteins, pumps, transport proteins, and ion channels.

[0055] In some embodiments of any composition or method provided herein, the tRNA therapeutic agent can be delivered as DNA (such as cDNA) or RNA. The one or more tRNAs can be in the form of any suitable recombinant plasmid, which comprises a heterologous nucleic acid sequence delivered to a target cell or subject in vitro or in vivo. Heterologous nucleic acid sequences encode gene products of interest (e.g., tRNA) for any use or method provided herein, including disease treatment, and can optionally be in the form of an expression cassette. The term "recombinant" refers to a polynucleotide that does not exist in nature, or a polynucleotide that is connected to another polynucleotide in an arrangement that does not exist in nature. As used herein, the term "heterologous" refers to a nucleic acid sequence obtained or derived from an entity that is genetically different from the remaining entities compared thereto.

[0056] In certain embodiments of any one of the compositions or methods provided, the tRNA is encoded in an expression cassette. Therefore, the present invention also provides an expression cassette comprising a sequence encoding a tRNA as provided herein. In certain embodiments, the expression cassette also contains a promoter. In certain embodiments, the promoter is a regulatable promoter. In certain embodiments, the promoter is a constitutive promoter. The promoter driving the expression of the sequence encoding the tRNA to be delivered can be any desired promoter, selected according to known considerations, such as the expression level of the nucleic acid functionally connected to the promoter and the cell type to be used of the vector therein. The promoter can be an exogenous or endogenous promoter.

[0057] As used herein, "expression cassette" means a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in an appropriate cell, which may include a promoter operably linked to a nucleotide sequence of interest, which may be operably linked to a termination signal. The expression cassette comprising the nucleotide sequence of interest may be chimeric. The expression cassette may also be a naturally occurring expression cassette that has been obtained in a recombinant form that can be used for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or a regulatable promoter. The expression cassette may be a vector or contained in a vector.

[0058] Typically, the expression cassette includes a heterologous gene and a promoter that controls the transcription of the new gene. The promoter is characterized by having a specific nucleotide sequence required for initiating transcription. Optionally, the expression cassette further includes additional sequences (i.e., enhancers) required for obtaining the desired gene transcription activity. The heterologous gene can be introduced into the nucleic acid immediately downstream of the promoter so that the promoter and the coding sequence are operably connected to allow transcription of the coding sequence.

[0059] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one sequence is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence encoding an RNA or polypeptide if the two sequences are positioned so that the regulatory DNA sequence affects the expression of the coding DNA.

[0060] In addition to at least one promoter and at least one heterologous nucleic acid, the expression cassette may contain a selection gene (e.g., green fluorescent protein (GFP)) for facilitating selection of cells transfected with the expression cassette. Alternatively, cells are transfected with two or more expression cassettes, at least one of which contains one or more genes encoding tRNAs and another of which contains a selection gene. The selection of suitable promoters, enhancers, selection genes, and / or signal sequences is considered to be within the knowledge of those of ordinary skill in the art without undue experimentation.

[0061] The tRNA therapeutic agent can be delivered in the form of a vector. Vectors include, but are not limited to, eukaryotic vectors, prokaryotic vectors (such as bacterial vectors), and viral vectors, including but not limited to retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors (human and others, including pigs), herpes virus vectors, Epstein-Barr viral vectors, SV40 viral vectors, poxvirus vectors, and pseudovirus vectors.

[0062] When in the form of a minicircle, the successful delivery and function of tRNA as provided herein can be successfully performed. Minicircle technology uses small excised, bacterial DNA-free circular DNA fragments, resulting in inserts of any size that can be effectively delivered for both in vitro and in vivo applications. As used herein, the term "minicircle" refers to a small circular DNA fragment that is substantially or completely free of non-essential prokaryotic elements. Minicircles include DNA in a circular form that has no prokaryotic elements and / or prokaryotic elements that have been removed. Minicircles can come from parent plasmids from which bacterial DNA sequences have been excised.

[0063] The use of minicircles has a strong safety profile, and the preparation of minicircles has been described in the art (e.g., in Nehlsen et al., Gene Ther. Mol. Biol. 10:233-244, 2006; and Kay et al., Nature Biotechnology. 28:1287-1289, 2010). For example, the preparation can follow a two-step procedure: (i) generation of a "parental plasmid" (a bacterial plasmid with a eukaryotic insert); and (ii) induction of a site-specific recombinase at the end of this process. These steps can be followed by excision of the prokaryotic vector portion via the recombinase target sequence and recovery by capillary gel electrophoresis.

[0064] As a non-limiting example, a minicircle can be produced as follows. An expression cassette contains a polynucleotide coding sequence and regulatory elements for its expression, flanked by attachment sites for a recombinase. The sequence encoding the recombinase is located outside the expression cassette and includes elements for inducing expression (e.g., an inducible promoter). Under the induction of recombinase expression, the vector DNA recombines to produce two different circular DNA molecules. One of the circular DNA molecules is relatively small and forms a minicircle containing the polynucleotide expression cassette; the minicircle DNA vector does not contain any bacterial DNA sequences. The second circular DNA sequence contains the remaining vector sequences, including bacterial sequences and sequences encoding the recombinase. The minicircle DNA containing the polynucleotide sequence can then be isolated and purified separately. In some embodiments, plasmids similar to pBAD.φ.C31.hFIX and pBAD.φ.C31.RHB can be used to produce minicircle DNA vectors. See, e.g., Chen et al. (2003) Mol.Ther.8:495-500 or as otherwise provided herein.

[0065] Examples of recombinases that can be used to create minicircles include, but are not limited to, Streptomyces phage φ31 integrase, Cre recombinase, and λ integrase / DNA topoisomerase IV complex. Each of these recombinases catalyzes recombination between different sites. For example, φ31 integrase catalyzes recombination between corresponding attP and attB sites, Cre recombinase catalyzes recombination between loxP sites, and λ integrase / DNA topoisomerase IV complex catalyzes recombination between phage λ attP and attB sites.

[0066] Published U.S. Application 20170342424 also describes a system utilizing a parent plasmid that is exposed to an enzyme that causes recombination at a recombination site to form (i) a minicircle comprising a polynucleotide sequence and (ii) a miniplasmid comprising the remainder of the parent plasmid. One recombination site is modified at the 5' end so that it reacts less efficiently with the enzyme than the wild-type site, and another recombination site is modified at the 3' end so that it reacts less efficiently with the enzyme than the wild-type site, and another recombination site is modified at the 3' end so that it reacts less efficiently with the enzyme than the wild-type site, and both modified sites are located within the recombined minicircle.

[0067] Ideally, the removal of prokaryotic sequences should be efficient, using the smallest possible excision site, while creating a supercoiled DNA minicircle consisting only of gene expression elements under appropriate (preferably mammalian) control regions. Some techniques for minicircle generation utilize bacterial phage lambda (λ) integrase-mediated recombination to generate minicircle DNA. See, e.g., Darquet et al., 1997 Gene Ther 4(12):1341-9; Darquet et al., 1999 Gene Ther 6(2):209-18; and Kreiss et al., 1998 Appl Micbiol Biotechnol 49(5):560-7.

[0068] Kits for producing minicircle DNA are known in the art and are commercially available (System Biosciences, Inc., Palo Alto, Calif.). For example, high-quality minicircle DNA can be obtained using the MC-EasyTM (Catalog No. MN920A-1, SBI System Biosciences) minicircle DNA production kit. Information about minicircle DNA is provided in Dietz et al., Vector Engineering and Delivery Molecular Therapy (2013); 218, 1526-1535 and Hou et al., Molecular Therapy-Methods & Clinical Development, Article No.: 14062 (2015) doi: 10.1038 / mtm.2014.62. More information on minicircles is in Chen ZY, He CY, Ehrhardt A, Kay M A. Mol Ther. 2003 Sep;8(3):495-500, and minicircle DNA vectors achieve constitutive expression as reflected by active chromatin and transcript levels. Gracey Maniar LE, Maniar JM, Chen ZY, Lu J, Fire AZ, Kay M A. Mol Ther. 2013 Jan;21(1):131.

[0069] Any composition provided herein can be contacted with cells, applied to cells or introduced into cells using genetic transfer methods (such as transfection). Therefore, any composition provided herein can be contained in a delivery vehicle or in a gene delivery vehicle. The composition provided herein can be contacted with cells or delivered or applied to a subject in a particle (such as a nanoparticle). Particles, such as nanoparticles (NPs), can be but are not limited to lipid-based nanoparticles (also referred to herein as lipid nanoparticles, i.e., nanoparticles whose major materials of its structure are lipids). Particles can also be polymer-based (also referred to herein as polymers or polymer nanoparticles, i.e., nanoparticles whose major materials of its structure are polymers) (e.g., polymer complexes). Lipids or polymer-based NPs can be formed by electrostatic interactions between negatively charged payloads and positively charged carrier materials (such as cationic lipids and cationic polymers).

[0070] In one embodiment of any composition or method provided herein, the nanoparticle is a silica nanoparticle (SNP), such as a glutathione (GSH) reactive silica nanoparticle. The SNP can have (1) high payload loading content and loading efficiency, (2) multifunctional surface chemistry to allow physical coating or chemical bonding of multiple parts to promote transport to the outer retina after intravitreal injection and achieve targeting, (3) small SNP size (e.g., dH<50nm), which is ideal for tissue penetration and diffusion, (4) excellent in vitro and in vivo stability, (5) effective endosome / lysosome escape, (6) once entering the cytosol, the stimulus-responsive payload can be quickly released, (7) excellent biocompatibility and negligible immunogenicity, (8) the ability to scale up, (9) easy to handle, store and transport, or a combination thereof. In one embodiment of any composition or method provided herein, the SNP is a negatively charged hyaluronic acid (HA) coated SNP, which can show enhanced outer retinal permeability after intravitreal injection. In another embodiment of any of the compositions or methods provided herein, in order to enhance the cellular uptake of SNP by target cells, a molecule such as 11cRal or ATRA can be bound to the surface of SNP for delivery.

[0071] The present disclosure provides a method for delivering one or more tRNAs or compositions provided herein to a subject or a subject's cell. One or more tRNAs or compositions as described herein can be delivered to cells in vivo or in vitro. Administration to cells can be accomplished in any manner, including simply contacting the cells. Contact with cells can be sustained for any desired length of time. Cells can include any desired cells in humans and other large (non-rodent) mammals (such as primates, horses, sheep, goats, pigs and dogs). Any subject provided herein can be a human or other mammal. The term "mammal" includes, but is not limited to, humans, mice, rats, guinea pigs, monkeys, dogs, cats, horses, cows, pigs and sheep. Any subject provided herein may suffer from any disease or condition provided herein and / or may benefit from treatment with any tRNA provided herein.

[0072] Also provided in the art or otherwise understood for delivery and introduction of a subject's suitable method. In one embodiment of any composition or method provided herein, the pharmaceutical composition comprises enough genetic material to produce one or more tRNAs of interest for treatment of an effective amount. The tRNA or genetic material encoding the one or more tRNAs can be delivered in an effective amount and delivered to the cell with, for example, an endogenous tRNA synthetase. If the tRNA synthetase is present in the cell into which the tRNA is introduced according to the present invention, the tRNA synthetase is considered to be "endogenous" to the cell. It is obvious to those of ordinary skill in the art that, for these purposes, the tRNA synthetase can be considered to be endogenous, whether it is naturally present in the cell of the relevant type, or whether the specific cell in question has been engineered or otherwise accommodated or expressed by artificial manipulation.

[0073] In some embodiments, the tRNA or composition as provided herein can be formulated into a pharmaceutical composition. The pharmaceutical composition may also contain a pharmaceutically acceptable excipient. Such excipients include any medicament that can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may be included, such as mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc. may be present in such vehicles. A comprehensive discussion of pharmaceutically acceptable excipients is provided in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0074] In view of the teachings of this specification, it will be apparent to those skilled in the art that the effective amount of the tRNA provided can be determined empirically. Administration can be achieved in a single dose, continuously or intermittently throughout the course of treatment. The administration can be substantially continuous over a preselected time period or can be performed in a series of spaced doses. The method of determining the most effective mode of administration and dosage can vary depending on the composition of the treatment, the target cell, and the subject being treated, etc. Single and multiple administrations can be performed, and the dosage level and pattern are selected by the treating physician.

[0075] The present invention can use vehicles including water, saline, artificial CSF or other known substances. In order to prepare the preparation, the purified composition can be isolated. The composition can then be adjusted to an appropriate concentration and packaged for use.

[0076] As used herein, the term "treat" or "treatment" refers to therapeutic treatments and measures that can relieve symptoms or provide some benefit to a subject. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in disease extent, stable (i.e., non-deterioration) disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. Subjects in need of treatment include those already suffering from a condition, disease, or illness.

[0077] The phrase "therapeutically effective amount" means an amount of one or more compounds or compositions of the invention that (i) treats a particular disease, condition, or disorder, (ii) reduces, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. One or more tRNAs or compositions described herein can be administered to alleviate at least one symptom associated with a disease or disorder. The amount administered will vary based on a variety of factors, including, but not limited to, the composition selected, the particular disease, body weight, physical condition, and age of the mammal. Clinicians can readily determine such factors using animal models or other test systems known in the art.

[0078] One or more suitable unit dosage forms having one or more tRNA or compositions of the present invention can be formulated and the unit dosage forms can be administered by a variety of routes. When the agents of the present invention are prepared for administration, they can be combined with pharmaceutically acceptable carriers, diluents or excipients to form pharmaceutical preparations or unit dosage forms. The total active ingredients in such preparations account for 0.1% to 99.9% of the weight of the preparation. "Pharmaceutically acceptable" is a carrier, diluent, excipient and / or salt that is compatible with the other ingredients of the preparation and harmless to its recipient. Pharmaceutical preparations can be prepared using well-known and readily available ingredients by procedures known in the art. The agents of the present invention can also be formulated into solutions suitable for administration. The pharmaceutical preparations of the agents of the present invention can also take the form of aqueous or anhydrous solutions or dispersions, or alternatively in the form of emulsions or suspensions.

[0079] Therefore, the one or more agents can be formulated for administration and can be present in an ampoule, a prefilled syringe, a small volume infusion container or a multi-dose container in a unit dose form and a preservative is added. The active ingredient can take the form of a suspension, solution or emulsion such as in an oily or aqueous vehicle, and can contain formulations such as suspending agents, stabilizers and / or dispersants. Alternatively, the active ingredient can be in a suitable vehicle (e.g., sterile pyrogen-free water) before use. It should be understood that the unit content of the active ingredient or ingredient contained in the individual dose of each dosage form does not itself have to constitute an effective amount for treating a specific indication or disease, because the necessary effective amount can be achieved by administering multiple dosage units. In addition, the effective amount can be achieved using less than the dosage in the dosage form alone or in a series of administration forms.

[0080] The pharmaceutical preparation of the present invention may include as optional ingredients a pharmaceutically acceptable carrier, diluent, solubilizer or emulsifier and salts of types well known in the art. Specific non-limiting examples of carriers and / or diluents that can be used in the pharmaceutical preparation of the present invention include water and physiologically acceptable buffered saline solutions (such as phosphate buffered saline solutions of pH 7.0-8.0) and water.

[0081] While the foregoing description and examples fully disclose and enable the invention, they are not intended to limit the scope of the invention, which is defined by the appended claims.

[0082] All publications, patents and patent applications are incorporated herein by reference. Although the present invention has been described in conjunction with certain embodiments of the present invention in the foregoing description, and many details have been set forth for illustrative purposes, it will be apparent to those skilled in the art that the present invention may have other embodiments, and that certain details described herein may vary considerably without departing from the basic principles of the present invention.

[0083] In the context of describing the present invention, the use of the terms "a" and "an" and "said" and similar referents should be interpreted as covering both the singular and the plural, unless otherwise specified herein or clearly contradictory to the context. The terms "comprising", "having", "including" and "containing" should be interpreted as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise specified herein. Unless otherwise specified herein, the enumeration of the value range herein is intended to be used as a shorthand method for individually referring to each individual value belonging to the scope, and each individual value is incorporated into this specification as it is individually listed herein. Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be carried out in any suitable order. The use of any and all examples or exemplary languages ​​(e.g., "such as") provided herein is intended only to better illustrate the present invention, and unless otherwise required, will not impose limitations on the scope of the present invention. The language in this specification should not be interpreted as indicating any non-required element as necessary for practicing the present invention.

[0084] Embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. After reading the foregoing description, variations of those embodiments may become apparent to those of ordinary skill in the art. The inventor hopes that the technician will adopt such variations as appropriate, and the inventor intends to practice the present invention in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter recited in the appended claims of the present invention as permitted by applicable law. In addition, unless otherwise specified herein or clearly contradictory to the context, the present invention encompasses any combination of the above-mentioned elements in all possible variations.

[0085] Example

[0086] Example 1: Restoring vision through high-fidelity nonsense codon correction

[0087] Inherited blindness is a serious health, social and economic burden affecting millions of patients worldwide. Although there are adeno-associated virus (AAV) gene therapy approaches available, such as But these approaches only address rare forms of blindness and have very limited indications. Unfortunately, many proteins that are critical for visual processing, such as ion channels, are beyond the payload size of currently available AAV delivery systems. In addition, mutations that produce premature stop codons (PTCs) (i.e., mutations that convert a "sense" codon to one of three nonsense codons (TGA, TAA, TAG), represent a unique challenge as they typically produce a more severe phenotype) are found throughout essential proteins and are more numerous in large genes. Therefore, nonsense codon PTCs themselves are a common molecular basis for inherited blindness.

[0088] Provided herein is a method for treating a high-fidelity in vivo repair of PTC using transfer RNA (tRNA) molecules. In some embodiments, the tRNA can be "ace-tRNA." Data show that such tRNAs can be expressed as DNA in retinal cells or by viral vectors (such as AAV), and they show potent biological PTC repair activity in retinal cells, and toxicological characteristics cannot be detected. Genes containing PTC can cause a variety of blindness symptoms-in total-affecting thousands of patients. Viral and non-viral delivery of tRNA in patient-derived iP SC-retinal pigment epithelium (RPE) cells (photoreceptor (PR) organoids) and in vivo repair activity, intravitreal injection in hereditary blindness mouse models, and biosafety and bioavailability in non-human primates have been evaluated or have been evaluated.

[0089] Importance of ion channels in inherited retinal diseases

[0090] Ion channels are expressed in retinal neurons, glial cells and epithelial cells to maintain membrane potential, signal transduction, ion transport and volume regulation. The interaction between PR and RPE is responsible for collecting and processing visual stimulation by the synchronous function of multiple ion channels. Ion channel-based disorders (collectively referred to as "channelopathies") are crucial to developing blindness therapeutic agents because they 1) affect both RPE and PR, 2) benefit from established experimental endpoints in vitro and in vivo readings (that is, channel function can be directly measured with electrophysiological methods-this is not easy under in vitro systems), 3) they are particularly sensitive to missense mutations, so they generally not only need to read through, but also need to insert wild-type AA at the PTC site, 4) generally represent a "class" of excellent diseases to test the therapeutic potential of tRNA.

[0091] Provided herein are therapeutic methods for repairing premature stop codons in any of the genes described herein. The method relies on transfer RNA (tRNA) to suppress premature stop codons. Data show that the method exhibits efficient, high-fidelity repair of PTCs regardless of the cell, gene, or repair site. High-fidelity repair: The correct amino acid is relocated at the PTC (as determined by mass spectrometry) and combined with minimal interactions at the 3′ protein termination stop codon, as shown by whole-exome ribosome analysis. Each ace-tRNA for a given amino acid is synthesized as an oligonucleotide containing nucleotide changes in the anticodon region and cloned into a single expression vector containing a nanoluciferase-STOP reporter gene, where STOP (TGA, TAA, TAG) corresponds to the inserted tRNA. Each construct was transfected into a 96-well plate and luciferase activity was measured 24 hours later on a SpectraMax i3 plate reader, as described in (16). Each bar represents a unique tRNA sequence. The screen identified multiple anticodon-editing ace-tRNAs for each of ten amino acids susceptible to stop codon conversion and stop codon type, with inhibition covering all three stop codons, providing therapeutic coverage for each human pathogenic PTC caused by a single nucleotide change.

[0092] The inhibitory activity of protein stop codons is very low

[0093] Riboprofiling was used to address the possibility that inhibitory tRNAs that exhibit potent repair activity for in-frame PTCs might also induce global readthrough of natural stop codons, which would be undesirable for therapeutic purposes. Riboprofiling, also known as ribosome profiling assays, allows for the simultaneous detection of the positions of actively transcribing ribosomes within a cell. If inhibitory tRNAs promote readthrough of protein stop codons, one would be able to measure this as ribosomes on the 3′UTR region of the RNA following the stop codon. To assess such “off-target” suppression of 3′ stop codons, transcriptome-wide quantitative analysis of actively engaged ribosomes on all cellular transcripts was performed by generating ribosome footprinting libraries from HEK293 cells expressing exogenous tRNAs or a control mock plasmid (puc57GG). Figure 3The ribosome footprint density of RNA purified from HEK cells exposed to G418 or expressing each of the five specified tRNA types is shown. Only transcripts with a minimum threshold of 5RPKM for coding sequences and a minimum threshold of 0.5RPKM for 3'UTR in two replicate libraries are included for quantitative comparison (254 transcripts in G418 and 495-748 transcripts in tRNA). The "sawtooth" graph represents the data of all transcripts. It has been averaged for ribosome positioning, making the reading frame obvious, because the ribosome pauses at each codon when translating mRNA, produces density at the tip of the spike, and then the ribosome stagnates at the end of the reading frame (observed as a significant spike). Please note that very low ribosome activity / no ribosome activity is observed after the "-10" coordinate, which marks the start of the stop codon region and 3'UTR (untranslated region), represented by the arrows below, respectively. The illustration on the right shows these data for each stop codon and ace-tRNA inhibitor type. These data were compared to controls (log2 fold change) in the 3'UTR region, i.e., evidence of ribosome presence on the 3'UTR region indicates read-through of the protein stop codon. The ribosome profiling data indicate that these engineered tRNAs suppress natural stop codons at low or undetectable efficiency and are significantly below the level of in-frame PTC suppression. One possibility for this observation is that eukaryotic translation termination factor 1 (eRF1) outcompetes the inhibitory ace-tRNA.

[0094] High fidelity encoding, decoding and PTC suppression

[0095] It was considered whether the ace-tRNA identified in the screen was functionalized to inhibit PTC while sacrificing the aminoacylation stringency of the cognate aminoacyl-tRNA synthetase. To this end, mass spectrometry was used to examine PTC inhibition in the model soluble protein histidinol dehydrogenase (HDH), Figure 4A. Here, an in-frame TGA codon was introduced at asparagine 94 (N94) and co-expressed in HEK293 cells in tandem with plasmids encoding Glychr19.trna2 or Trpchr17.trna39 ace-tRNA (two representative tRNAs from the screen). Searching the MS data found that Asn was modified to Trp (+72Da) in Trp chrl7.trna39 and Asn was modified to Trp (-57Da) in Glychrl9.trna2, confirming the accurate coding of the cognate amino acid of each ace-tRNA type in >98% of the identified peptides. Codon-edited tRNAs show selectivity for the UGA stop codon over UAA and UAG, Figure 4B, left panel. Inhibitor tRNAs outperform read-through compounds gentamicin and G418, Figure 4B, right panel. In addition, missense mutations in ion channels have been determined to cause changes in gating and expression profiles, and are often the cause of disease phenotypes. Examples of this are shown in Figures 4C and 4D, where the tolerance of Kir7.1 potassium channels (subtypes) to W53E and W53Q mutations was tested, where W53X is a known LCA PTC mutation, and Gln and Glu are "near homologous" amino acids that are encoded when using read-through drugs (such as atalulum). Here, channels containing missense reduce current density and disrupt voltage-dependent gating.

[0096] Robust amino acid coding and protein rescue efficiency (regardless of PTC or PTC position)

[0097] To experimentally evaluate the ability of the tRNAs identified in the screen to inhibit bona fide human pathogenic PTCs, SCN2A sodium channels transiently expressed in HEK cells were used. Five different types of human PTC mutations found in the SCN2A gene that are known to cause autism and developmental delay were selected. For each mutation, tRNAs from the expression library were matched for activity against the desired PTC stop codon and amino acid type. SCN2A PTC channel cDNAs were then expressed alone or together with constructs expressing the designated tRNA type and a GFP-X reporter gene to mark expressing cells for patch clamp analysis. SCN2A PTC cDNAs expressed with WT GFP did not show sodium channel activity consistent with a truncated, non-functional Nav1.2 protein. However, co-expression with the designated ace-tRNA constructs for each PTC produced robust sodium channel activity with current density (a measure of the number of channels expressed) and gating properties indistinguishable from those of the WT SCN2A channel ( Figure 5 , top left). In each case, the rescue current was similar to WT expression, regardless of the PTC context (beginning, middle, or end of the gene), PTC class (TGA or TAG), or encoded amino acid.

[0098] Preparation and characterization of SNPs

[0099] SNPs are synthesized using an oil-in-water microemulsion method. An aqueous solution containing a payload (e.g., pDNA, cDNA encoding ace-tRNA, mRNA, or Cas9 RNP) is mixed with N-(3-(triethoxysilyl)propyl)-1H-imidazole-4-carboxamide (TESPIC), tetraethyl orthosilicate (TEOS), and bis[3-(triethoxysilyl)propyl]disulfide (BTPD), and then added to an organic solution consisting of Triton X-100, n-hexanol, and cyclohexane. Then, an aqueous ammonia solution is added to the mixture to initiate the reaction and stirred overnight. The SNPs are precipitated with acetone, recovered by centrifugation, and purified by washing with ethanol and water. Polyethylene glycol (PEG)-conjugated SNPs are prepared by reacting SNPs with PEG-silane and purified by washing with water and centrifugation. The resulting PEGylated SNPs ( Fig. 6A to Figure 6C).

[0100] The morphology (spherical NPs with a size of approximately 40-50 nm) and zeta potential (i.e., surface charge) of the SNPs were evaluated by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The loading efficiency (~95%) and loading content (~10 wt %) of nucleic acids were determined by Nanodrop and / or bicinchoninic acid assay (BCA assay). The SNPs were prepared using an oil-in-water microemulsion process, resulting in high encapsulation efficiency (~95%) and high nucleic acid loading content (~10 wt %). In the microemulsion process, the size of the SNPs can also be conveniently controlled to produce SNPs with different sizes, which is very important for determining the optimal size for retinal penetration via intravitreal delivery. The hydrodynamic diameter of the SNPs ranges from 40 to 50 nm. In general, smaller NPs have higher diffusion rates and can be more easily absorbed by target cells. Due to their covalent nature, SNPs also have excellent stability in the extracellular space. Imidazole functional groups can be incorporated into the SNPs, which can promote the escape of the SNPs from endo / lysosomes via the proton sponge effect. Disulfide crosslinkers are incorporated into the silica network, which can be decomposed in the cytosol due to high GSH concentration (2-10 mM), triggering the rapid release of the payload.

[0101] SNPs showed high in vitro transfection efficiency for pDNA and mRNA delivery in HEK 293T cells and high editing efficiency for CRISPRRNP delivery. SNPs also showed significant cellular uptake in iPSC-RPE cells. In addition, subretinal injection of Cre mRNA or Cas9 RNP-loaded SNPs combined with all-trans retinoic acid (ATRA, targeting RPE cells) or 11-cis retinal (11cRal, targeting PR) induced significant gene editing in RPE cells or PR in vivo. Photoreceptor inter-retinoic acid binding protein is the main protein in the inter-photoreceptor matrix, which selectively transports 11cRal to PR and ATRA to RPE (58,59). SNP-ATRA can also deliver cDNA encoding tRNA in vivo, resulting in strong ace-tRNA transfection in mouse RPE cells. In addition, SNP treatment did not induce significant cytotoxicity or tissue damage / inflammation in cells or animals, indicating that SNPs have good biocompatibility.

[0102] The versatility of SNP is reflected in: (1) the types of payloads it can deliver (e.g., tRNA minicircles, plasmid DNA, and proteins); (2) the types of surface functionalization it can achieve; (3) its basic properties (e.g., high encapsulation efficiency and high payload loading content, small and controllable size, excellent biocompatibility, high stability in the extracellular space, and GSH-triggered payload release in the cytosol); and (4) it has been shown to be able to induce high transfection / editing efficiency of multiple types of payloads or their combinations in various cells both in vivo and in vitro, making it an ideal nanoplatform that can be further engineered for intravitreal delivery of tRNA, such as in the form of minicircles to target PR or RPE cells.

[0103] Efficiency of in vitro delivery of nucleic acids and CRISPR RNPs by SNPs

[0104] The SNP formulation was optimized in vitro using plasmid DNA and mRNA encoding red fluorescent protein (RFP) as payloads. The incorporation ratio of imidazole-containing TESPIC and the surface charge of SNP in the formulation were optimized in HEK 293T cells, and the transfection efficiency was significantly higher than that of the commercially available transfection agent Lipofectamine 2000 (Lipo 2000) ( Fig. 7A and Figure 7B ), indicating that SNPs can efficiently deliver nucleic acids. To investigate the genome editing efficiency of SNPs loaded with Cas9 RNPs, sgRNA targeting the GFP gene in a HEK293T cell line expressing transgenic GFP was used. Figure 7C As shown, SNP exhibited significantly higher GFP knockdown efficiency than Lipo2000. To evaluate the biocompatibility of SNP, HEK 293T cells were treated with SNP and cell viability was investigated by MTT assay ( Fig.7D ). These results indicate that SNPs can effectively deliver a variety of hydrophilic payloads (including nucleic acids) in vitro with minimal cytotoxicity.

[0105] Efficiency of in vivo delivery of nucleic acids and CRISPR RNPs via SNPs

[0106] SNPs are suitable for in vivo nucleic acid delivery due to their small size, versatile surface modifications, and excellent biocompatibility. The genome editing efficiency of subretinal injected RNP-loaded SNPs was evaluated in vivo in transgenic Ai14 mice ( FIG. 8A to FIG. 8E The Ai14 mouse contains a LoxP-flanked stop cassette (3 repeats of the Sv40-poly A sequence) that prevents expression of the tdTomato fluorescent protein. CRISPR RNP-directed excision or Cre-lox recombination of the stop cassette results in tdTomato expression ( Fig. 8A ). Therefore, the Ai14 mouse model provides a robust, high-throughput quantitative readout of site-specific genomic modifications at the loxP-flanked stop box locus, and generates a functional gain fluorescent signal in cells edited by delivering Cre encoding DNA / mRNA for Cre-lox recombination or targeted excision of the Sv40-poly A block RNP (60, 61). Cre mRNA was loaded in SNP-ATRA (for RPE cell targeting) and SNP-11cRal (for PR targeting) respectively, and subretinal injection was performed. Four days after injection, the RPE layer and retinal layer were then separated and laid flat, and tdTomato expression was evaluated by CLSM. The retinal layer was imaged with the PR facing up, and the plane contained 99% of the PR.

[0107] SNP-ATRA induced strong tdTomato expression in RPE ( Figure 8C ). tdTomato expression induced by SNP-11cRal was also detected in the retinal layer ( Figure 8B ). These data show that the SNP successfully delivered Cre mRNA, leading to Cre expression. Fig. 8E As shown, strong tdTomato was also observed around the injection site in RPE cells 12 days after injection, indicating that high gene editing efficiency can be achieved by delivering Cas9 RNPs via ATRA-conjugated SNPs.

[0108] The 5-copy Arg-UGAace-tRNA suppressor and the fluorescent reporter gene eGFP were Y1 52X The cDNA plasmid of the (TGA) construct (i.e., 5XtRNA) was loaded into SNP-ATRA and injected subretinaly ( FIG. 9A to FIG. 9B ). GFP expression was assessed four days after injection. Fig. 9B As shown in Figure 2, SNP-ATRA induced GFP expression in RPE, indicating efficient delivery of 5XtRNA into RPE cells and subsequent inhibitory activity, which then repaired GFP. Y152x Construct.

[0109] Testing the cargo delivery efficiency of SNPs in iPSC-RPE cells

[0110] iPSC-RPE is a promising alternative to human RPE in genetic research and has been shown to exhibit the same characteristics as mature human RPE (62, 63). The delivery efficiency of SNP was also tested in iPSC-RPE cells (Figure 10A to Figure 10D). RNP and donor ssODN (RNP+ssODN) were loaded into SNP-CPP (TAT) at a molar ratio of 1:1. The donor sequence ssODN was labeled with green fluorescent dye ATTO-488. iPSC-RPE was treated with SNP-CPP loaded with different doses of RNP+ssODN, and the cellular uptake of the payload was evaluated by confocal laser scanning microscopy (CLSM) four days after treatment. Significant cellular uptake was observed in iPSC-RPE, and the uptake efficiency was dose-dependent (Figure 10B to Figure 10D). In addition, no signs of cytotoxicity were observed, indicating that SNP is an effective and biocompatible nanoplatform that can be used to deliver RNP+ssODN to iPSC-RPE.

[0111] Patient-derived iPSC-RPE as a cellular test bed for therapeutic development

[0112] Studies in mouse models have made important contributions to our understanding of human retinal diseases (64). Several attempts have been made to generate Kcnj13 knockout mouse models, but this mutation results in P0 lethality (35, 65). The hiPSC line was derived from an LCA-16 patient with the W53X mutation (identified as iPSC-RPEW53X), an unaffected family member (defined as iPSC-RPEKir7.1), and another from an unrelated healthy donor reprogrammed via lentiviral-mediated pluripotency factor transfer (iPSC-RPEWT) ( FIG. 11A to FIG. 11I )(8). As confirmed by Western blotting and immunohistochemistry, iPSC-RPEW53X cells have comparable karyotype, morphology, and expression of RPE cell molecular markers (except Kir7.1 protein expression) ( Fig.11A , Fig. 11B , Fig.11D , Fig.11E ).

[0113] When iPSC-RPE cells were grown in cell culture inserts and enzymatically digested with papain, pigmented, polarized cells with prominent apical process extensions were obtained. Whole-cell recordings from single cells showed that the current-voltage (IV) curves were characterized by inward and outward K + The current is composed of two components, the resting membrane potential is -55mV, Rb + The current increases by more than 10 times ( Fig. 11C , Fig.11F), which is in stark contrast to recordings from iPSC-RPEW53X cells, which showed spontaneous depolarizations of at least 15 mV and Rb + There is no increase in current ( Fig. 11C , Fig.11F ). Kir7.1 channel to Rb + The results are consistent with previous observations that truncated protein products do not form Kir7.1 channels and that Kir7.1 current recordings are a reliable measure of LCA-16 disease status using iPSC-RPE cells as a model system.

[0114] Gene therapy was also tested by transducing LCA16 patient-derived iPSC-RPE cells with lentiviral particles carrying GFP-fused human KCNJ13. The open reading frame was under the control of the EF1a and VMD2 promoters. Normal Kir7.1 currents were recorded in LCA16 iPSC cells after transduction ( FIG. 11G to FIG. 11I ). It was found that ion channel protein expression was polarized ( Fig.11I ) and are at normal levels ( Fig.11G , 11H ).

[0115] Restoration of currents in the LCA16 Kir channel PTC mutation by ace-tRNA

[0116] As previously described, the LCA16 nonsense mutations W53X and R166X render the channel nonfunctional. We used W53X fused to GFP alone or in combination with a codon-edited ace-tRNA to Trp HEK293 cells transfected with W53X showed GFP localization in the cytoplasm, but ace-tRNA Trp The induced GFP is mainly localized in the plasma membrane, the site of Kir7.1 protein expression ( Fig. 12A , Figure 121B). Similarly, with no expression of tRNA Trp Non-functional linear IV plot of cells ( Fig. 12C , Fig.12D ), whole-cell current recordings revealed a significant inwardly rectifying Kir7.1 channel ( Fig. 12C , Figure 2 D). Compared with -7mV and -62mV membrane potentials of cells expressing W53X and wild-type Kir7.1 channels ( Fig.12D ) compared to ace-tRNA rrpThe treated W53X channel recovered to -53 mV. The nearly 10-fold increase in inward current through Kir7.1 channels using Rb as a charge carrier also revealed that ace-tRNA Trp Evidence for rescue channel function ( Fig.12E , Fig.12F ).

[0117] Retinal organoids from human pluripotent stem cells (hPSCs), photoreceptor differentiation, and subsequent transduction with AAV vectors

[0118] Retinal cell differentiation from hPSCs can be performed using previously established protocols (2-5)(66-68), with recent modifications greatly improving the generation and long-term maintenance of the outer nuclear (i.e., photoreceptor) layer (ONL) from hPSC-OVs (1). FIG. 13A to FIG. 13B ). Briefly, hPSCs will be raised using ReLeSR (STEMCELL Technologies) and grown in neural induction medium (NIM) for 6 days. On day 6 (D6), suspended cell aggregates will be treated with 1.5nM BMP4 and reattached to Matrigel-coated plates in medium containing BMP4 one day later. BMP4 will then be gradually diluted by replacing half of the medium every three days until D16, when the cultures are switched to retinal differentiation medium (RDM). Between D27-35, optic vesicle-like neural retinal structures (OVs) become clearly visible and are dissected into 3D-RDMs, which also contain 5% FBS, taurine, and defined lipids (Thermo Fisher) to enhance survival and retinal differentiation. Thereafter, hPSC-OVs will be maintained in 3D-RDM+retinoic acid until D100, when retinoic acid is removed to enhance outer segment formation. The resulting retinal organoids were further differentiated in 3D-RDM until ≥D180, a time point when well-developed ONL and mature optic stem with outer segments and cone photoreceptors were clearly visible ( FIG. 14A to FIG. 14L Differentiated hPSC retinal organoids are then routinely analyzed for gene and protein expression by PCR and immunocytochemistry to verify retinal identity and differentiation status (1-6).

[0119] For AAV experiments, hPSC-derived retinal organoids (ROs) in RDM were plated with 1.5x10 11 -2.5x10 12 VP / mL of AAV transduction, half of the medium was replaced 24 hours after transduction. Thereafter, the entire medium of the culture was replaced every day. Based on preliminary AAV-GFP experiments ( FIG. 15A to FIG. 15G), transgene expression is expected to begin seven days after transduction, with maximal expression levels reached approximately 12 days after transduction. To date, superior transduction of hPSC-RO has been observed using AAV7m8 and AAV5 ( FIG. 15A to FIG. 15G ).

[0120] AAV2.7m8-mediated cellular and posterior retinal transduction by intravitreal injection

[0121] To demonstrate AAV-mediated gene transduction ( FIG. 16A to FIG. 1 6I), AAV packaging using ace-tRNA ( Fig.16A , Fig. 16B ). Preliminary tests using lentiviral constructs showed higher ace-tRNA expression in iPSC neuronal models ( Fig. 16C ). Then, use 1 μl of 1X 10 11 rAAV2.7m8 virus titer / 6min transwell display field, each iPSC-RPE cell expressed GFP without damaging cell health ( Fig.16D ). By intravitreal injection (2μl 1X10 11 The same rAAV2.7m8 vector was delivered into the mouse eye ( Fig.16E Eight days later, GFP expression in the posterior retina was imaged by fundus autofluorescence imaging ( Fig.16F ), and the OCT images of the retina showed normal ( Fig.16H ). Examination of RPE cell flat films showed that GFP was expressed in RPE cells, and the morphology of RPE cells was normal cobblestone-shaped. This result indicates that intravitreal delivery of AAV2.7m8 has lower cytotoxicity and higher GFP transduction expression in vivo.

[0122] Generation of nonsense mutation clones

[0123] Site-directed mutagenesis was performed to generate W53X and R166X clones of the human KCNJ13 gene (8, 29) and a constitutively expressing HEK-FRT cell line was generated using the Flip-In system (Thermo Fisher). R200 、CACNA1F R680 and KCNV2 E148 Human mutant clones are available for mammalian expression plasmids. These nonsense mutant clones can be expressed in HEK293T cells and the respective protein function can be quantified by whole-cell patch clamp electrophysiology compared to wild-type clones (positive control), untreated cells, and mock-transfected cells (negative control, using empty plasmid). Experiments can be repeated at least six times to determine statistical differences between groups.

[0124] For the experimental groups, the corresponding ace-tRNA constructs (tRNA Trp 、tRNE Arg and tRNA Glu ) were packaged in silica nanocomplexes with mutant clones for delivery to HEK cells. Two days after transfection, electrophysiology can confirm the functional rescue of the channel after ace-tRNA delivery. The expression of full-length protein can be confirmed by immunolocalization and western blot assays using the corresponding antibodies. For KCNJ13 W53X and R166X HEK-FRT cell lines, ace-tRNA packaged in silica nanocomplexes can be used Trp and ace-tRNE Arg , followed by electrophysiology and expression studies. The results can be compared with cell lines expressing the mutant channel or with control HEK-FRT cells.

[0125] Generation of minicircles containing the ace-tRNA ORF

[0126] Minicircle episomal DNA vectors are minimized eukaryotic expression cassettes without prokaryotic plasmid DNA backbones (6, 75). When using equimolar reporter gene expression cassettes for in vitro experiments, minicircle DNA expression increased by 3 to 10 times in the absence of cytotoxicity (76), and in vivo expression increased by at least 10-13 times (77). The smaller size of DNA plasmids is conducive to diffusion through the plasma membrane, escaping endonuclease / lysosomal degradation and passing through the nuclear membrane for long-term gene expression (78, 79). The plasmid maintains a stable supercoil structure and can be combined with cell-specific promoters for cell targeting of PR or RPE cells. Minicircle technology has been found to be widely used in the field of stem cells (for reprogramming because of the non-transgenic expression of factors) and hereditary retinopathy (80, 81). Minicircles are very suitable for the expression of tRNA, because the coding region length of a single tRNA is about 75 bases, and the promoter (A and B box regions) of tRNA expression is contained in this 75 base sequence. The minimal "expression cassette" for ace-tRNA expression has been found to also include about 70 bases of 5' genomic sequence upstream of the tRNA and a short (6 base) "terminator" sequence 3' to the tRNA sequence. Thus, about 150 bases represent the entire genetic package.

[0127] The promoter (for the reporter gene) and the coding sequence of the ace-tRNA can be subcloned into the parent plasmid (SystemBioSciences, Palo Alto, CA). For example, MC-Easy TM(Catalog No. MN920A-1, SBI System BioSciences) minicircle DNA production kit to obtain high-quality minicircle DNA. The purified sterile minicircle DNA can be packaged in SNPs for delivery to iPSC cells or subretinal injection.

[0128] AAV packaging and delivery of tRNA ORFs

[0129] AAV2.7m8 can be used for outer retinal transduction. In addition, existing AAV systems show a maximum "payload" amount of about 4.5 kb, which limits the size of genes that can be delivered to retinal cells. Transfer RNA is small, about 75 bases, and requires limited surrounding sequences for biological processing: a short genomic "leader sequence" of about 100 bases at the 5' and a 6-base termination sequence at the 3'.

[0130] The coding sequence for the ace-tRNA (leader and terminator sequences) can be packaged within the AAV payload region between the ITR sequences. The tRNA "cassettes" can be interleaved with non-identical cDNA sequences to minimize recombination. For NHP experiments, the AAV can have the following payloads: VMD2-GFP for RPE cell expression Arg -ace-tRNA Arg or hGRK1-GFP for expression in PR Arg -acetRNA Arg . High titer (10^14 genome copies / mL), affinity purified, ready-to-use AAV2.7m8 can be specific for the PR (OPSIN) or RPE (VMD2) depending on the choice of cell type-specific promoter. A minimized VMD2 promoter and optimized intravitreal delivery have been used to avoid toxicity (83). The transgene is optimized for expression in hiPSC-RPE and in vivo RPE. In addition, T2A-GFP can be included so that whole-cell electrophysiological analysis of transduced cells can be performed individually. AAV5 also transduces the PR in hiPSC organoids and mouse RPE cells, providing an alternative method for expressing ace-tRNA.

[0131] Design, synthesis and optimization of SNPs for intravitreal injection of PR or RPE targeted ace-tRNA microcircle delivery

[0132] SNPs targeting PR or RPE can be used for tRNA minicircle delivery for intravitreally injected. SNPs coated with HA and conjugated with various targeting ligands in HEK293, iPSC-RPE, iPSC-PR cells can be first tested in vitro.

[0133] Design and synthesis of SNPs for tRNA minicircle delivery

[0134] Subretinal injection of SNP can effectively deliver many payloads to RPE and PR cells in vivo, but intravitreal delivery is less invasive. Surface functionalization of SNP with hyaluronic acid (SNP-HA) can not only promote retinal penetration, but also enhance the cellular uptake of SNP by PR or RPE cells through receptor-mediated endocytosis, making it an ideal nanocarrier for intravitreal delivery of nucleic acids in PR and RPE cells. In addition, both 11cRal and ATRA have high binding affinity for interphotoreceptor retinoic acid binding protein (IRBP) (90, 91). IRBP is the main protein in the interphotoreceptor matrix (IPM) that selectively transports 11cRal to PR and ATRA to RPE cells (58, 59, 92). Therefore, SNP-HA conjugated with 11cRal and ATRA can further improve the transfection efficiency and specificity of ace-tRNA microcircles to PR and RPE cells, respectively, through such selective transport mechanisms.

[0135] HA modified SNP can be engineered for intravitreal delivery of ace-tRNA microcircles, or co-delivery of ace-tRNA microcircles and GFP plasmids as a reporter system for initial formulation optimization. In addition, the potential synergistic effects of mixed targeting ligands can be studied (e.g., HA combined with 11cRal (for PR) or ATRA (for RPE cells)). Since SNP is small (e.g., 40-50nm) and coated with hyaluronic acid, its penetration depth and diffusion area are expected to be higher than other nucleic acid nanocarriers (55, 87-89, 93).

[0136] Fig.17A SNPs reengineered for intravitreal injection are shown. SNP-HA as well as 11cRal-conjugated SNP-HA (i.e., SNP-HA-11cRal targeting PR) and ATRA-conjugated SNP-HA (i.e., SNP-HA-ATRA targeting RPE) were prepared. 3-Aminopropyl)triethoxysilane (APTES) is a cationic silane monomer that can be used as an additional reactant in the manufacturing process of SNPs to change the surface charge of the original SNP (i.e., not PEGylated) from anionic to cationic through a microemulsion process. The size of the SNP is not significantly affected because it is controlled by the water-in-oil microemulsion process. As Fig. 17B As shown, after purification, cationic SNPs can be coated with HA (anionic polysaccharide) or ligand-conjugated HA through electrostatic interactions at a ratio of HA sugar unit / APTES = 10. Uncoated HA can be easily removed by washing and centrifugation.

[0137] To conjugate the targeting ligand to HA, e.g. Fig. 17CAs shown, HA will first react with 2-aminoethanethiol to generate HA-thiol through amidation. At the same time, the targeting ligand 11cRal can be conjugated with NH2-oligo(ethylene glycol) (OEG)-maleimide (Mal) by forming an imine bond, and ATRA can be conjugated with NH2-OEG-Mal by amidation. Thereafter, Mal-OEG-11cRal / ATRA can be conjugated with HA-thiol to form HA-OEG-11cRal / ATRA through a Michael addition reaction. The chemical structures of all intermediates and final polymer products can be characterized by nuclear magnetic resonance spectroscopy (NMR). HA with a low molecular weight (i.e., 10 kDa) will be used because it can produce a thinner HA coating, thereby producing a smaller SNP-HA size, and provide higher reactivity for ligand conjugation. OEG500 will be used as a spacer for ligand conjugation because it can enhance the binding efficiency of ligand-conjugated SNPs to IRBP.

[0138] Since negatively charged surfaces may affect cellular uptake, the molar ratio of HA sugar units / APTES was optimized (i.e., 5, 10, and 15). The number of targeting ligands on HA and the molecular weight of HA and OEG can be optimized in vitro and in vivo. The amount of targeting ligands can be controlled by the degree of substitution of the ligands on HA. HA and OEG of different molecular weights can be ordered directly from Lifecore (Chaska, MN). The degree of substitution of HA-OEG-11cRal or HA-OEG-ATRA can vary from 0%, 10%, 20%, 30%, to 40% (0% represents unmodified HA). The molecular weight of HA can vary from 10kDa to 1MDa. The molecular weight of OEG can also vary from 200Da to 1kDa. A family of SNPs coated with ligand-modified HA of different types / degrees of substitution can be prepared. Their morphology and zeta potential can be characterized by TEM and DLS.

[0139] In one study, a 10% molar ratio of a cationic silane monomer (i.e., APTES) was successfully incorporated into SNPs and resulted in positively charged SNPs (zeta potential of +25 mV) and effective nucleic acid delivery in HEK 293T cells. The HA coating process and parameters will be optimized by varying the following aspects: (1) HA / SNP ratio, (2) HA molecular weight, and (3) ligand-to-HA conjugation ratio.

[0140] In terms of targeting ligands, in addition to using 11cRal / ATRA to target PR / RPE cells, other types of targeting ligands can also be explored to optimize in vivo delivery efficacy (e.g., aptamers (AS1411)). AS1411 is a G-quadruplex DNA aptamer that targets nucleolin, an RNA and protein-binding protein expressed on the surface of PR and RPE cells (94). AS1444 can promote the transport of SNPs to PR / RPE cells and enhance their uptake by PR / RPE cells. In order to conjugate the aptamer to SNP, a thiol group (Integrated DNA Technologies, IA) can be used at its 5′ end and then connected to HA-OEG-Mal by Michael addition reaction. The resulting HA-OEG-aptamer can coat the SNP through electrostatic interaction, thereby producing a SNP-HA-aptamer. The density of AS1411 can be controlled by changing the feed molar ratio of AS1411 and HA-OEG-Mal during the reaction (eg, 10%, 20%, and 30% molar ratio to HA sugar units, respectively).

[0141] In vitro evaluation of cellular uptake, biocompatibility, and transfection efficiency of non-viral ace-tRNA minicircle-delivered SNPs

[0142] The cellular uptake, biocompatibility, and transfection efficiency of SNPs engineered for delivery of ace-tRNA microcircles can be evaluated in vitro first using human embryonic kidney cells (HEK 293T cells), and then induced pluripotent stem cell-derived RPE cells (iPSC-RPE) and iPSC-derived PR (iPSC-PR), respectively. For cellular uptake studies, SNPs can be conjugated with fluorescent dyes (e.g., AlexaFluor488 or 594) by OEG conjugation. Both flow cytometry and fluorescence microscopy can be used to monitor cellular uptake at different time points. The biocompatibility of SNPs can be evaluated using a colorimetric MTT assay. To facilitate the preliminary evaluation and optimization of SNPs, ace-tRNA microcircles and GFP-encoding plasmid DNA (molar ratio of 1:1) in SNPs can be encapsulated. Successful transfection results in GFP expression; therefore, it will serve as a fluorescent reporter system for SNP formulation optimization. Using this reporter system, the in vitro delivery efficacy of SNPs can be observed by CLSM and quantified by counting GFP-positive cells by flow cytometry. However, for most in vitro and subsequent in vivo studies in cells and animals with target mRNA, the ace-tRNA minicircle can be delivered using the SNP alone. In this case, the editing efficiency can be measured by deep sequencing. At the same time, full-length protein expression and electrophysiological studies can be performed with the treated cells to evaluate ACE-tRNA minicircle-mediated restoration of ion channel function.

[0143] The following SNP preparations can be tested: (1) In HEK293T cells, cationic SNPs (with different cationic APTES ratios, i.e., 10%, 20%, and 30% molar ratios to the total silica reagent (i.e., TEOS and BTPD)) and SNP-HA (with different HA / APTES molar ratios (e.g., 5, 10, and 15), and HA molecular weights ranging from 10 kDa to 1 MDa). (2) In iPSC-RPE, SNP-HA (without ligand) and SNP-HA-ATRA (with 10%, 20%, and 30% molar ratios to HA sugar units). (3) In iPSC-PR, SNP-HA (without ligand) and SNP-HA-11cRal (with 10%, 20%, and 30% molar ratios to HA sugar units, respectively). For control groups to be tested in three cell types, one can compare SNPs with untreated cells, SNPs with Lipofectamine 2000 treated cells (containing payload), and SNP preparations loaded with a negative control payload (eg, non-coding nucleic acid).

[0144] In vitro transfection method

[0145] HEK293T cells can be cultured in 24-well plates. SNP formulations can be treated with cells at 3-12 μg payload dose per well. The ace-tRNA microcircle delivery efficiency can be analyzed four days after treatment. Similarly, iPSC-RPE and PR experiments can be evaluated and the results of the study evaluated.

[0146] Optimization of SNP formulation for ACE-tRNA microcircle delivery in PR and RPE cells in vivo via intravitreal injection

[0147] SNP can encapsulate ace-tRNA microcircle. For optimization, before being applied to SNP, the ligand (ie, ATRA and 11cRal) can be conjugated to HA at a molar ratio of 10%, 20% and 30% to the HA sugar unit. HA of different molecular weights can be tested, including 10, 100 and 1000 kDa. The length of the OEG conjugated to HA by the ligand can be initially maintained at 500 Da. SNP-HA without any ligand can be used as one of the controls. Several controls other than non-ligand SNPs can also be included in the study. Specifically, the delivery efficiency of the above-mentioned SNP preparations with PBS, naked ace-tRNA microcircle, Lipofectamine+ace-tRNA microcircle, non-coding ace-tRNA, and finally the delivery efficiency of the best SNP with the coding Ace-tRNA microcircle can be compared. SNP preparations encapsulating ace-tRNA minicircles and 8-16ug of control (2ul solution, delivered intravitreally) can be injected into Rd1 and Rd12 mouse disease (RPE / PR) models.

[0148] To evaluate the delivery efficiency of ace-tRNA microcircles (ace-tRNA preparations as well as reporter genes) by imaging, mice were euthanized and eyes were collected four days after injection. The eyes removed from these mice were rinsed twice with PBS, punctured at the ora serrata with an 18-gauge needle, and the eyes were opened along the corneal incision. The lens was then carefully removed. The optic cup was flattened and radial incisions were made toward the center to present the final "starfish" appearance. The retina was gently separated from the RPE layer. The separated RPE and retina were placed flat on a coverslip and imaged using a Nikon C2 confocal microscope (Nikon Instruments Inc.) by NIS-Elements. The delivery efficacy of ace-tRNA can be determined by PCR amplification and detection of ace-tRNA sequences. Any decreased vision, weakened ERG response, and retinal structural changes in the RPE, PR, or inner retina (whether by mock delivery of SNPs or SNPs containing ace-tRNA microcircles) may be signs of toxicity.

[0149] Ultrasound (US)-enhanced intravitreal delivery of various SNP-HAs can also be tested. Briefly, microbubbles (i.e., An FDA-approved ultrasound enhancer) will be injected into the vitreous along with the ligand-conjugated SNP-HA formulation. We will use a therapeutic ultrasound (US) machine and a 2 cm 2 The US exposure parameters were as follows: frequency, 1 MHz; intensity, 2 W / cm 2 ; Duty cycle, 50%; Pulse repetition frequency, 100 Hz; Duration, 5 min (95).

[0150] In addition, preliminary data show that SNPs conjugated with appropriate targeting ligands (such as 11cRal and ATRA) can successfully deliver ace-tRNA microcircles to PR and RPE cells in vivo. In addition, cell permeable peptides (CPPs) can be used to enhance the delivery of NPs to PR and RPE cells. Examples of CPPs include TAT (a peptide rich in arginine (97)) and POD (a novel peptide for enhancing the efficiency of DNA delivery to retinal cells in vivo (98, 99)). Because CPPs lack cell type specificity, they can be used for subretinal injections together with PR or RPE cell-specific ligands to enhance the specificity of PR or RPE cell targeted delivery and the cellular uptake of SNPs.

[0151] 18A to 18C The SNP design for subretinal injection is listed. GSH-responsive SNPs were synthesized and ligands or CPPs were targeted and conjugated to the surface of SNPs using PEG (SNP-PEG) as a spacer. Fig.18B and18C The SNP surface modification process and synthesis scheme of 11cRal or ATRA modified SNP or CPP modified SNP are shown. 11cRal or ATRA are conjugated to the PEG end by imine bond or amidation, respectively. CPP (e.g., TAT and POD) is modified with cysteine ​​with a thiol group at the end and then conjugated to the SNP-PEG surface by Michael addition reaction. The morphology and zeta potential of SNP are characterized by TEM and DLS. SNP preparations with different PEG densities (i.e., PEG / SNP weight ratio) and different types (11cRal, ATRA, CPP) and ligand / CPP amounts (i.e., molar ratio of ligand / CPP to total surface PEG) injected subretina were evaluated in HEK293T, iPSC-RPE and iPSC-PR cells. Two factors can be studied to achieve optimal in vivo efficacy, including: (1) the density of PEG on the SNP, which can be adjusted by changing the weight ratio of PEG in the SNP (there are three levels, i.e., 1 / 10, 2 / 10, and 3 / 10), and (2) the density of targeting ligand / CPP on the SNP, which can be adjusted by changing the molar ratio of targeting ligand / CPP to PEG on each SNP (there are three levels, i.e., 0%, 20%, 40%, and 60% molar ratio to PEG).

[0152] Rescue of phenotype in iPSC-RPE and iPSC-PR models of blindness

[0153] For monogenic diseases, iPSC technology provides a unique opportunity to create human disease models with patient-specific genetic features (19). Channelopathies are a key focus when conducting any iPSC-based disease modeling effort. Standard electrophysiological techniques are used to determine the effects of ace-tRNA therapeutics.

[0154] Cell lines and ace-tRNA delivery. Differentiation of iPSCs into retinal organoids and RPE and subsequent characterization of mature cultures

[0155] Retinal organoids from LCA16 and multiple Best patients, as well as their genetically corrected isogenic controls, have been generated (8, 74, 101). R200 A wild-type iPSC line was gene-edited to generate an appropriate nonsense mutation model in BEST1 (74). R628 and CACNA1F R680 All iPSC lines were used. All patient-specific and genetically corrected iPSC lines were differentiated into RPE or stage 3 organoids (which contain numerous PRs with outer segments visible under light microscopy) as previously described (8, 67, 74, 100, 101).

[0156] Characterization of mutant retinal organoid PR phenotypes and initiation of drug read-through experiments

[0157] Overall, the iPSC tool allows testing the ability of read-through ace-tRNA drugs to restore full-length protein production in differentiated KCNJ13 and BEST1 in iPSC-RPE and CACNA1F and CACNA2D4 in iPSC-PR. For iPSC-RPE cells, ace-tRNAs were delivered via SNPATRA minicircles or AAV2.7m8. For iPSC-PRs, ace-tRNAs were delivered using SNP11-cis-retinal minicircles and AAV2.7m8. Expression of ion channel currents and expression of full-length proteins could be determined in KCNJ13, BEST1, CACNA1F, and CACNA2D4 cells. Patch clamp measurements of enzymatically dissociated cells at single-cell resolution produce highly accurate real-time electrophysiological measurements of channel activity. The details of the voltage-dependency and current response waveforms, combined with the specialized composition of the salt solution, allow identification of the activity of individual channel types. TotalSeq TM For fusion protein analysis and scRNA sequencing, libraries were constructed using specific antibody-derived TAGs after ace-tRNA treatment (Biolegend) (102, 103).

[0158] Whole-cell electrophysiological analysis will reveal characteristic ion currents (Kir7.1, Ca2+ activated Cl-, Ca2+) and pharmacological features (8, 74, 104). Measured channel dynamics (such as voltage dependence and pharmacological drug sensitivity) can indicate subtle changes after therapeutic intervention. Previous studies have shown that Kir7.1 and Ca2+ activated Cl- currents were successfully recorded in KCNJ13 and BEST1 mutant iPSC-RPE after gene therapy (74, 105). Protein expression analysis is expected to show membrane localization of channel proteins and full-length protein products compared to untreated cells, as previously shown in KCNJ13 and BEST1 mutant iPSC-RPE. TotalSeqTM validation can determine any differences between RNA and protein expression, as well as any effects that ace-tRNA may have on the overall transcriptional profile (74). AAV9 or AAV5 vectors can be considered.

[0159] Restoration of visual function in a PTC mouse model with PR and RPE dystrophy

[0160] Several mouse mutants with established molecular underpinnings of human blindness phenotypes are available through the Translational Vision Research Models (TVRMs) and can be used to test drugs targeting PR or RPE cells. The rdl mouse carries an autosomal recessive natural mutation 347C-T that switches the mRNA codon from UAC to UAA in the PRPde6b gene, resulting in early-onset severe retinal degeneration (106, 107). AAV-mediated gene therapy restores retinal structure and function in rdl mice (108).

[0161] Similarly, the Cacna1fG305X loss-of-function mouse is a model for human CSNB2A, and the electroretinogram and retinal morphology have been well characterized (109, 110). Functional restoration of the Cav1.4 channel is a good model for PR-targeted ace-tRNA. The nob2 mouse has a spontaneous loss-of-function null mutation. To target RPE cells, the rd12 mouse is a spontaneously generated LCA mouse with an R44X mutation in the Rpe65 gene (111). Therefore, targeting RPE cells with ace-tRNA can rescue retinal morphology, protein expression, and visual function.

[0162] The mice can be bred and maintained. Mice approximately 2-3 weeks old were used for intravitreal injection targeting PR or RPE ( Fig.19 ) to conduct the following experiments.

[0163] 1) PR-rd1 / Cacna1f eyes receive tRNA via AAV2 / 9.

[0164] 2) RPE-rd12 eyes received tRNA via AAV2.7m8.

[0165] 3) A Kcnv2 E314X mouse colony was established by mutation knock-in mediated by CRISPR genome editing. These Kcnv2 mutant mice can be used as a model for targeted PR to study vision (112). The mouse Kcnv2 gene (GenBank accession number: NM183179.1; Ensembl: ENSMUSG00000047298) is located on mouse chromosome 19. Two exons have been identified, the ATG start codon in exon 1 and the TAA stop codon in exon 2. Exon 1 was selected as the target site. A gRNA targeting vector and donor oligonucleotides (with the targeting sequence flanked by 64 bp of homologous sequences combined on both sides) were designed. The E314X (GAA to UAA) mutation site in the donor oligonucleotide can be introduced into exon 1 by homology-directed repair. Silent mutations can be introduced upstream to prevent the gRNA from re-cutting the sequence. Cas9 mRNA, two gRNAs generated by in vitro transcription, and donor oligonucleotides can be co-injected into fertilized eggs to produce KI mice. Pups are genotyped by PCR followed by sequence analysis. Positive founders can be propagated to produce further homozygous colonies. One set of experiments per strain can use AAV-mediated expression, and one set per strain can receive minicircle-based expression. For each experiment, wild-type mice and mock injections serve as experimental controls.

[0166] Visual acuity was assessed by 1) ERG every two weeks for up to 12 weeks post-injection, 2) fundus photographs and OCT images at 4 weeks post-injection, and 3) visual motor responses at 8 weeks. Imaging of marker expression was performed 4-8 days later to confirm delivery. After 3 weeks, some mice were sacrificed to image targeted delivery to the PR or RPE. At the end of the experiment, eyes were removed from control and experimental animals for retinal image comparison and protein expression studies. Using our recently acquired 3View series block face scanning electron microscope (SBFSEM), cellular architecture can be reconstructed in 3D to determine the expression of full-length protein products of the PR and RPE at the finest resolution (113, 114). Images can determine the exact expression of corrected protein products within RPE or PR microdomains (115). Morphological analysis of outer nuclear layer thickness and RPE cell counts was performed using frozen tissue histology. Correlations between proteins and transcriptomes can be established using the TotalSeqTM method.

[0167] Heterozygous (E3 14X / +) and homozygous (E314X / E314X) G to T mutant mice can be detected compared to wild-type mouse (+ / +) sequences. Cas-CLOVER (Herabiolabs) or iG ONAD technology can be used to generate homozygous (E3 14X / E314X) mice (116). These mice are viable after birth and all breeding will follow normal Mendelian inheritance. The ERG waveforms of the mice are severely attenuated for both photopic and scotopic responses, and the outer retinal structure is also altered. Truncated proteins can show altered localization compared to wild-type and heterozygous mice. Phenotypic characterization can reveal cone dystrophies with abnormal rod responses.

[0168] Photopic and scotopic ERG responses as well as measured c-wave amplitudes and implicit times all show normal or near-normal values ​​(35, 117). The effects are likely localized and may result in suboptimal full-field results. The Celeris system can be used to measure multifocal ERGs in mice to reveal any localized changes. Ex vivo ERG or whole-cell recordings can be used to demonstrate expression of channel currents using pharmacological blockers. Fundus photographs and OCT can both show preservation of structures similar to wild-type or heterozygous control mice. Three-dimensional volumes of RPE cells, PRs, and PR synapses can indicate rescue of protein expression and structural phenotypes (same as wild-type retina) compared to untreated ones. For Rd1 and Rd12 mice, PR phagocytosis (118) is expected to be normal. In Cacna1f mice, treatment with ace-tRNA resulted in a crescent-shaped localization of Ca2+ channels in 3-D reconstructed images of the mouse OPL (119). In general, full-length protein products were not associated with changes in the cellular transcriptome profile between treated and untreated cells. Morphology, inflammation, and physiology are expected to be consistent.

[0169] Example 2: Preliminary results of high-fidelity nonsense codon correction

[0170] Generation of ace-tRNA and viral minicircles

[0171] Testing of 2X Arg tRNA in minicircle format transfected into HEK cells together with mOrange2 carrying the TAG mutation showed similar results to the negative control ( Fig. 20B , tRNA sequence randomized 2XArgRAN) compared to the robust mOrange2 expression ( Fig. 20A ). 3XArg tRNA was produced by lentiviral and plasmids were verified, and mOrange2 was also edited using TGA mutation ( Fig. 20C ) and GFP( Fig.20DResults of GFP expression in RPE florets delivered by microcircular GFP packaged in silica nanoparticles (SNPs) and injected via the subretinal route are provided ( FIG. 20E to FIG. 20F ).

[0172] Preparation of hyaluronic acid-coated silica nanoparticles (HA-SNP)

[0173] like Fig.21 As shown, silica nanoparticles (SNPs) can be synthesized by a water-in-oil microemulsion method. An aqueous phase consisting of a mixture of a payload and a silica reagent is emulsified in an oil phase. A silica network is constructed using tetraethyl orthosilicate (TEOS) as a basic building block, while bis[3-(triethoxysilyl)propyl]-disulfide (BTPD) with a disulfide bond is incorporated into the silica network to obtain glutathione (GSH)-reactive SNPs. By adding (3-aminopropyl)triethoxysilane (APTES), the surface of SNP nanoparticles can be modified with amine groups to obtain positively charged SNPs (SNP-NH2). SNP-NH2 can be purified by precipitation in acetone and washing with ethanol and deionized (DI) water. To obtain hyaluronic acid-coated nanoparticles, SNP-NH2 is redispersed in deionized water and thoroughly mixed with an equal volume of HA aqueous solution. The size and surface charge of HA-SNP can be adjusted by the molar ratio of HA sugar unit / APTES. Through electrostatic interaction, HA-SNPs with a size of approximately 50-60 nm and a surface charge of -15 mV can be prepared at a ratio of HA sugar unit / APTES = 0.5.

[0174] In vivo RPE genome editing via subretinal injection of SNPs

[0175] The nucleic acid delivery / genome editing efficiency of SNPs conjugated to the retinal pigment epithelium (RPE) targeting ligand all-trans retinoic acid (ATRA) (SNP-PEG) was tested in transgenic Ai14 mice ( Fig. 22 )(Wang et al., PMID: 34174352; PMCID: PMC8383466). SNP-PEG-ATRA loaded with Cre-mRNA or Cas9 / sgRNA ribonucleoprotein (RNP) induced strong tdTomato expression in the RPE around the subretinal injection site, indicating that SNP can be a reliable nanoplatform for in vivo biomacromolecule delivery.

[0176] In vitro nucleic acid and genome editor delivery via SNPs

[0177] To investigate the DNA and RNP delivery efficiency of HA-SNP in cells, RPF (red fluorescent protein) DNA or RNP was loaded into SNP. Both SNP-NH2 and HA-SNP showed significantly higher gene transfection / editing efficiency than the Lipo 2000 control group. In addition, there was no significant difference between the SNP-NH2 group and the HA-SNP group, indicating that the HA coating did not impair the transfection ability of SNP.

[0178] Kir current and protein expression after ace-tRNA treatment in HEK-FRT cells

[0179] The majority of Kir current analysis in HEK-FRT cells revolved around setting up high-throughput patch clamp electrophysiology. Patch solutions, cell numbers, cell dissociation, and APC protocols were successfully standardized, increasing whole-cell success rates from 51% to 81%. Unlike previously reported by several groups using automated patch systems, Kir7.1 currents were obtained using inwardly rectifying current-voltage plots without the use of enhancers.

[0180] Kv8.2 currents and protein expression in HEK-293 cells

[0181] Kv8.2 is present in photoreceptors and, as a heteromeric channel with Kv2.1, it can shift the steady-state membrane potential and reduce the dark current. Several point mutations or deletions in the KCNV2 gene encoding the Kv8.2 channel lead to altered rod electroretinograms, "cone dystrophy with supernormal rod responses (CDSRR)". Using HEK293 cells stably expressing Kv2.1 channels ( Fig.23A ) Overexpression of Kv8.2 wild type ( Fig. 23B ) or Kv8.2E148*( Fig.23C ). The current recording was optimized using the high-throughput automated patch clamp device QPatchII. Expression of Kv8.2 reduced the current amplitude ( Fig. 23B , Fig.23D ) and changes the activation curve ( Fig.23E Co-expression of Kv8.2E148*, which likely results in a truncated protein product, did not affect the currents or activation curves of Kv2.1 channels, confirming the dominant negative effect of Kv8.2 on Kv2.1 currents to recapitulate the disease phenotype.

[0182] Establishment and characterization of BEST1R200 iPSC-RPE

[0183] The delivery of payload to photoreceptors (PR) in stage 3 3D retinal organoids was tested ( Fig.24). In this example, 3D organoids were treated with 15ug / well of SNPs conjugated to a cell-permeable peptide (CPP) carrying a 10% payload of nuclear-localized Cas9 / gRNA complex RNPs conjugated to ATTO 550. Due to osmotic toxicity, half of the culture medium was replaced after 24 hours. After removing excess SNPs, the organoids were fixed and imaged. The upper panels in the figure represent images of untransduced organoids and the lower panels represent images of transduced organoids. In the figure, ATTO 550 appears to be localized in the interphotoreceptor matrix and in the nuclei of CRX+ and CRX- cells.

[0184] Generation of Kcnv2 and Cacna1f mutant mice

[0185] Nonsense mutations in human KCNV2 and CACNA1f cause CDSSR and congenital stationary night blindness (CSNB), respectively. Mouse models have been generated to investigate the mechanisms linking gene mutations to retinal dystrophies and to test the effectiveness of ace-tRNA therapeutics for channelopathies. These models were developed by genome editing using Cas9, the corresponding guide RNA, and a 64-nucleotide donor DNA containing the desired mutation. Fertilized eggs were microinjected into surrogate mothers and the pups were genotyped. Fig.25 As shown, sequencing of PCR products identified heterozygous mice Kcnv2 pE156* (GAG to TGA) with a silent mutation (GAC to GAT) and Cacna1f pR830* (CGA to TGA) with a silent mutation (GCT to GCG). PCR products confirmed the gene mutation of the founder.

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[0319] While the foregoing description and examples fully disclose and enable the invention, they are not intended to limit the scope of the invention, which is defined by the appended claims.

[0320] All publications, patents and patent applications are incorporated herein by reference. Although the present invention has been described in conjunction with certain embodiments of the present invention in the foregoing description, and many details have been set forth for illustrative purposes, it will be apparent to those skilled in the art that the present invention may have other embodiments, and that certain details described herein may vary considerably without departing from the basic principles of the present invention.

[0321] In the context of describing the present invention, the use of the terms "a" and "an" and "said" and similar referents should be interpreted as covering both the singular and the plural, unless otherwise specified herein or clearly contradictory to the context. The terms "comprising", "having", "including" and "containing" should be interpreted as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise specified herein. Unless otherwise specified herein, the enumeration of the value range herein is intended to be used as a shorthand method for individually referring to each individual value belonging to the scope, and each individual value is incorporated into this specification as it is individually listed herein. Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be carried out in any suitable order. The use of any and all examples or exemplary languages ​​(e.g., "such as") provided herein is intended only to better illustrate the present invention, and unless otherwise required, will not impose limitations on the scope of the present invention. The language in this specification should not be interpreted as indicating any non-required element as necessary for practicing the present invention.

[0322] Embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. After reading the foregoing description, variations of those embodiments may become apparent to those of ordinary skill in the art. The inventor hopes that the technician will adopt such variations as appropriate, and the inventor intends to practice the present invention in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter recited in the appended claims of the present invention as permitted by applicable law. In addition, unless otherwise specified herein or clearly contradictory to the context, the present invention encompasses any combination of the above-mentioned elements in all possible variations.

Claims

1. A method for reading through one or more premature stop codons (PTCs) in cells of the eye, comprising delivering one or more tRNAs, one or more oligonucleotides encoding one or more tRNAs, one or more vectors encoding one or more tRNAs, one or more expression cassettes encoding one or more tRNAs, or one or more compositions comprising any of the foregoing provided herein, in an amount effective to read through the one or more PTCs to cells of the eye.

2. A method for restoring protein function in cells of the eye, comprising delivering one or more of the tRNAs, one or more of the oligonucleotides encoding one or more of the tRNAs, one or more of the vectors encoding one or more of the tRNAs, one or more of the expression cassettes encoding one or more of the tRNAs, or one or more of the compositions comprising any of the foregoing provided herein to the cells of the eye in an amount effective to restore protein function in the cells.

3. A method for restoring vision in a subject, comprising administering one or more of the tRNAs, one or more of the oligonucleotides encoding one or more of the tRNAs, one or more of the vectors encoding one or more of the tRNAs, one or more of the expression cassettes encoding one or more of the tRNAs, or one or more of the compositions comprising any of the foregoing provided herein to cells of the eye in an amount effective to restore vision to the subject.

4. The method of claim 3, wherein the administration is by subretinal injection.

5. The method of claim 3, wherein the administering is by intravitreal injection.

6. The method of any one of claims 3-5, wherein the subject is a human.

7. The method of any one of the preceding claims, wherein the cells are photoreceptor cells.

8. The method of any one of claims 1-6, wherein the cell is a retinal pigment epithelial cell.

9. The method of any one of the preceding claims, wherein the cells are iPSCs.

10. The method of any one of the preceding claims, wherein the one or more oligonucleotides or one or more expression cassettes is DNA, such as cDNA.

11. The method of any one of the preceding claims, wherein the one or more oligonucleotides or one or more expression cassettes are in the form of a plasmid.

12. The method of any one of the preceding claims, wherein the one or more oligonucleotides or the one or more expression cassettes are in the form of a minicircle.

13. The method of any one of claims 1-9, wherein the vector is a viral vector.

14. The method of claim 13, wherein the viral vector is an AAV vector.

15. The method of claim 14, wherein the AAV vector is an AAV2, AAV5, AAV9, AAV2 / 9, AAV7m8 or AAV2.7m8 vector.

16. The method of any of the preceding claims, wherein at least two or at least three of any of the tRNAs provided herein are delivered or administered.

17. The method of any one of claims 1-15, wherein the at least two or at least three tRNAs provided herein are expressed encoded by the one or more oligonucleotides, one or more vectors, one or more expression cassettes, or are contained in one or more of the compositions.

18. The method of any one of the preceding claims, wherein the one or more tRNAs are one or more ArgtRNAs, one or more TrptRNAs and / or one or more GlutRNAs.

19. The method of any of the preceding claims, wherein the one or more tRNAs, the one or more oligonucleotides encoding the one or more tRNAs, the one or more vectors encoding the one or more tRNAs, the one or more expression cassettes encoding the one or more tRNAs, or the one or more compositions comprising any of the foregoing provided herein are contained in a delivery vehicle.

20. The method of claim 19, wherein the delivery vehicle is a particle.

21. The method of claim 20, wherein the particle is a lipid-based particle, such as a liposome.

22. The method of claim 20, wherein the particles are polymer-based particles.

23. The method of claim 20, wherein the particles are silica nanoparticles (SNPs).

24. The method of claim 23, wherein the SNPs are delivered or administered as a population of SNPs.

25. The method of claim 24, wherein the SNP is conjugated to HA.

26. The method of claim 25, wherein the HA has a molecular weight in the range of 10 kDa-1 MDa, such as any one of the specific molecular weights provided herein.

27. The method of any one of claims 23-26, wherein the one or more SNPs have surface amine groups, such as APTES amine groups.

28. The method of claim 27, wherein the molar ratio of APTES to total silica of the one or more SNPs is in the range of 10-30%, such as any one of the specific molar ratios provided herein.

29. The method of claim 27 or 28, wherein the molar ratio of HA carbohydrate units to APTES of the one or more SNPs is 5-15, such as any one of the specific molar ratios provided herein.

30. The method of any one of claims 23-29, wherein the one or more SNPs are conjugated to a targeting ligand or a cell penetrating peptide (CPP), such as any of the targeting ligands or CPPs provided herein.

31. The method of claim 30, wherein the molar ratio of targeting ligand or CPP to HA is 10-30%, such as any one of the specific molar ratios provided herein.

32. The method of any one of claims 25-31, wherein the degree of substitution of HA to a targeting ligand or CPP conjugate is 1-40%, such as any one of the specific degrees of substitution provided herein.

33. The method of any one of claims 23-32, wherein the one or more SNPs are conjugated to PEG.

34. The method of claim 33, wherein the PEG links the targeting ligand or CPP to the one or more SNPs.

35. The method of claim 33 or 34, wherein the weight ratio of PEG / SNP is 1 / 10-3 / 10, such as any of the specific weight ratios provided herein.

36. The method of any one of claims 33-35, wherein the ratio of the targeting ligand or CPP to PEG is 1-60, such as any one of the specific ratios provided herein.

37. The method of any one of claims 30 or 31, wherein the targeting ligand or CPP is conjugated to the one or more SNPs via a spacer such as OEG.

38. The method of claim 37, wherein the OEG has a molecular weight of 200 Da-1 kDa, such as any one of the specific molecular weights provided herein.

39. The method of any one of claims 23-38, wherein the average size of the one or more SNPs is between 40-60 nm, such as any one of the specific average sizes provided herein.

40. A composition comprising one or more tRNAs, one or more oligonucleotides, one or more vectors, one or more expression cassettes, or one or more compositions of any one of the preceding claims.

41. A composition comprising the delivery vehicle or population of delivery vehicles, such as the particles, of any one of claims 19-40.

42. The composition of claim 40 or 41, further comprising a pharmaceutically acceptable carrier.

43. A cell comprising the one or more tRNAs, one or more oligonucleotides, one or more vectors, one or more expression cassettes, or one or more compositions of any one of the preceding claims.

44. The cell of claim 43, wherein the cell is a photoreceptor cell.

45. The cell of claim 43, wherein the cell is a retinal pigment epithelial cell.

46. ​​The cell of any one of claims 43-45, wherein the cell is an iPSC.

47. The cell of any one of claims 43-26, which is of a mouse model, such as provided herein.

48. The cell of claim 47, wherein the mouse model is a Kcnv2 and Cacna1f mutant mouse, such as provided herein.

49. A Kcnv2 mutant mouse.

50. A Cacna1f mutant mouse.

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