Endoproteins and uses thereof

Abstract

The present invention relates to endoproteins and uses thereof. The present invention relates to constructs, vectors, related host cells and pharmaceutical compositions that allow for effective gene therapy, in particular for genes greater than 5 Kb.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on October 15, 2019, with application number 201980081288.0 and invention title "Endoprotein and its use". Technical Field

[0002] This invention relates to constructs, vectors, associated host cells, and pharmaceutical compositions that enable effective gene therapy, particularly for diseases caused by mutations in genes with coding sequences (CDS) greater than 5 Kb. Background Technology

[0003] Gene therapy using adeno-associated virus (AAV) vectors is safe and effective for humans. AAV-based gene therapy products have been approved in the United States and Europe in recent years for hereditary metabolic and blinding diseases, and clinical trials of AAV-based gene therapy approaches targeting various therapeutic areas are continuously increasing, ranging from ophthalmological to hematological to musculoskeletal and metabolic diseases.

[0004] However, the limitation of AAV vector cargo capacity prevents the development of AAV-based therapies for diseases caused by mutations in genes with coding sequences (CDS) greater than 5 kb (also referred to as large genes in this paper).

[0005] Genetic diseases caused by major gene mutations (listed in Table 1 below) include Duchenne muscular dystrophy caused by DMD gene mutation, cystic fibrosis caused by CFTR gene mutation, hemophilia A caused by F8 gene mutation, dysferlinopathies caused by DYSF gene mutation, polycystic kidney disease caused by PKD gene mutation, Wilson's disease caused by ATP7B gene mutation, Huntington's disease caused by HTT gene mutation, and Niemann-Pick type C disease caused by NPC1 gene mutation.

[0006] Table 1: Genetic Diseases Caused by Major Gene Mutations

[0007]

[0008] In addition, some inherited retinal degenerations (IRDs) are caused by the large gene mutations listed in Table 2 below. In Europe and the United States, approximately 1 in 3,000 people are affected by IRD (58).

[0009] The most common and severe IRDs are retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and STGD, which are usually inherited as single-gene conditions with a global prevalence of 1 in 2,000 (1) and are the leading cause of blindness worldwide. Most mutations leading to IRDs occur in genes expressed in the photoreceptors (PR), rods, and / or cones of neurons in the retina (2).

[0010] Gene therapy holds great promise for treating IRD. The first gene therapy product based on an adeno-associated virus (AAV) vector for a form of inherited blindness was approved in December 2017 (3). In addition, many other AAV-based products are currently in clinical development for gene therapy of both rare and common forms of blindness (4). Although AAV is now recognized as the most effective gene therapy vector for retinal diseases to date (4,5), its limited cargo capacity hinders its use in treating conditions that require the delivery of DNA sequences larger than 5 kb (6), which include not only transgenes but also cis-regulatory elements necessary for their expression.

[0011] Table 2 below summarizes examples of disease genes that are larger than 5kb in size.

[0012] Table 2: Disease genes larger than 5kb

[0013]

[0014] Staggart disease (STGD; MIM#248200) is the most common form of inherited macular degeneration caused by a mutation in the ABCA4 gene (CDS: 6822 bp), which encodes an all-trans retinal transporter located in the outer segment of the PR (7); Usher syndrome type IB (USH1B; MIM#276900) is the most severe form of RP and deafness caused by a mutation in the MYO7A gene (CDS: 6648 bp) that encodes an unconventional MYO7A, an actin-based kinase expressed in both the PR and RPE in the retina (9-11).

[0015] Type 3 cone-rod dystrophy, fundus flavimaculatus, type 2 age-related macular degeneration, early-onset severe retinal dystrophy, and retinitis pigmentosa type 19 are also associated with ABCA4 mutations (referred to as ABCA4-related diseases in this article).

[0016] The inventors and others have demonstrated that this limitation can be overcome by using dual (up to 9 kb) (6, 12, 13) or triple (up to 14 kb) (14) AAV vectors, each containing a fragment of the coding sequence (CDS) of a large transgene expression cassette. Dual and triple AAV vectors utilize the cloning and recombination of the AAV genome to reconstruct the full-length genome in cells co-infected by multiple AAV vectors. However, for photoreceptors, which are the primary therapeutic targets for most inherited retinal diseases, transgene expression efficiency achieved using dual or triple AAV vectors is lower than that achieved using a single AAV vector (6, 14, 15). This is likely due to various limiting steps required for efficient transduction, including proper DNA polymerization, heterogeneous mRNA stability, and splicing efficiency at the transvector junction.

[0017] The inventors demonstrated in WO2014 / 170480 and Colella et al. (15) that dual AAV vectors reconstruct large genes through splicing (trans-splicing), homologous recombination (overlap), or a combination of both (hybridization), and found that dual trans-splicing and hybridization vectors are particularly effective for treating hereditary retinal degeneration. Furthermore, Maddalena et al. (14) demonstrated a triple AAV vector approach for genes up to 14 kb. However, transgene expression efficiency achieved using dual or triple AAV vectors is lower than that achieved using a single AAV vector (6, 13, 14). This is likely due to various limiting steps required for effective transduction, including proper DNA polymer formation, heterogeneous mRNA stability, and splicing efficiency at the transvector junction. Additionally, the gene expression levels produced by the triple AAV vector strategy are below the threshold required for therapeutic treatment.

[0018] Therefore, constructs and vectors that can be used to reconstruct large gene expression for effective gene therapy are still needed.

[0019] The inventors have now discovered that multiple AAV vectors, each delivering a fragment encoding a reporter or a large therapeutic protein and flanked by short cleavage-type endoproteins, can achieve protein trans-splicing and full-length protein reconstruction both in vitro and in vivo.

[0020] Intrins are genetic elements transcribed and translated within host proteins. They self-excise in a manner similar to protein introns without energy supply, exogenous host-specific proteases, or cofactors, leaving no amino acid modifications in the final protein product (16, 17, 27, 28). The activity of intrins is background-dependent, with certain peptide sequences (called N- and C-exenes) surrounding their junctions necessary for efficient trans-splicing, most importantly the thiol- or hydroxyl-containing amino acids (e.g., cysteine, serine, or threonine) that serve as the first residue in C-exenes (18). Split-type intrins are a subset of intrins that are expressed as two separate polypeptides at the ends of two host proteins and catalyze their trans-splicing to produce a single, larger polypeptide (19). Introns, including split-type introns, are widely used in biotechnological applications, including protein purification and labeling steps (19, 20) and the reconstruction of widely used CRISPR / Cas9 genome editing nucleases (21, 22).

[0021] Several attempts have been made to reconstruct the expression of therapeutic genes, including factor VIII genes, using endonucleoprotein-based protein splicing. Among these, it has been demonstrated that the fusion of heavy and light chain genes with the factor VIII ... Furthermore, a protein transsplicing strategy mediated by splitting endorphins (i.e., *N. punctiforme* DnaE splitting endorphins) is alleged to reconstruct macropore-forming subunits of L-type calcium channels from two separate fragments in heart cells (26). US 6,544,786 further reports the use of splitting endorphins to deliver small genes for dystrophin.

[0022] The inventors utilize the intrinsic ability of cleavage-type endogenous proteins to mediate protein trans-splicing, fragmenting large full-length proteins into two or three polypeptides flanked by cleavage-type endogenous proteins, and then reconstructing the large full-length protein, wherein the coding sequence of the polypeptide is suitable for a single AAV vector.

[0023] Therefore, the present invention achieves the reconstruction of cellular macroproteins by providing target cells with two or more fragments of the macroproteins, wherein the fragments of the macroproteins are fused with mitotic endoproteins to promote endoprotein-mediated trans-splicing and reconstruct functional proteins. Summary of the Invention

[0024] This invention provides gene therapy using an AAV vector for diseases caused by gene mutations, particularly gene mutations with coding regions exceeding 5 kb.

[0025] Based on the discovery that single-celled organisms use protein trans-splicing mediated by cleaving endoproteins for protein remodeling, the inventors constructed multiple AAV vectors, each encoding either a reporter fragment flanked by a short cleaving endoprotein or a fragment of a large therapeutic protein, thereby inducing protein trans-splicing and full-length protein remodeling in vivo and in vitro.

[0026] Advantageously, compared to known AAV-based large protein methods in the art, the AAV-based protein trans-splicing-mediated reconstruction of disease proteins achieved by the present invention provides higher target protein expression levels. This is likely due to overcoming various limiting steps required for efficient transduction of dual-vector-based systems, including: formation of correct DNA multiply, stability of heterogeneous mRNA, and splicing efficiency at the transvector junction.

[0027] This invention provides a vector system for expressing a coding sequence in cells, the coding sequence comprising a first part (CDS1), a second part (CDS2), and an optional third part (CDS3), the vector system comprising:

[0028] a) A first carrier, the first carrier comprising:

[0029] - The first portion (CDS1) of the encoded sequence,

[0030] - The first nucleotide sequence encoding the N-endoprotein, said sequence being located at the 3' end of CDS1; and

[0031] b) A second carrier, the second carrier comprising:

[0032] - The second part (CDS2) of the encoded sequence,

[0033] - A second endonuclein nucleotide sequence encoding C-endon, the sequence being located at the 5' end of CDS2;

[0034] When the first and second vectors are inserted into the cell, the protein product encoding the sequence is generated by protein splicing.

[0035] Or the carrier system may include:

[0036] a') A first carrier, the first carrier comprising:

[0037] - The first portion (CDS1) of the encoded sequence,

[0038] - A first intraprotein nucleotide sequence encoding a first N-endoprotein, said sequence being located at the 3' end of CDS1; and

[0039] b') A second carrier, the second carrier comprising:

[0040] - The second part (CDS2) of the encoded sequence,

[0041] - A second endonuclein nucleotide sequence encoding the first C-endon, the sequence being located at the 5' end of CDS2;

[0042] - A nucleotide sequence encoding a third inner protein of the second N-intrin, said sequence being located at the 3' end of CDS2; and

[0043] c') A third carrier, the third carrier comprising:

[0044] - The third part of the encoded sequence (CDS3)

[0045] - A fourth nucleotide sequence encoding the second C-endoprotein, located at the 5' end of CDS3.

[0046] The first endonucleoprotein nucleotide sequence is different from the third endonucleoprotein nucleotide sequence, and the second endonucleoprotein sequence is different from the fourth endonucleoprotein nucleotide sequence. When the first vector, the second vector, and the third vector are inserted into the cell, the protein product encoding the sequence is generated by protein trans-splicing.

[0047] Preferably, in the vector system, the first endonucleoprotein, the second endonucleoprotein, the third endonucleoprotein, and the fourth endonucleoprotein encode a split-type endonucleoprotein. Preferably, the maximum length of the split-type endonucleoprotein is 150 amino acids. More preferably, the split-type endonucleoprotein is a DnaE or DnaB endonucleoprotein.

[0048] According to the present invention, an endoprotein is a fragment of a protein that is capable of excising itself and connecting the remaining portion (exoprotein) with peptide bonds through a process called protein splicing. The fragment is called an "endoprotein," representing an internal protein sequence, and an "exoprotein," representing an external protein sequence, wherein the upstream exoprotein is called an "N-exoprotein" and the downstream exoprotein is called a "C-exoprotein," and the upstream endoprotein is called an "N-endoprotein" and the downstream endoprotein is called a "C-endoprotein."

[0049] Therefore, in the context of this invention, the N-endoprotein is an endonucleoprotein fragment located at the N-terminus of the first polypeptide (and fused with the first polypeptide), and the C-endoprotein is an endonucleoprotein fragment located at the C-terminus of the second polypeptide (and fused with the second polypeptide), wherein after expression of the two polypeptides, the two endonucleoprotein fragments undergo protein trans-splicing and join to form a complete endonucleoprotein, and link the two polypeptides together, wherein the full-length protein is reconstructed when the two polypeptides form a full-length protein.

[0050] According to the present invention, the first endonucleoprotein sequence is an N-endonucleoprotein sequence and the second endonucleoprotein sequence is a C-endonucleoprotein sequence, wherein the N-endonucleoprotein and the C-endonucleoprotein preferably originate from the same endonucleoprotein or split-type endonucleoprotein gene. Alternatively, the N-endonucleoprotein and the C-endonucleoprotein originate from two different endonucleoprotein genes capable of naturally undergoing trans-splicing or modified to undergo the trans-splicing reaction. Accordingly, the same gene can originate from the same organism or different organisms. For example, widely used split-type endonucleoproteins originate from the DnaE gene of different organisms. According to the present invention, when the coding sequence of the target protein is split into two parts, the N-endonucleoprotein coding sequence is fused in-frame with the sequence encoding the N-terminal portion of the target protein; the C-endonucleoprotein coding sequence is fused in-frame with the sequence encoding the C-terminal portion of the target sequence. After expression of the two precursor fusion proteins, the endonucleoprotein undergoes autocatalytic excision and forms a connected exonucleoprotein, such as the reconstructed target protein.

[0051] According to the present invention, the coding sequence of the target protein can be split into three parts. Accordingly, the first endonucleoprotein sequence is an N-endonucleoprotein sequence and the second endonucleoprotein sequence is a C-endonucleoprotein sequence, wherein the first endonucleoprotein coding sequence is fused in a frame at its C-terminus to the sequence encoding the N-part of the target protein, and the second endonucleoprotein coding sequence is fused in a frame at its N-terminus to the sequence encoding the middle part of the target protein. Accordingly, the N-endonucleoprotein and the C-endonucleoprotein preferably originate from the same endonucleoprotein or split-type endonucleoprotein gene. Alternatively, the N-endonucleoprotein and the C-endonucleoprotein originate from two different endonucleoprotein genes, which are capable of naturally undergoing trans-splicing or being modified to undergo the trans-splicing. Accordingly, the same gene can come from the same organism or different organisms. In the configuration of the present invention, the third endonucleoprotein is an N-endonucleoprotein coding sequence fused in a frame to the C-terminal sequence encoding the middle part of the target protein, and the fourth endonucleoprotein is a C-endonucleoprotein coding sequence fused in a frame to the N-terminus sequence encoding the C part of the target protein. Accordingly, the third and fourth endonucleoproteins preferably originate from the same endonucleoprotein or split-type endonucleoprotein gene. Alternatively, the N-endonucleoprotein and the C-endonucleoprotein originate from two different endonucleoprotein genes, which are capable of natural trans-splicing or modified to perform the trans-splicing reaction. Accordingly, the same gene can originate from the same organism or different organisms. Within the scope of the invention, the first and second endonucleoproteins, as well as the third and fourth endonucleoproteins, originate from different endonucleoprotein genes, and the first endonucleoprotein selectively binds to the second endonucleoprotein, while the third endonucleoprotein selectively binds to the fourth endonucleoprotein.

[0052] In this invention, when a first vector, a second vector, and optionally a third vector are inserted into a cell, at least two or three fusion proteins are formed, and upon contact with the two or three fusion proteins, a protein product encoding the said sequence is generated. The contact step is performed under conditions that allow N-endoprotein to bind to C-endoprotein.

[0053] In this invention, when the first, second, and third carriers are inserted into cells, three independent polypeptides are generated, and full-length proteins are produced through trans-splicing. The key to the formation of the three AAV intraprotein carriers is the use of different intraproteins, namely DnaE and DnaB, which do not cross-react, thereby preventing improper trans-splicing between the polypeptides generated by the first and third carriers.

[0054] According to a preferred embodiment of the present invention, a vector system for expressing the coding sequence of a target gene in cells comprises two vectors, each vector comprising a portion of the coding sequence flanked by an intraprotein sequence, wherein the 5' end of the coding sequence is flanked by an N-intraprotein sequence at the 3' end, and the 3' end of the coding sequence of the target gene is flanked by a C-intraprotein sequence, such that when the two vectors are expressed in cells, two fusion proteins are produced, and the target full-length protein is generated due to a spontaneous trans-splicing reaction.

[0055] According to another preferred embodiment of the present invention, a vector system for expressing the coding sequence of a target gene in cells comprises three vectors, each vector comprising a portion of the coding sequence flanked by an endonuclein sequence, wherein the coding sequence is divided into three parts such that the 5' end of the coding sequence is flanked by a first N-endonuclein sequence at the 3' end; the middle portion of the coding sequence is flanked by a first C-endonuclein at the 5' end and by a second N-endonuclein at the 3' end; and the 3' portion of the coding sequence is flanked by a second C-endonuclein at the 5' end, such that when all three vectors are expressed in cells, three fusion proteins are produced, and the target full-length protein is generated due to a spontaneous trans-splicing reaction, wherein the first N-endonuclein reacts with the first C-endonuclein, and the second N-endonuclein reacts with the second C-endonuclein.

[0056] The fissile endonucleoprotein of the present invention can be encoded by a single gene, which is then engineered to encode two separate endonucleoprotein fragments, such as fissile endonucleoproteins; alternatively, naturally occurring fissile endonucleoproteins are encoded by two separate genes; for example, in cyanobacteria, DnaE, ​​the catalytic subunit α of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. Preferred endonucleoproteins in the present invention are endonucleoproteins derived from endonucleoproteins (e.g., small endonucleoproteins) or fissile endonucleoproteins formed through trans-splicing reactions, having a length of 150 aa or less.

[0057] The splitting endonucleoprotein of the present invention may be 100%, 98%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical to naturally occurring endonucleoproteins or SEQ ID Nos. 1 to 14 (homophores), wherein the endonucleoprotein retains the ability to perform trans-splicing reactions. Fragments or variants of naturally occurring or modified endonucleoproteins that retain trans-splicing activity are within the scope of the present invention.

[0058] Conveniently, the fission-type endonucleoproteins of the present invention can be derived from the same gene isolated from different organisms. Preferred endonucleoprotein genes are DnaB and DnaE.

[0059] In a preferred embodiment, the endonucleoprotein of the present invention is a fission-type endonucleoprotein derived from the DnaE gene (e.g., DNA polymerase III subunit α) of cyanobacteria, including *Nostoc punctiforme* (Npu), *Synechocystis* sp. PCC6803 (Ssp), *Fischerella* sp. PCC 9605, *Scytonema tolypothrichoides*, cyanobacteria SW_9_47_5, *Nodularia spumigena*, *Nostoc flagelliforme*, *Crocosphaera watsonii* WH 8502, *Chroococcidiopsis cubana* CCALA 043, and *Trichodesmium*. erythraeum); preferably, the endoprotein of the present invention is derived from the Dna E gene isolated from Nostoc punctata or Synechocystis PCC6803.

[0060] In another preferred embodiment, the endogenous protein of the present invention is a fission-type endogenous protein derived from the DnaB gene of cyanobacteria, including, for example, the marine red halophilic bacterium (R. marinus, Rma), Synechocystis PC6803 (Ssp), and Porphyra purpurea chloroplast (Ppu) described in (59).

[0061] Preferably,

[0062] - The first intraprotein nucleotide sequence encodes an intraprotein selected from the group consisting of: SEQ. ID. No. 1, 3, 5, 7, 9, 11, 13 or variants thereof, fragments thereof or homologues thereof;

[0063] - The second intraprotein nucleotide sequence encodes an intraprotein selected from the group consisting of: SEQ. ID. No. 2, 4, 6, 8, 10, 12, 14 or variants thereof, fragments thereof or homologues thereof;

[0064] - The third endonucleoprotein nucleotide sequence encodes an endonucleoprotein selected from the group consisting of: SEQ. ID. No. 1, 3, 5, 7, 9, 11, 13 or variants thereof, fragments thereof or homologues thereof;

[0065] - The fourth endonucleoprotein nucleotide sequence encodes an endonucleoprotein selected from the group consisting of: SEQ. ID. No. 2, 4, 6, 8, 10, 12, 14 or variants thereof, fragments thereof or homologues thereof;

[0066] Preferably, when the first or third endonucleoprotein is SEQ ID No. 1, the second or fourth endonucleoprotein is SEQ ID 2; or when the first or third endonucleoprotein is SEQ ID 3, the second or fourth endonucleoprotein is SEQ ID 4; or when the first or third endonucleoprotein is SEQ ID 5, the second or fourth endonucleoprotein is SEQ ID 6; or when the first or third endonucleoprotein is SEQ ID 7, the second or fourth endonucleoprotein is SEQ ID 8; or when the first or third endonucleoprotein is SEQ ID 9, the second or fourth endonucleoprotein is SEQ ID 10; or when the first or third endonucleoprotein is SEQ ID 11, the second or fourth endonucleoprotein is SEQ ID 12.

[0067] Preferably, when the first endonucleoprotein is SEQ ID 1 and the second endonucleoprotein is SEQ ID 2, the third endonucleoprotein is not SEQ ID 1 and the fourth endonucleoprotein is not SEQ ID 2; preferably, when the first endonucleoprotein is SEQ ID 3 and the second endonucleoprotein is SEQ ID 4, the third endonucleoprotein is not SEQ ID 3 and the fourth endonucleoprotein is not SEQ ID 4; preferably, when the first endonucleoprotein is SEQ ID 5 and the second endonucleoprotein is SEQ ID 6, the third endonucleoprotein is not SEQ ID 5 and the fourth endonucleoprotein is not SEQ ID 6; preferably, when the first endonucleoprotein is SEQ ID 7 and the second endonucleoprotein is SEQ ID 8, the third endonucleoprotein is not SEQ ID 7 and the fourth endonucleoprotein is not SEQ ID 8; preferably, when the first endonucleoprotein is SEQ ID 9 and the second endonucleoprotein is SEQ ID 10, the third endonucleoprotein is not SEQ ID 9 and the fourth endonucleoprotein is not SEQ ID 10; preferably, when the first endonucleoprotein is SEQ ID 11 and the second endonucleoprotein is SEQ ID 12, the third endonucleoprotein is not SEQ ID 10. 11. And the fourth endonucleoprotein is not SEQ ID 12.

[0068] In one specific implementation, the first endonucleoprotein is SEQ ID 1, the second endonucleoprotein is SEQ ID 2, the third endonucleoprotein is SEQ ID 3, and the fourth endonucleoprotein is SEQ ID 4; or, the first endonucleoprotein is SEQ ID 5, the second endonucleoprotein is SEQ ID 6, the third endonucleoprotein is SEQ ID 3, and the fourth endonucleoprotein is SEQ ID 4.

[0069] In a preferred embodiment, the first, second, and third vectors further include a promoter sequence operatively connected to the 5' end portion of the first portion (CDS1) of the coding sequence, or the 5' end portion of the second portion (CDS2) of the coding sequence, or the 5' end portion of the third portion (CDS3) of the coding sequence.

[0070] Preferred promoters are ubiquitous, artificial, or tissue-specific promoters, including fragments and variants that retain transcription promoter activity. Particularly preferred promoters are photoreceptor-specific promoters, including photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1), interreceptor photopigment-like protein promoter (IRBP), rhodopsin promoter (RHO), uveal dystrophy 2 promoter (VMD2), and rhodopsin kinase promoter (RK); further particularly preferred promoters are muscle-specific promoters, including MCK and MYODI; liver-specific promoters, including thyroxine-binding globulin (TBG) and heterozygous liver-specific promoter (HLP) (67); neuron-specific promoters, including hSYN1 and CaMKIIa; and kidney-specific promoters, including Ksp-cadherin 16 and NKCC2. The ubiquitous promoters according to the invention are, for example, ubiquitous cytomegalovirus (CMV) (32) and short CMV (33) promoters. More preferred promoters within the scope of this invention are GRK1, TBG, CaMKIIa, and Ksp-cadherin 16.

[0071] In a more preferred embodiment, the first, second, and third vectors further comprise a 5'-terminal repeat (5'-TR) nucleotide sequence and a 3'-terminal repeat (3'-TR) nucleotide sequence, preferably, the 5'-TR is a 5'-inverted terminal repeat (5'-ITR) nucleotide sequence, and the 3'-TR is a 3'-inverted terminal repeat (3'-ITR) nucleotide sequence.

[0072] In a more preferred embodiment, the first, second, and third vectors further comprise a polyadenylation signal nucleotide sequence.

[0073] In a more preferred embodiment, the coding sequence is split into a first portion, a second portion, and optionally a third portion at positions consisting of nucleophilic amino acids that do not fall into the structural or functional domains of the encoded protein product, wherein the nucleophilic amino acids are selected from serine, threonine, or cysteine.

[0074] Preferably, at least one of the first vector, the second vector, and the third vector further includes at least one enhancer or regulatory nucleotide sequence operatively linked to the coding sequence.

[0075] Preferred enhancer or regulatory nucleotide sequences are globin IgG chimeric introns and post-transcriptional regulatory elements of marmot hepatitis virus.

[0076] Optionally, at least one of the first, second, and third vectors further includes at least one degradation signal to reduce the stability of the reconstructed protein.

[0077] Preferably, the degradation signal is a CL1 degron or a PB29 degradation determinant. More preferably, the degradation signal is ecDHFR or a fragment thereof, preferably, the ecDHFR degradation signal is a DHFR variant that functions as an internal degradation determinant as described herein. Most preferably, the fragment retains the degradation characteristics of ecDHFR, preferably, the characteristics of a variant DHFR that functions as an internal degradation determinant, preferably, this fragment is a small ecDHFR, wherein the small ecDHFR is a variant that functions as an internal degradation determinant.

[0078] Preferably, the coding sequence encodes a protein that can correct a pathological state or symptom, and preferably, the symptom is retinal degeneration, metabolic disease, blood disease, neurodegenerative disease, hearing loss, channel disease, lung disease, myopathy, heart disease, or muscular dystrophy.

[0079] More preferably, the coding sequence encodes a protein capable of correcting a pathological state or symptom; preferably, the symptom is retinal degeneration; preferably, the retinal degeneration is hereditary; preferably, the pathology or disease is selected from the group consisting of: retinitis pigmentosa (RP), Lieber's congenital amaurosis (LCA), Stargardt disease (STGD), Usher syndrome (USH), Alstrom syndrome, congenital stationary night blindness (CSNB), macular dystrophy, latent macular dystrophy, and diseases caused by mutations in the ABCA4 gene. More preferably, the coding sequence is the coding sequence of a gene selected from the group consisting of: ABCA4, MYO7A, CEP290, CDH23, EYS, PCDH15, CACNA1, SNRNP200, RP1, PRPF8, RP1L1, ALMS1, USH2A, GPR98, HMCN1, or fragments thereof or orthologs thereof, or small genes thereof with a coding sequence length exceeding 5kb, i.e., the smallest gene fragment comprising one or more exons and the regulatory elements necessary for the gene to express itself in the same manner as the wild-type gene fragment.

[0080] More preferably, the coding sequence encodes proteins that can correct muscular dystrophy, such as Dechené muscular dystrophy, cystic fibrosis, hemophilia A, Wilson's disease, etc.

[0081] Phenylketonuria, dysferlinopathies, Rett's syndrome, polycystic kidney disease, Niemann-Pick type C, and Huntington's disease.

[0082] More preferably, the coding sequence is the coding sequence of a gene selected from the group consisting of: ABCA4, MYO7A, CEP290, CDH23, EYS, PCDH15, CACNA1, SNRNP200, RP1, PRPF8, RP1L1, ALMS1, USH2A, GPR98, HMCN1 or fragments thereof or orthologs thereof or small genes with a coding sequence length exceeding 5kb, i.e., including one or more of the smallest gene fragments containing the control regions necessary for the gene to express itself in the same manner as the wild-type gene fragment.

[0083] More preferably, the coding sequence is the coding sequence of a gene selected from the group consisting of: DMD, CFTR, F8, ATP7B, PAH, DYSF, MECP2, PKD, NPC1, HTT or fragments thereof or orthologs thereof, or a small gene thereof with a coding sequence length exceeding 5kb, i.e., the smallest gene fragment comprising one or more of the regulatory elements necessary for the gene to express itself in the same manner as the wild-type gene fragment.

[0084] In a particularly preferred embodiment of the invention, the coding sequence encodes the ABCA4 gene. Preferably, the coding sequence splits at nucleotides corresponding to aa Cys1150, Ser1168, and Ser 1090 of the ABCA4 protein, and the mitotic protein is inserted at the splitting point.

[0085] In another preferred embodiment, the coding sequence encodes the CEP290 gene. Preferably, the coding sequence splits at nucleotides corresponding to aa Cys1076; Ser1275. More preferably, the coding sequence splits at nucleotide sequences corresponding to aa Cys 929 and 1474; Ser 453 and Cys 1474 of the CEP290 protein, and two mitotic endoproteins are inserted at the splitting point.

[0086] EGFP SEQ ID No. 15

[0087] The first amino acid of the C-exon protein is highlighted in the sequence. (Cys.71, bold)

[0088]

[0089] ABCA4 SEQ ID No. 16

[0090] The first amino acid of the C-exon protein is highlighted in the sequence.

[0091] Split group 1 Cys.1150 (bold)

[0092] Split group 2 Ser.1168 (underlined)

[0093] Split group 3 Ser.1090 (italicized)

[0094]

[0095]

[0096] CEP290 SEQ ID No. 17

[0097] The first amino acid of the c-exon protein is highlighted in the sequence.

[0098] Split group 1 Cys.1076 (bold)

[0099] Split group 2-3 Ser.1275 (underlined)

[0100] Split groups 4 Cys.929 and Cys.1474 (italicized)

[0101] Split groups 5 Ser.453 and Cys.1474 (Double underline)

[0102]

[0103]

[0104] F8 SEQ ID No. 18

[0105] The first amino acid of the c-exon protein is highlighted in the sequence. Split group 1 Cys.1312 (Underlined) Group 2, Ser.984 (Bold)

[0106]

[0107]

[0108] In a preferred embodiment, the carrier system of the present invention includes:

[0109] a) A first carrier, the first carrier comprising, in the 5'-3' direction:

[0110] - 5'-inverted terminal repeat (5'-ITR) sequence;

[0111] - Starter sequence;

[0112] - The 5' end portion (CDS1) of the encoded sequence, the 5' end portion being operatively connected to and under the control of the promoter;

[0113] - The first endonuclein nucleotide sequence encoding the N-endonuclein; and

[0114] - 3'-inverted terminal repeat (3'-ITR) sequence; and

[0115] b) A second carrier, the second carrier comprising, in the 5'-3' direction:

[0116] -5'-inverted terminal repeat (5'-ITR) sequence;

[0117] - Starter sequence;

[0118] - The second endonuclein nucleotide sequence encoding the C-endonuclein;

[0119] - The 3' end portion (CDS2) of the encoded sequence; and

[0120] - 3'-inverted terminal repeat (3'-ITR) sequence;

[0121] Or include:

[0122] a') A first carrier, the first carrier comprising, in the 5'-3' direction:

[0123] - 5'-inverted terminal repeat (5'-ITR) sequence;

[0124] - Starter sequence;

[0125] - The 5' end portion of the encoded sequence (CDS1'), said 5' end portion being operatively connected to and under the control of the promoter;

[0126] - The nucleotide sequence of the first N-endoprotein encoding the first N-endoprotein; and

[0127] - 3'-inverted terminal repeat (3'-ITR) sequence; and

[0128] b') A second carrier, the second carrier comprising, in the 5'-3' direction:

[0129] - 5'-inverted terminal repeat (5'-ITR) sequence;

[0130] - Starter sequence;

[0131] - The nucleotide sequence of the second endonuclein encoding the first C-endonuclein;

[0132] - The second part (CDS2') of the encoded sequence; and

[0133] - The nucleotide sequence of the third endonuclein encoding the second N-endonuclein;

[0134] - 3'-inverted terminal repeat (3'-ITR) sequence; and

[0135] c') A third carrier, said third carrier comprising, in the 5'-3' direction:

[0136] - 5'-inverted terminal repeat (5'-ITR) sequence;

[0137] - Starter sequence;

[0138] - The nucleotide sequence of the fourth inner protein encoding the second C-inner protein;

[0139] - The third portion (CDS3') of the encoded sequence; and

[0140] - 3' inverted terminal repeat (3'-ITR) sequence.

[0141] Preferably, the first vector, the second vector, and the third vector are independently viral vectors, preferably adenovirus vectors or adeno-associated virus (AAV) vectors. Preferably, the first vector, the second vector, and the third AAV vector are selected from the same or different AAV serotypes. Preferably, the serotypes are selected from serotype 2, serotype 8, serotype 5, serotype 7 or serotype 9, serotype 7m8, serotype sh10; serotype 2 (quad YF).

[0142] The present invention also provides host cells transformed using a vector system as defined above.

[0143] Preferably, the vector system or the host cell is used for medical purposes, preferably in gene therapy, and preferably for the treatment and / or prevention of pathologies or diseases characterized by retinal degeneration, metabolic disorders, hematologic disorders, neurodegenerative diseases, hearing loss, channel diseases, lung diseases, myopathy, heart diseases, and muscular dystrophy.

[0144] Preferably, the retinal degeneration is hereditary, and preferably, the pathology or disease is selected from the group consisting of: retinitis pigmentosa (RP), Lieber congenital amaurosis (LCA), Staggart disease (STGD), Usher syndrome (USH), Alstrém syndrome, congenital stationary night blindness (CSNB), macular dystrophy, latent macular dystrophy, and diseases caused by mutations in the ABCA4 gene.

[0145] Preferably, the carrier system or the host cell is used for the prevention and / or treatment of Dichenne muscular dystrophy, cystic fibrosis, hemophilia A, Wilson's disease, phenylketonuria, dysferlin lesions, Rett syndrome, polycystic kidney disease, Niemann-Pick disease type C, and Huntington's disease.

[0146] The present invention also provides a pharmaceutical composition comprising the carrier system or host cell of the present invention and a pharmaceutically acceptable carrier. Attached Figure Description

[0147] Figure 1 The AAV protein reconstructs EGFP at levels higher than those achieved with dual AAV and up to those achieved with single AAV in vitro and in mouse and porcine retinas.

[0148] (A) Schematic diagram of protein trans-splicing mediated by AAV proteins. ITR: AAV2 inverted terminal repeat; CDS: coding sequence; : 3xflag tag; PolyA: polyadenylation signal.

[0149] (B) Western blot (WB) analysis of lysates from HEK293 cells transfected with the full-length or AAV intraprotein CMV-EGFP plasmid. pEGFP: full-length EGFP plasmid; pAAV I+II: AAV-EGFP I+II intraprotein plasmid; pAAV I: single AAV-EGFP I intraprotein plasmid; pAAV II: single AAV-EGFP II intraprotein plasmid; Neg: untransfected cells. Arrows indicate the full-length EGFP protein (EGFP), the N-terminal and C-terminal halves of the EGFP protein (B and A, respectively), and the reconstructed intraprotein excised from the full-length EGFP protein (C). WB indicates n=3 independent experiments.

[0150] (C) Western blot analysis of lysates from HEK293 cells infected with single, intraprotein, or dual AAV2 / 2-CMV-EGFP vectors. WB represents n=5 independent experiments.

[0151] (D) Cryosection of the retina of C57BL / 6J mice injected subretinally with the AAV2 / 8-CMV-EGFP intracellular protein vector. Scale bar: 50 μm. RPE: retinal pigment epithelium; OS: outer segment; ONL: outer nuclear layer.

[0152] (EF) Cryosections of the retina of C57BL / 6J mice (E) or Large White pigs (F) injected subretinically with single, intraprotein, or dual AAV2 / 8-GRK1-EGFP vectors. Scale bar: 50 μm (E); 200 μm (F). OS: outer segment; ONL: outer nuclear layer.

[0153] (G) Fluorescence analysis of retinal organoids infected with the AAV2 / 2-GRK1-EGFP-intraprotein vector at day 293 of culture. Scale bar: 100 μm.

[0154] Figure 2 Optimization of AAV proteins allows for the correct reconstruction of large ABCA4 and CEP290 proteins.

[0155] (AB) Western blot (WB) analysis of lysates of HEK293 transfected with different groups of AAV-shCMV-ABCA4 or -CEP290 protein particles (group 1 and group 5, respectively). Figure 16A schematic diagram of the various groups used is shown. WB represents n=3 independent experiments.

[0156] (CD) Representative images of HeLa cells transfected with AAV-shCMV-ABCA4(C) or AAV-shCMV-CEP290(D) intraprotein granules for immunofluorescence analysis. pABCA4(C) or pCEP290(D): plasmid including the full-length expression cassette; pAAV intraprotein: AAV-intraprotein granule (group 1 in C or group 5 in D); I+II+III: AAV I+II+III intraprotein granule; I+II: AAV I+II intraprotein granule; I+III: AAV I+III intraprotein granule; II+III: AAV II+III intraprotein granule; I: Single AAV I intraprotein granule; II: Single AAV II intraprotein granule; III: Single AAV III intraprotein granule; Neg: Untransfected cells.

[0157] Cells were stained in C for 3xFLAG and VAP-B (endoplasmic reticulum markers) and TGN46 (transporting Golgi network marker), or in D for acetylated tubulin (microtubule markers). White arrows point to... Figure 18 Cells shown at a higher magnification.

[0158] Figure 3 AAV intramolecular proteins reconstruct large ABCA4 and CEP290 proteins more efficiently than dual AAV vectors.

[0159] Western blot analysis of lysates from HEK293 cells infected with dual or intraprotein AAV2 / 2-shCMV-ABCA4 (A) or -CEP290 (B) vectors.

[0160] AAV intraprotein: AAV-ABCA4 (Group 1, A) or -CEP290 (Group 5, B) intraprotein vector; I+II+III: AAV+II+III intraprotein vector; I+II: AAV I+II intraprotein vector; I+III: AAV I+III intraprotein vector; II+III: AAV II+III intraprotein vector; I: Single AAV I intraprotein vector; II: Single AAV II intraprotein vector; III: Single AAV III intraprotein vector; Dual AAV: Dual AAV vector; Neg: AAV-EGFP vector.

[0161] (A) The arrow indicates the full-length ABCA4 protein, and A: protein product derived from AAV I; B: protein product derived from AAV II. Protein products with potentially different post-translational modifications.

[0162] (B) Arrows indicate the full-length CEP290 protein, and A: protein product derived from AAV II+III; B: protein product derived from AAV I+II; C: protein product derived from AAV II; D: protein product derived from AAV III; E: protein product derived from AAV I. WB indicates n=3 independent experiments.

[0163] Figure 4 AAV proteins reconstruct large proteins in mouse, pig, and human photoreceptors at therapeutic levels.

[0164] (AC) Western blot analysis of retinal lesions from wild-type mice (A, B) or Large White pigs (C) injected with dual or intraprotein AAV2 / 8-GRK1-ABCA4 (A, C) or -CEP290 (B) vectors (groups 1 and 5, respectively). AAV intraprotein: AAV intraprotein vector; Dual AAV: Dual AAV vector; Neg: AAV-EGFP vector or PBS.

[0165] (D) Western blot analysis of lysates of human iPSC-derived 3D retinal organoids infected with the AAV2 / 2-GRK1-ABCA4 intraprotein carrier. AAV intraprotein: AAV-ABCA4 intraprotein carrier; Neg: uninfected organoids; - / -: organoids derived from STGD1 patients.

[0166] (A, C, D) The arrows represent the full-length ABCA4 protein (ABCA4), where A: protein product derived from AAV I; B: protein product derived from AAV II. Protein products with potentially different post-translational modifications.

[0167] (B) The arrow indicates the full-length CEP290 protein (CEP290); A: protein product derived from AAV II+III; and D: protein product derived from AAV III.

[0168] Figure 5 Subretinal administration of AAV protein improved the retinal phenotype in a mouse model of hereditary retinal degeneration.

[0169] (A) Abca4 treated with AAV endogenous protein - / - Quantification of the average area occupied by lipofuscin in the RPE of mice. Each point represents the mean measured for each eye. The mean lipofuscin area for each group is shown in the figure. + / + or + / -: Abca4 of the injected control. + / + or + / -Eye (PBS); - / -: Abca injection of negative control - / - Eye (AAV I ABCA4 or AAV II ABCA4 or PBS); - / - AAV intraprotein: Injection of AAV intraprotein carrier (Group 1) Abca4 - / - Eye. ANOVA p-value < 0.05; ANOVA p-value < 0.001.

[0170] (B) Representative images of retinal sections from wild-type uninjected mice and rd16 mice injected subretinally with the AAV2 / 8-GRK1-CEP290 intraretinal protein carrier (AAV intraretinal protein, group 5) or injected with negative controls (Neg; i.e., AAV I+II or AAV II+III or PBS). Scale bar: 25 μm. The thickness of the ONL measured in each image is indicated by a vertical black line. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.

[0171] (C) Representative images of eyes from wild-type uninjected mice and rd16 mice injected subretinally with the AAV2 / 8-GRK1-CEP290 intraprotein carrier (AAV intraprotein, group 5) or injected with negative controls (Neg; i.e., AAV I+II or AAV II+III or PBS). White circles define the pupils.

[0172] Figure 6 : Schematic diagram of large protein remodeling mediated by protein transsplicing.

[0173] The coding sequence (CDS) of the large gene splits into two halves (5' and 3'), flanked by inverted terminal repeats (ITRs), each packaged in a separate AAV capsid. Following co-transduction in the same cell, different mechanisms were explored to reconstruct full-length protein expression by linking these two halves at the protein level. The 5'-vector comprises the 5'CDS, the 5' endonuclease (n-endonuclease), and a degradation determinant, while the 3'-vector comprises the 3'CDS and the 3' endonuclease (c-endonuclease); both vectors include a promoter and a polyA. Pairing of these two hemipeptides is mediated by endonuclease self-recognition; subsequent self-cleavage of the endonuclease from the host protein leads to full-length protein reconstruction. The degradation determinant is now embedded within the cleaved endonuclease, which is rapidly ubiquitinated and degraded by the proteasome.

[0174] Figure 7 : In vitro EGFP expression from AAV intraprotein vectors with and without degradation signals.

[0175] Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intraprotein particles containing ecDHFR (+) or without ecDHFR (-). Arrows indicate full-length EGFP protein (EGFP), excised intraprotein containing the degradation determinant (DnaE + ecDHFR), or without this degradation determinant (DnaE).

[0176] Figure 8 : In vitro ABCA4 expression from AAV intraprotein carriers with and without degradation signals.

[0177] Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intraprotein particles containing ecDHFR (+) or without ecDHFR (-). Arrows indicate full-length ABCA4 protein (ABCA4), excised intraprotein containing the degradation determinant (DnaE + ecDHFR), or without this degradation determinant (DnaE).

[0178] Figure 9 The expression of the endogenous protein DnaE-ecDHFR depends on TMP.

[0179] Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV_ABCA4 endoprotein plasmids and treated with increasing doses of Trimetrophin (1 to 50 m), wherein the plasmids contained ecDHFR (pAAV endoprotein + ecDHFR) or did not contain ecDHFR (pAAV endoprotein). Arrows indicate excised endoproteins containing or without the degradation determinant (DnaE + ecDHFR).

[0180] Figure 10 : In vitro EGFP expression from AAV intraprotein vectors with and without degradation signals.

[0181] Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intraprotein particles containing small ecDHFR (+) or without small ecDHFR (-). Arrows indicate full-length EGFP protein (EGFP), excised intraprotein containing the degradation determinant (DnaE+ small ecDHFR), or without this degradation determinant (DnaE).

[0182] Figure 11 : In vitro ABCA4 expression from AAV intraprotein carriers with and without degradation signals.

[0183] Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intraprotein particles containing small ecDHFR (+) or without small ecDHFR (-). Arrows indicate full-length ABCA4 protein (ABCA4), excised intraprotein containing the degradation determinant (DnaE + small ecDHFR) or without this degradation determinant (DnaE).

[0184] Figure 12 EGFP fluorescence analysis in HEK293 cells transfected with AAV I+II, but not with a single AAV I or AAV II protein plasmid.

[0185] Fluorescence analysis of HEK293 cells transfected with full-length or intraprotein CMV-EGFP plasmids. pEGFP: plasmid containing the full-length EGFP expression cassette; pAAV I+II: intraprotein cassette of AAV I+II; pAAV I: intraprotein cassette of AAV I alone; pAAVII: intraprotein cassette of AAV II alone; Neg: untransfected cells. Scale bar: 100 μm.

[0186] Figure 13 : The variation of intracellular proteins relative to full-length proteins across species.

[0187] Western blot analysis (WB) of lysates from HEK293 cells (A), C57BL / 6J mice (B), and Large White pig retinas (C) infected with AAV-CMV-EGFP (A) or AAV-GRK1-EGFP intraprotein vector (BC). AAV intraprotein: cells infected with AAV intraprotein vector (A) or injected eyes (B, C); Neg: uninfected cells (A) or injected eyes with PBS (B, C). Arrows indicate full-length EGFP protein (EGFP) and excised intraprotein (DnaE).

[0188] Figure 14 Characterization of 3D retinal organoids derived from human iPSCs.

[0189] (A) Optical microscopic analysis of retinal organoids cultured for 183 days.

[0190] (B) Immunofluorescence analysis using antibodies against markers of mature photoreceptors. Scale bar: 100 μm.

[0191] (C) Fluorescence analysis of retinal organoids infected with AAV2 / 2-CMV-EGFP and AAV2 / 2-IRBP-DsRed vectors. Scale bar: 100 μm.

[0192] (D) At 230 days of culture, an outer segmental structure protruding from the surface of the retinal organoid was observed. The inset shows the presence of the outer segmental (OS) structure with radial structure. NR: neuroretina; RPE: retinal pigment epithelium.

[0193] (E) Scanning electron microscopy analysis revealed the presence of inner segments (IS), connecting cilia (CC), and outer segments (OS). Scale bar: 4 μm.

[0194] (F) Electron microscopy analysis reveals a sketch of the outer membrane (*), centrioles (C), matrix (BB), connecting cilia (CC), and outer segments (OS).

[0195] The illustration shows disordered membrane disks within the OS. Scale bar: 500 nm.

[0196] D: Number of days for cultivation.

[0197] Figure 15 : Low endogenous protein relative to the full-length protein in human 3D retinal organoids.

[0198] Western blot (WB) analysis of lysates from human iPSC-derived 3D retinal organoids infected with the AAV2 / 2-GRK1-EGFP intraprotein vector. AAV intraprotein: AAV intraprotein vector; Neg: uninfected organoid. Arrows indicate full-length EGFP protein (EGFP) and excised intraprotein (DnaE).

[0199] Figure 16 Schematic diagram of AAV-ABCA4 and -CEP290 proteins in each group.

[0200] (A) AAV-ABCA4-intraprotein construct. (Groups 1-2 use constructs as examples) n-DnaE: n-intraprotein of DnaE from Npu; c-DnaE: c-intraprotein of DnaE from Npu; (Group 3) n-mDnaE: n-intraprotein of mutant DnaE from Npu (mNpu); c-mDnaE: c-intraprotein of DnaE from mNpu.

[0201] (B) AAV-CEP290-intraprotein construct. (Group 1) n-DnaE: n-intraprotein of DnaE from Npu; c-DnaE: c-intraprotein of DnaE from Npu; shPolyA: short synthetic polyA; (Group 2) n-DnaE: n-intraprotein of DnaE from mNpu; c-DnaE: c-intraprotein of DnaE from mNpu; (Group 3) n-mDnaE: n-intraprotein of DnaE from mNpu; c-mDnaE: c-intraprotein of DnaE from mNpu; (Group 4) n-DnaE: n-intraprotein of DnaE from Npu; c-DnaE: c-intraprotein of DnaE from Npu between AAV I and AAV II; n-DnaB: N-intraprotein of DnaB from Rhodothermus marinus (Rma); c-DnaB: AAV II and AAV c-intraprotein of DnaE from Rma between III; wpre: post-transcriptional regulatory element of marmot hepatitis virus. (Group 5) n-mDnaE: n-intraprotein of DnaE from mNpu; c-mDnaE: AAV I c-intraprotein of DnaE from mNpu between AAV II and AAV III; n-DnaB: n-intraprotein of DnaB from Rma; c-DnaB: c-intraprotein of DnaE from Rma between AAV II and AAV III; wpre: post-transcriptional regulatory element of marmot hepatitis virus. (AB) ITR: AAV2 inverted terminal repeat; : 3xflag tag; promoter: short CMV for in vitro experiments and human G protein-coupled receptor (GRK1) promoter for in vivo experiments; PolyA: simian virus 40 polyadenylation signal (A for ABCA4) and bovine growth hormone polyadenylation signal (B for CEP290). The amino acids at the split point of each group are shown in the figure. The predicted protein molecular weight is shown under each AAV vector.

[0202] Figure 17 The combination of heterologous N- and C-endoproteins does not lead to detectable EGFP protein remodeling in vitro.

[0203] Fluorescence analysis of HEK293 cells transfected with the full-length or intraprotein AAV-CMV-EGFP plasmid. N+C-DnaE: AAV I+II fused with an endonuclein from DnaE; N+C-DnaB: AAV I+II fused with an endonuclein from DnaB; N+C-mDnaE: AAV I+II fused with a splitting endonuclein from mDnaE; N-DnaE+C-DnaB: AAV I fused with an n-endonuclein from DnaE and AAV II fused with a c-endonuclein from DnaB; N-DnaB+C-DnaE: AAV I fused with an n-endonuclein from DnaB and AAV II fused with a c-endonuclein from DnaE; N-mDnaE+C-DnaB: AAV I fused with an n-endonuclein from mDnaE and AAV II fused with a c-endonuclein from DnaB; N-DnaB + C-mDnaE: AAV I fused with an n-endonuclein from DnaB. I and AAV II fused with c-endoprotein from mDnaE; pEGFP: plasmid containing the full-length EGFP expression cassette; Neg: untransfected cells. Scale bar: 100 μm.

[0204] Figure 18 CEP290 is arranged along the microtubules.

[0205] Figure 2 Magnification of single cells in D. Immunofluorescence analysis of HeLa cells transfected with plasmids containing the full-length CEP290 expression cassette (pCEP290) or with protein plasmids within CEP290 (group 5, pAAV I+II+III). Cells were stained for 3xFLAG and acetylated tubulin (microtubule markers). Scale bar: 50 μm.

[0206] Western blot (WB) analysis of lysates from HEK293 cells transfected with full-length or AAV intraprotein particles encoding short CMV-ABCA4 (Group 1, A) or -CEP290 (Group 5, B).

[0207] (A) pABCA4: Full-length ABCA4 expression cassette; Group 1: ABCA4 (Cys.1150)-intraprotein granule.

[0208] (B) pCEP290: Full-length CEP290 expression cassette; Group 5: CEP290 (Ser.453 and Cys.1474)-intraprotein granules.

[0209] Neg: AAV EGFP plasmid. WB indicates n=3 independent experiments.

[0210] Figure 19Compared to transfection with a single plasmid containing a full-length expression cassette, transfection with AAV protein cassettes can reconstruct ABCA4 and CEP290 proteins in lower quantities.

[0211] Western blot (WB) analysis of lysates from HEK293 cells transfected with full-length or AAV intraprotein cassettes encoding short CMV-ABCA4 (A) or -CEP290 (B). (A) pABCA4: Full-length ABCA4 expression cassette; Group 1: ABCA4 (Cys.1150)-intraprotein cassette. (B) pCEP290: Full-length CEP290 expression cassette; Group 5: CEP290 (Ser.453 and Cys.1474)-intraprotein cassette. Neg: AAV EGFP plasmid. WB represents n=3 independent experiments.

[0212] Figure 20 Subretinal delivery of the AAV protein carrier leads to ABCA4 expression in the mouse retina.

[0213] Western blot (WB) analysis of retinal lesions from wild-type mice injected with dual or intraprotein AAV2 / 8-GRK1-ABCA4 vector (Group 1). AAV intraprotein: AAV intraprotein vector; Dual AAV: Dual AAV vector; Neg: AAV-EGFP vector.

[0214] Figure 21 AAV proteins can recombinant approximately 10% of endogenous Abca4.

[0215] Abca4 was injected with the AAV2 / 8-GRK1-ABCA4 intraprotein carrier (group 1). + / - Or Abca4 - / - Western blot (WB) analysis of retinal lesions from mice. mAbca4:Abca4 + / - Retina; AAV intraprotein: Retina injected with AAV intraprotein; Neg: Uninjected retina. Retinal lysates from Abca4+ / - loaded on gels #2 and #3 were identical. The percentage of AAV intraprotein ABCA4 expression relative to endogenous expression is plotted below each lane.

[0216] Figure 22 The AAV protein reconstructs the full-length ABCA4 protein in human retinal organoids.

[0217] Western blot analysis of lysates from human iPSC-derived 3D retinal organoids infected with the AAV2 / 2-GRK1-ABCA4 intraprotein carrier (Group 1). AAV intraprotein: AAV intraprotein carrier; Neg: uninfected organoid; - / -: organoids derived from STGD1 patients; + / -: organoids derived from healthy donors.

[0218] Figure 23 Subretinal administration of AAV intracellular protein carriers led to the formation of lipofuscin in Abca4. - / - Accumulation in mice is reduced.

[0219] Representative images analyzed by transmission electron microscopy show wild-type and Abca4 cells injected with either a negative control (Neg) or an AAV intraprotein carrier (group 1). - / - Lipofuscin granules in mouse RPE. White arrows indicate lipofuscin granules; M: mitochondria.

[0220] Figure 24 Subretinal delivery of AAV intraretinal protein carriers in mice does not modify ONL thickness.

[0221] Spectral domain optical coherence tomography analysis of eyes of C57BL / 6J mice injected subretinally with AAV intracellular protein carrier, unrelated AAV carrier (AAV neg), or PBS. Black bars represent eyes 6 months after injection of AAV-ABCA4 intracellular protein carrier (group 1) and its corresponding control; white bars represent eyes 4.5 months after injection of AAV-CEP290 intracellular protein carrier (group 5) and its corresponding control. Data are expressed as mean ± se. Mean values ​​are shown above the corresponding bars.

[0222] Figure 25 AAV intraprotein vectors can deliver full-length wild-type F8.

[0223] A) Schematic diagram of the single AAV B domain deletion variant factor VIII (F8-V3) and the AAV F8 intraprotein vector.

[0224] The coding sequence of the F8 gene is divided into two halves (5' and 3' F8), flanked by inverted terminal repeats (ITRFs), each encapsulated in a separate AAV capsid. The 5'-vector includes 5' F8 and the 5' endonucleoprotein (n-DnaE), while the 3'-vector includes 3' F8 and the 3' endonucleoprotein (c-DnaE); both vectors include the HLP promoter and a synthetic polyA. V3: Variant 3; SS: Signal sequence.

[0225] B) Unlike a single super-large AAV F8-V3, the F8 protein is correctly packaged into an AAV capsid with a defined vector genome.

[0226] Southern blot analysis of vector genome integrity using HLP promoter-specific probes showed that the truncated product in the super-large AAV F8-V3 was absent in the AAV F8 intraprotein vector. Neg: negative control.

[0227] The AAV F8 intraprotein carrier showed a slight correction to the bleeding phenotype in hemophilia A knockout mice at 8 weeks post-injection.

[0228] Compared with the PBS-injected control group, aPTT analysis of plasma samples from hemophilia A knockout mice 8 weeks after injection of AAV F8 endogenous protein (two cleavage points) showed slight phenotypic correction. aPTT: activated partial prothrombin kinase time. Detailed Implementation

[0229] Gene therapy

[0230] Over the past decade, gene therapy has been applied to the treatment of diseases in hundreds of clinical trials. Various tools have been developed to deliver genes into human cells; among them, genetically engineered viruses, including adeno-associated viruses (AAVs), are one of the most popular gene delivery tools. Most systems include a vector capable of containing the target gene and helper cells that provide viral structural proteins and enzymes to allow the generation of infectious viral particles containing the vector. AAVs are a family of viruses with diverse nucleotide and amino acid sequences, genome structures, pathogenicity, and host ranges. This diversity provides opportunities to develop different therapeutic applications using viruses with different biological characteristics. As with any delivery tool, efficiency, the ability to target specific tissues or cell types, expression of the target gene, and the safety of AAV-based systems are crucial for the successful application of gene therapy. Numerous efforts have been made in these research areas in recent years. Various modifications have been made to AAV-based vectors and helper cells to alter gene expression, target delivery, increase viral titers, and enhance safety. This invention represents an improvement in this design process because it is used as an effective delivery method for target genes whose cargo size exceeds the limitations of a single AAV-based vector. Viruses are the logical tool for gene delivery. They replicate within cells and have thus evolved mechanisms to enter cells and use cellular mechanisms to express their genes. The concept of virus-based gene delivery involves engineering viruses to express target genes. Depending on the specific application and virus type, most viral vectors contain mutations that hinder their ability to replicate freely in the host as wild-type viruses. Viruses from several different families have been modified to generate viral vectors for gene delivery. These viruses include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, piconemaviruses, and alpha viruses. This invention preferably uses adeno-associated viruses. Therefore, virus-based vectors for gene delivery include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, pseudotyped AAV vectors, herpesvirus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors.

[0231] Ideal adeno-associated virus (AAV)-based vectors for gene delivery must be efficient, cell-specific, regulated, and safe. Delivery efficiency is crucial as it determines therapeutic efficacy. Current efforts aim to achieve cell-type-specific infection and gene expression via AAV vectors. Furthermore, AAV vectors are being developed to regulate the expression of target genes, as treatment may require long-term or regulated expression. Safety is a major concern in viral gene delivery because most viruses are pathogens or have pathogenic potential.

[0232] Adeno-associated virus (AAV) is a small virus that can infect humans and some other primates. It is currently unknown whether AAV causes disease, therefore the virus elicits a very mild immune response. Gene therapy vectors using AAV can infect both dividing and dormant cells and remain in an extrachromosomal state without integrating into the host cell genome. These properties make AAV a very attractive candidate for creating viral vectors for gene therapy and for creating gene-related human disease models.

[0233] Wild-type AAV has attracted considerable interest from gene therapy researchers due to its many characteristics. Most notably, this virus is remarkably lacking in pathogenicity. It can also infect non-dividing cells and is able to stably integrate into the host cell genome at a specific site on human chromosome 19 (designated AAVS1). This characteristic makes it more predictable than retroviruses, which are threatened by random insertion and mutagenesis, sometimes leading to cancer development. The AAV genome integrates most frequently into the mentioned site, while the frequency of random integration into the genome is negligible. However, in the development of AAV as a gene therapy vector, this integration capability has been eliminated by removing rep and cap from the vector DNA. The desired gene is inserted between inverted terminal repeats (ITRs) along with the promoter driving gene transcription. These ITRs facilitate the formation of episodic polyhedra in the cell nucleus after the single-stranded vector DNA is converted into double strands by the host cell's DNA polymerase complex. AAV-based gene therapy vectors form episodic polyhedra in the host cell nucleus. In non-dividing cells, these polyhedra remain intact throughout the host cell's lifespan. In dividing cells, AAV DNA is lost during cell division because the episome DNA does not replicate along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable, but occurs very infrequently. AAVs also exhibit very low immunogenicity, appearing to be limited to producing neutralizing antibodies, and they do not induce well-defined cytotoxic responses. This characteristic, along with the ability to infect dormant cells, suggests that AAVs are superior to adenoviruses as vectors for human gene therapy.

[0234] AAV genomics, transcriptomics, and proteomics

[0235] The AAV genome is constructed from single-stranded deoxyribonucleic acid (ssDNA) of either positive or negative strands, and is approximately 4.7 kilobases in length. The genome includes inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs): rep and cap. The former consists of four overlapping genes that encode the Rep protein, required for the AAV life cycle, while the latter includes overlapping nucleotide sequences of capsid proteins: VP1, VP2, and VP3, which interact to form an icosahedral capsid.

[0236] ITR sequence

[0237] Each inverted terminal repeat (ITR) sequence comprises 145 bases. They are so named because of their symmetry, which has been shown to be essential for efficient amplification of the AAV genome. Another characteristic of these sequences is their ability to form hairpins, which facilitates so-called self-initiation, allowing for primase-independent synthesis of the second DNA strand. ITRs have also been shown to be essential for both the integration and rescue of AAV DNA into and from the host cell genome (human chromosome 19) and for the efficient capsidation of AAV DNA with the generation of fully assembled, deoxyribonuclease-resistant AAV particles.

[0238] Regarding gene therapy, the ITR appears to be the only sequence required for cis-acting recombinant AAV (rAAV) vectors near the therapeutic gene: both the cap and rep genes can be trans-acted. Based on this hypothesis, numerous methods have been developed to efficiently generate recombinant AAV vectors containing reporter or therapeutic genes. However, it has also been published that the ITR is not the only element required for efficient replication and capsidation in cis-acting recombinant AAV. Several research groups have identified sequences within the coding sequence of the rep gene called cis-acting rep-dependent elements (CAREs). When present in cis-acting recombinant AAV, CAREs are shown to enhance replication and capsidation.

[0239] AAV serotype

[0240] To date, dozens of different AAV variants (serotypes) have been identified and classified (60). All known serotypes can infect cells from a wide variety of tissue types. Tissue specificity is determined by the capsid serotype, and pseudotyping of AAV vectors to alter their tropism range may be crucial for their use in therapy. Pseudotyped AAV vectors are vectors that contain the AAV serotype genome within the capsid of a second AAV serotype; for example, the AAV2 / 8 vector contains an AAV8 capsid and the AAV2 genome (61). Such vectors are also called chimeric vectors.

[0241] Serotype 2

[0242] To date, serotype 2 (AAV2) has been the most extensively examined. AAV2 exhibits natural tropism for skeletal muscle, neurons, vascular smooth muscle cells, and hepatocytes. Three cellular receptors for AAV2 have been described: heparan sulfate proteoglycan (HSPG), avβ5 integrin, and fibroblast growth factor receptor 1 (FGFR-1). The first functions as the primary receptor, while the latter two have co-receptor activity, enabling AAV to enter cells via receptor-mediated endocytosis. These findings have been challenged by Qiu, Handa, and others. HSPG functions as the primary receptor, although its abundance in the extracellular matrix can clear AAV particles and impair infection efficiency.

[0243] The study showed that serotype 2 (AAV-2) of the virus clearly kills cancer cells without harming healthy cells. "Our findings suggest that adeno-associated virus type 2, which has infected most people without known adverse effects, kills multiple types of cancer cells but has no effect on healthy cells," said Craig Meyers, professor of immunology and microbiology at Pennsylvania State University School of Medicine. This could lead to a new anticancer agent.

[0244] Other serotypes

[0245] Although AAV2 is the most commonly used serotype in various AAV-based studies, other serotypes have been shown to be potentially more effective as gene delivery vectors. For example, AAV6 performs better in infecting airway epithelial cells; AAV7 exhibits very high transduction rates in mouse skeletal muscle cells (similar to AAV1 and AAV5); AAV8 performs well in transducing hepatocytes and photoreceptors; and AAV1 and 5 have demonstrated high efficiency in gene delivery to vascular endothelial cells. In the brain, most AAV serotypes show neuronal tropism, while AAV5 also transduces astrocytes. AAV6 (a hybrid of AAV1 and AAV2) shows lower immunogenicity than AAV2.

[0246] Serotypes can differ in terms of the receptors they bind. For example, transduction of AAV4 and AAV5 can be inhibited by soluble sialic acid (different forms of each of these serotypes), and AAV5 has been shown to enter cells via platelet-derived growth factor receptors. Novel AAV variants, such as the tetratyrosine mutant or AAV2 / 7m8, have been shown to transduce the outer retina from the vitreous in small animal models (62, 63). Another AAV mutant, ShH1O, is an AAV6 variant that exhibits improved glial tropism upon intravitreal administration (64). Another AAV mutant with particularly favorable tropism for the retina is AAV2 (quad YF) (65).

[0247] The gene delivery vector of the present invention can be administered to a patient. The administration can be "in vivo" or "ex vivo". A skilled person can determine the appropriate dose rate. The term "administration" includes delivery via viral or non-viral techniques. Viral delivery mechanisms include, but are not limited to, the adenovirus vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors described above.

[0248] Non-viral delivery systems include DNA transfection such as electroporation, lipid-mediated transfection, and compressed DNA-mediated transfection; liposomes, immunoliposomes, lipofectin, cationic surface amphiphilic products (CFAs), and combinations thereof.

[0249] The delivery of one or more therapeutic genes via the vector system according to the invention can be used alone or in combination with other therapies or therapeutic components.

[0250] Pharmaceutical Composition

[0251] This invention also provides pharmaceutical compositions for treating individuals via gene therapy, wherein the compositions comprise a therapeutically effective amount of the vector / construct or host cell of the invention, said vector / construct or host cell comprising one or more deliverable therapeutic and / or diagnostic transgenic or viral particles produced or obtained therefrom. The pharmaceutical compositions may be used for human or animal purposes. Typically, a physician will determine the practical dose best suited for an individual subject, and it varies depending on the individual's age, weight, and response. The compositions may optionally include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The choice of drug carrier, excipient, or diluent may be selected based on the intended route of administration and standard pharmaceutical practice. As a carrier, excipient, or diluent, or in addition to a carrier, excipient, or diluent, the pharmaceutical composition may be any suitable binder, lubricant, suspending agent, coating agent, solubilizer, and other carrier agents (e.g., lipid delivery systems) that can help or increase viral entry into the target site. Where appropriate, the pharmaceutical composition may be administered by any one or more of the following methods: inhalation, in the form of suppositories or pessaries; topically in the form of lotions, solutions, creams, ointments, or powders; by use of skin patches; orally, in the form of tablets containing excipients such as starch or lactose, or as capsules or ovules alone or in combination with excipients, or as elixirs, solutions, or suspensions containing flavoring or coloring agents; preferably, they may be administered by parenteral injection, such as intracavernosal, intravenous, intramuscular, or subcutaneous injection. For parenteral administration, the composition is preferably used in the form of a sterile aqueous solution, which may contain other substances, such as sufficient salt or monosaccharides, to make the solution isotonic with blood. For buccal or sublingual administration, the composition may be administered in the form of tablets or lozenges formulated in a conventional manner.

[0252] The preferred formulation is when the carrier system is applied topically in the conjunctival sac or subconjunctival region, preferably 1 to 10 times daily, preferably for 1 day to 6 months, and preferably for 1 day to 30 days.

[0253] Preferred methods of administration include anterior chamber injection, intravitreal injection, subretinal injection, periorbital and / or retroorbital injection, and intrastromal corneal injection.

[0254] Preferably, the pharmaceutical composition of the present invention is for topical ocular use, and is therefore an ophthalmic composition.

[0255] The carrier system according to the invention can be applied by any convenient means, but the preferred method of application is local application to the ocular surface, particularly to the cornea. An even more preferred method is instillation into the conjunctival sac.

[0256] A particular object of the present invention is the use of a carrier system for generating ophthalmic compositions, which are applied topically to the eye for medical use.

[0257] More generally, a preferred embodiment of the invention is to formulate a composition for topical application to local, superficial, or restricted areas of the eye and / or ocular appendages, said composition comprising a carrier system, optionally together with one or more pharmaceutically acceptable additives (e.g., diluents or carriers).

[0258] As used herein, the terms “medium,” “diluent,” “carrier,” and “additive” are interchangeable.

[0259] The ophthalmic compositions of the present invention may be in the form of solutions, emulsions or suspensions (eye washes), ointments, gels, aerosols, nebulizers or liniments, which together comprise a pharmaceutically acceptable, ophthalmally tolerable and compatible ophthalmic carrier with the active ingredient.

[0260] Specific ocular application routes for delayed release are also within the scope of this invention, for example, as ocularly corrosive inserts or polymer film “reservoir” systems to be positioned in the conjunctival sac or contact lenses.

[0261] The ophthalmic compositions of the present invention can be applied topically, for example, by delivering the composition and directly contacting the eye and / or ophthalmic appendages.

[0262] Pharmaceutical compositions containing at least the carrier system of the present invention can be prepared by any conventional technique, such as that described in Remington: The Science and Practice of Pharmacy, edited by EW Martin, Mack Publishing Company, 19th edition, Easton, Pa.

[0263] In one embodiment, the composition is formulated as a liquid, wherein the carrier system can be a solution or a suspension. The composition can be formulated into any liquid form suitable for topical application, such as eye drops, artificial tears, eye washes, or contact lens absorbents containing a liquid carrier such as a cellulose ether (e.g., methylcellulose).

[0264] Preferably, the liquid is an aqueous liquid. More preferably, the liquid is sterile. Sterility can be imparted by any conventional method, such as filtration, radiation, or heating, or by manufacturing processes under sterile conditions.

[0265] The liquid may include one or more lipophilic carriers.

[0266] In one embodiment of the invention, the composition is formulated as an ointment. Preferably, one of the carriers in the ointment may be a petrolatum carrier.

[0267] Pharmaceutically acceptable mediators can generally be any conventionally used pharmaceutically acceptable mediator, and the selection should be based on the specific formulation, intended route of administration, etc. Furthermore, pharmaceutically acceptable mediators can be any acceptable additive listed in the FDA's "List of Inactive Ingredients," which can be obtained, for example, via the internet address http: / / www.fda.gov / cder / drug / iig / default.htm.

[0268] At least one pharmaceutically acceptable diluent or carrier may be a buffer. For certain purposes, it is generally desirable that the composition includes a buffer capable of buffering the solution to a pH range of 5 to 9, such as pH 5 to 6, pH 6 to 8, or pH 7 to 7.5.

[0269] However, in other embodiments of the invention, the pharmaceutical composition may be completely free of buffers, or may contain only micromolar amounts of buffers. Buffers may be selected, for example, from the group consisting of: TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, borate, carbonate, glycine, histidine, glycine, succinate, and triethanolamine buffers. Therefore, the buffer may be K₂HPO₄, Na₂HPO₄, or sodium citrate.

[0270] In a preferred embodiment, the buffer is a TRIS buffer. TRIS buffers are known by various other names, such as tromethamine, including tromethamine USP, THAM, Trizma, Trisamine, Tris amino, and trometamol. The name TRIS encompasses all of the above names.

[0271] Furthermore, the buffer can be selected, for example, from USP-compatible buffers used for parenteral administration, particularly when the pharmaceutical preparation is intended for parenteral use. For example, the buffer can be selected from the group consisting of: monobasic acids, such as acetic acid, benzoic acid, gluconic acid, glyceric acid, and lactic acid; dibasic acids, such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, and tartaric acid; polybasic acids, such as citric acid and phosphoric acid; and bases, such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.

[0272] The composition may include preservatives such as thimerosal, chlorobutanol, benzalkonium chloride, or chlorhexidine; buffers such as phosphates, borates, carbonates, and citrates; and thickeners such as high molecular weight carboxyvinyl polymers, such as polymers sold under the trademark Carbopol of BFGoodrich Chemical Company, hydroxymethyl cellulose, and polyvinyl alcohol, all of which are in accordance with the prior art.

[0273] In some embodiments of the invention, pharmaceutically acceptable additives include stabilizers. Stabilizers may be, for example, detergents, amino acids, fatty acids, polymers, polyols, metal ions, reducing agents, chelating agents, or antioxidants, but any other suitable stabilizer may also be used with the invention. For example, stabilizers may be selected from the group consisting of: poloxamer, Tween-20, Tween-40, Tween-60, Tween-80, Brij, metal ions, amino acids, polyethylene glycol, Triton, and ascorbic acid.

[0274] In addition, stabilizers can be selected from the group consisting of: amino acids, such as glycine, alanine, arginine, leucine, glutamic acid, and aspartic acid; surfactants, such as polysorbate 20, polysorbate 80, and poloxamer 407; fatty acids, such as phosphatidylcholine ethanolamine and acetyltryptophan; polymers, such as polyethylene glycol and polyvinylpyrrolidone; polyols, such as sorbitol, mannitol, glycerol, sucrose, glucose, propylene glycol, ethylene glycol, lactose, and trehalose; antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, thioglycerol, mercaptoacetic acid, thiosorbitol, and glutathione; reducing agents, such as several thiols; and chelating agents, such as EDTA salts, glutamic acid, and aspartic acid.

[0275] Pharmaceutically acceptable additives may include one or more of the following groups: isotonic salts, hypertonic salts, hypotonic salts, buffers, and stabilizers.

[0276] In a preferred embodiment, other pharmaceutical excipients are present, such as preservatives. In one embodiment, the preservative is a paraben, such as, but not limited to, methylparaben or propylparaben.

[0277] In some embodiments of the invention, pharmaceutically acceptable additives include mucolytics (e.g., N-acetylcysteine), hyaluronic acid, cyclodextrin, and petroleum.

[0278] Exemplary compounds that may be included in the pharmaceutical compositions of the present invention to promote and accelerate the transdermal delivery of the topical composition to the eye or adnexal tissues include, but are not limited to, alcohols (ethanol, propanol, and nonanol), fatty alcohols (laurate), fatty acids (valeric acid, hexanoic acid, and decanoic acid), fatty acid esters (isopropyl myristate and isopropyl hexanoate), alkyl esters (ethyl acetate and butyl acetate), polyols (propylene glycol, propylene glycol, and glycerol), sulfoxides (dimethyl sulfoxide and decylmethyl sulfoxide), amides (urea, dimethylacetamide, and pyrrolidone derivatives), surfactants (sodium lauryl sulfate, hexadecyltrimethylammonium bromide, poloxamer, Spans, Tweens, bile salts, and lecithin), terpenes (d-limonene, alpha-terpeneol, 1,8-cineole, and menthone), and ketones (n-heptane and n-nonane). In addition, the composition for topical application may include surface adhesion molecule modifiers, including but not limited to cadherin antagonists, selective protein antagonists, and integrin antagonists.

[0279] In addition, ophthalmic solutions may contain thickeners such as hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, methylcellulose, polyvinylpyrrolidone, etc., to improve drug retention in the conjunctival sac.

[0280] In one embodiment, the carrier system used according to the invention can be combined with ophthalmologically acceptable preservatives, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride, and water to form an aqueous, sterile, ophthalmic suspension or solution. The ophthalmic solution may also include an ophthalmologically acceptable surfactant to aid in dissolving the carrier system. The ophthalmic solution formulation can be prepared by dissolving the carrier system in a physiologically acceptable isotonic buffer.

[0281] To prepare sterile ophthalmic ointment formulations, a carrier system can be combined with a preservative in a suitable carrier, such as mineral oil, liquid lanolin, or white petrolatum. Sterile ophthalmic gel formulations can be prepared by suspending a carrier system in a hydrophilic matrix, which is prepared according to published formulations of similar ophthalmic formulations using a combination of, for example, carbopol-940; preservatives and tensile agents may be added.

[0282] Preferably, the formulation of the present invention is an aqueous, non-irritating ophthalmic composition for topical ocular application, the composition comprising: a therapeutically effective amount of a carrier system for topical treatment; a xanthine derivative present in an amount between the amount of a water-soluble derivative of the composition and 0.05% by weight / volume of the composition, which effectively reduces discomfort associated with the carrier system after topical application of the composition, the xanthine derivative being selected from the group consisting of theophylline, caffeine, theobromine, and mixtures thereof; an ophthalmic preservative; and a buffer to provide an isotonic, aqueous, non-irritating ophthalmic composition.

[0283] Drug delivery device

[0284] In one embodiment, the invention includes a drug delivery device comprising at least a carrier system and a pharmaceutically compatible polymer. For example, a composition is incorporated into or coated onto said polymer. The composition is chemically bound or physically trapped by the polymer. The polymer is hydrophobic or hydrophilic. The polymer device comprises a plurality of physical arrangements. Exemplary physical forms of the polymer device include, but are not limited to, membranes, scaffolds, chambers, spheres, microspheres, or other structures. The polymer device has an inner surface and an outer surface. The device has one or more inner chambers. These chambers comprise one or more compositions. The device contains a polymer with one or more chemically distinguishable monomers. The subunits or monomers of the device are polymerized in vitro or in vivo.

[0285] In a preferred embodiment, the invention includes a device comprising a polymer and a bioactive composition incorporated into or on the polymer, wherein the composition includes a carrier system, and wherein the device is implanted or injected into ocular surface tissue, adnexal tissue in contact with ocular surface tissue, fluid-filled ocular cavity or adnexal cavity, or ocular cavity or adnexal cavity.

[0286] Exemplary mucosal adhesive polyanionic natural or semi-synthetic polymers that can form the device include, but are not limited to, polygalacturonic acid, hyaluronic acid, carboxymethyl amyl starch, carboxymethyl chitosan, chondroitin sulfate, heparin sulfate, and mesoglycosaminoglycans. In one embodiment, the device includes a biocompatible polymer matrix that may optionally be wholly or partially biodegradable. Hydrogels are an example of suitable polymer matrix materials. Examples of materials that can form hydrogels include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids such as peptides, especially poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon-caprolactone, polyanhydrides; polyphosphazene, polyvinyl alcohol, poly(oxyethylene oxide), especially poly(ethylene oxide), poly(allylamine)(PAM), poly(acrylate), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamer, poly(uronic acid), poly(vinylpyrrolidone), and copolymers of the above substances, including graft copolymers. In another embodiment, the scaffold can be made from a variety of synthetic and naturally occurring polymers, such as, but not limited to, collagen, fibroin, hyaluronic acid, agarose, and gels rich in laminin.

[0287] A preferred hydrogel material is alginate or modified alginate. Alginate molecules consist of (1-4)-linked β-D-mannuronic acid (M units) and L-guluronic acid (G units) monomers, which vary in proportion and sequence along the polymer chain. Alginate polysaccharides are polyelectrolyte systems with a strong affinity for divalent cations (e.g., Ca²⁺, Mg²⁺, Ba²⁺) and form stable hydrogels upon exposure to these molecules.

[0288] The device can be administered locally, subconjunctivally, or extrascleral, subcutaneously, or intraductally. Specifically, the device is placed on or directly beneath the surface of ocular tissue. Alternatively, the device is placed within the lacrimal duct or gland. The composition incorporated into or on the polymer is released or diffused from the device.

[0289] In one embodiment, the composition is incorporated into or coated onto a contact lens or drug delivery device, from which one or more molecules diffuse away from the lens or device or are released in a time-controlled manner. In this embodiment, the contact lens composition remains on the ocular surface, for example, if the lens is necessary for vision correction, or if the contact lens dissolves over time, while the composition is released into closely juxtaposed tissues. Similarly, in various embodiments, the drug delivery device is optionally biodegradable or permanent.

[0290] For example, the composition is incorporated into or coated onto the eyeglasses. The composition is chemically bound or physically trapped by the contact lens polymer. Alternatively, a color additive is chemically bound or physically trapped by the polymer composition, which is released at the same rate as the therapeutic composition, such that a change in the intensity of the color additive indicates a change in the amount or dosage of the therapeutic composition still bound or trapped in the polymer. Alternatively, or additionally, an ultraviolet (UV) absorber is chemically bound or physically trapped within the contact lens polymer. The contact lens polymer is hydrophobic or hydrophilic.

[0291] Exemplary materials for manufacturing hydrophobic eyeglasses having a means of delivering the compositions of the present invention include, but are not limited to, Amefocon A, Amsilfocon A, Aquilafocon A, Arfocon A, Cabufocon A, Cabufocon B, Carbosilfocon A, Crilfocon A, Crilfocon B, Dimefocon A, Enflufocon A, Enflofocon B, Erifocon A, Flurofocon A, Flusilfocon A, Flusilfocon B, Flusilfocon C, Flusilfocon D, and Flusilfocon E. E), Hexafocon A, Hofocon A, Hybufocon A, Itabisfluorofocon A, Itafluorofocon A, Itafocon A, Itafocon B, Kolfocon A, Kolfocon B, Kolfocon C, Kolfocon D, Lotifocon A, Lotifocon B, Lotifocon C, Melafocon A, Migafocon A, Nefocon A, Nefocon B, Nefocon C, Onsifocon A A) Oprifocon A, Oxyfluflocon A, Paflufocon B, Paflufocon C, Paflufocon D, Paflufocon E, Paflufocon F, Pasifocon A, Pasifocon B(pasifocon B), Pasifocon C, Pasifocon D, Pasifocon E, Pemufocon A, Porofocon A, Porofocon B, Roflufocon A, Roflufocon B, Roflufocon C, Roflufocon D, Roflufocon E, Rosilfocon A, Satafocon A, Siflufocon A, Silafocon A, Sterafocon A, Sulofocon A, Sulofocon B, Telafocon A, Tisilfocon A A), tolofocon A, trifocon A, unifocon A, vinafocon A and wilofocon A. Exemplary materials for manufacturing hydrophilic eyeglasses having means of delivering the compositions of the present invention include, but are not limited to: abafilcon A, acofilcon A, acofilcon B, acquafilcon A, alofilcon A, alphafilcon A, amfilcon A, astifilcon A, atlafilcon A, balafilcon A, bisfilcon A, bufilcon A, comfilcon A, crofilcon A, cyclofilcon A, darfilcon A, deltafilcon A, deltafilcon B, dimefilcon A, and droxfilcon A. A) Elastofilcon A, Epsilonilcon A(esterifilcon A), etafilcon A, focofilcon A, galyfilcon A, genfilcon A, goovafilcon A, hefilcon A, hefilcon B, hefilcon C, hilafilcon A, hilafilcon B, hioxifilcon A, hioxifilcon B, hioxifilcon C, hydrofilcon A, lenefilcon A, licryfilcon A, licryfilcon B, lidofilcon A, lidofilcon B, lotrafilcon A A) Lotrafilcon B, Mafilcon A, Mesafilcon A, Metafilcon B, Mipafilcon A, Nelfilcon A, Netrafilcon A, Ocufilcon A, Ocufilcon B, C, Ocufilcon D, Ocufilcon E, Ofilcon A, Oomafilcon A, Oxyfilcon A, Pentafilcon A, Perfllcon A, Pevafilcon A, Phemfilcon A, Polymacon, Senofilcon A, Silafilcon A A) Western European brands including siloxyfilcon A, surfilcon A, tefilcon A, tetrafilcon A, trilfilcon A, viflcon A, viflcon B, and xylofilcon A.A).

[0292] Compositions formulated as gels or gel-like substances, emulsions, or viscous emulsions are within the scope of this invention. Preferably, the composition includes at least one gelling component, polymer, or other suitable agent to increase the viscosity of the composition. Any gelling component known to those skilled in the art can be used, which has no adverse effect on the area to be treated and is suitable for formulating compositions and pharmaceutical compositions for topical application to the skin, eyes, or mucous membranes. For example, the gelling component may be selected from the group consisting of: acrylic acid, carbomer, carboxylated polymethyl methacrylate, such materials sold by BF Goodrich under the trademark Carbopol (e.g., Carbopol 940), polyethylene-polypropylene glycol, such materials sold by BASF under the trademark Poloxamer (e.g., Poloxamer 188), cellulose derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxyethylene cellulose, methyl cellulose, carboxymethyl cellulose, propylene glycol alginate, polyvinylpyrrolidone, veegum (magnesium aluminum silicate), Pemulen, Simulgel (e.g., Simulgel 600, Simulgel EG and Simulgel NS), Capigel, Colafax, plasdones, and mixtures thereof.

[0293] The gel or gel-like substance according to the invention comprises, for example, less than 10% w / w water, for example, less than 20% w / w water, for example, at least 20% w / w water, for example, at least 30% w / w water, for example, at least 40% w / w water, for example, at least 50% w / w water, for example, at least 75% w / w water, for example, at least 90% w / w water, for example, at least 95% w / w water. Preferably, the water is deionized water.

[0294] The gel-like substances of this invention include hydrogels, colloidal gels formed as dispersions in water or other aqueous media. Thus, a hydrogel is formed after the formation of a colloid, wherein the dispersed phase (colloid) combines with the continuous phase (i.e., water) to produce a viscous, gel-like product; for example, condensed silica. The hydrogel is a three-dimensional network of hydrophilic polymer chains cross-linked by chemical or physical bonding. Due to the hydrophilicity of the polymer chains, the hydrogel absorbs water and swells. The swelling process is the same as the dissolution of a non-cross-linked hydrophilic polymer. By definition, water constitutes at least 10% of the total weight (or volume) of the hydrogel.

[0295] Examples of hydrogels include synthetic polymers such as polyhydroxymethyl acrylate, and chemically or physically crosslinked polyvinyl alcohol, polyacrylamide, poly(N-vinylpyrrolidone), polyethylene oxide, and hydrolyzed polyacrylonitrile. Examples of hydrogels as organic polymers include covalently or ionicly crosslinked polysaccharide-based hydrogels such as alginate, pectin, carboxymethyl cellulose, heparin, polyvalent metal salts of hyaluronic acid, and hydrogels derived from chitin, chitosan, amylopectin, gelling sugar, and xanthan gum. The specific hydrogels used in our experiments were cellulose compounds (i.e., hydroxypropyl methylcellulose [HPMC]) and high molecular weight hyaluronic acid (HA).

[0296] Hyaluronic acid is a polysaccharide produced by various body tissues. U.S. Patent 5,166,331 discusses different fractions of purified hyaluronic acid for use as an alternative to intraocular fluids and as a carrier for topical ophthalmic drugs. Other U.S. patent applications discussing the ophthalmic uses of hyaluronic acid include Serial Nos. 11 / 859,627; 11 / 952,927; 10 / 966,764; 11 / 741,366; and 11 / 039,192. Large molecular formulations for intraocular use are known. See, for example, U.S. Patent Application Serial Nos. 11 / 370,301; 11 / 364,687; 60 / 721,600; 11 / 116,698 and 60 / 567,423; and 11 / 695,527. Various active agents are used with high-viscosity hyaluronic acid and are known. See, for example, U.S. patent application serial numbers 10 / 966,764; 11 / 091,977; 11 / 354,415; 60 / 519,237; 60 / 530,062 and 11 / 695,527.

[0297] Sustained-release formulations as described in WO2010048086 are within the scope of this invention.

[0298] Those skilled in the art are familiar with standard methods for incorporating polynucleotides or vectors into host cells, such as transfection, lipid transfection, electroporation, microinjection, viral infection, heat shock, transformation after chemical permeation of membranes, or cell fusion.

[0299] As used herein, the term "host cell or genetically engineered host cell" refers to a host cell that has been transduced, transformed, or transfected using the aforementioned constructs or vectors.

[0300] Representative examples of suitable host cells include bacterial cells such as *Escherichia coli*, *Streptomyces*, and *Salmonella typhimurium*; fungal cells such as yeast; insect cells such as Sf9; animal cells such as CHO or COS; plant cells, etc. The selection of a suitable host is considered to be within the scope of those skilled in the art in accordance with the teachings herein. Preferably, the host cell is an animal cell, and most preferably, a human cell. The invention further provides host cells comprising any recombinant expression vector described herein. The host cell can be a cultured cell or a primary cell, i.e., a cell directly isolated from an organism, such as a human. The host cell can be an adherent cell or a suspension cell, i.e., a cell grown in a suspension. Suitable host cells are known in the art and include, for example, DH5α, *Escherichia coli* cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc.

[0301] In the case of ex vivo gene therapy, the host cell can be a cell isolated from the patient, such as a hematopoietic stem cell, which is reintroduced into the patient in need after the introduction of the transgene.

[0302] AAV-based virus delivery system

[0303] The construction of AAV vectors can be carried out according to procedures and using techniques known to those skilled in the art. Several scientific and patent publications have described the theory and practice of adeno-associated virus vector construction and its use in treatment (the following references are incorporated herein by reference: Flotte TR. Adeno-associated virus-based gene therapy for inherited disorders. Pediatr Res. 2005 Dec;58(6):1143-7; Goncalves MA. Adeno-associated virus: from defective virus to effective vector, Virol J. 2005 May 6;2:43; Surace EM, Auricchio A. Adeno-associated viral vectors for retinal gene transfer. Prog Retin Eye Res. 2003 Nov;22(6):705-19; Mandel RJ, Manfredsson FP, Foust KD, Rising A, Reimsnider S, Nash K, Burger C. Recombinant adeno-associated viral vectors as therapeutic agents to treat neurological disorders. Mol Ther. 2006). Mar;13(3):463-83).

[0304] Suitable forms of administration for pharmaceutical compositions containing AAV vectors include, but are not limited to, injectable solutions or suspensions, eye washes, and ophthalmic ointments. In a preferred embodiment, the AAV vector is administered via intrathecal injection. In a particularly preferred embodiment, the AAV vector is administered subretinally into the anterior chamber or posterior space of the eye and into the vitreous body. Preferably, the viral vector is delivered via a subretinal method (e.g., as described in Bennicelli J, et al Mol Ther. 2008 Jan 22; Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer).

[0305] The viral load used for treatment should be determined on a case-by-case basis, depending on the route of administration, the severity of the disease, the patient's general condition, and other clinical parameters. Generally, an appropriate dose range is 10. 8 Up to 10 13 vg (vector genome) / eye.

[0306] Intraprotein

[0307] Intraproteins are protein fragments capable of shearing themselves and connecting with their remaining parts (exoproteins) via peptide bonds in a process called protein splicing. This fragment is called an "intraprotein," representing the internal protein sequence, and an "exoprotein," representing the external protein sequence. The upstream exoprotein is called an "N-exoprotein," and the downstream exoprotein is called a "C-exoprotein." The products of protein splicing are two stable proteins: the mature protein and the intraprotein.

[0308] Endoproteins can also exist as two segments encoded by two separately transcribed and translated genes, which we refer to as "split-type endoproteins" in this paper.

[0309] The endonucleoproteins of the present invention include, but are not limited to, the splitting endonucleoproteins listed in the New England Biolabs endonucleoprotein database disclosed in (66).

[0310] Small endonucleases can be generated by first removing the homing endonuclease domain sequence, and then cleavage-type endonucleases can be produced from these endonucleases. Subsequently, the small endonucleases can cleave at one or more sites designed to generate cleavage-type endonucleases by comparing the protein sequence of endonucleases with known crystal structures, and trans-splicing activity can be determined according to the protocols included in this disclosure.

[0311] Desired properties of splitting endonucleases, including activity, efficiency, versatility, and stability, can be further improved through site-directed mutagenesis or modification based on rationally designed endonucleoprotein sequences, and / or through directed evolution using methods such as functional selection, phage display, and ribosome display.

[0312] An example of a fissile endonucleoprotein is the endonucleoprotein derived from DnaE, ​​which is the catalytic subunit α of DNA polymerase III in cyanobacteria and is encoded by two separate genes, dnaE-n and dnaE-c. The endonucleoprotein encoded by the dnaE-n gene is referred to herein as the "N-endonucleoprotein." The endonucleoprotein encoded by the dnaE-c gene is referred to herein as the "C-endonucleoprotein." Generally, the N-part of the fissile endonucleoprotein is referred to as the "N-endonucleoprotein," and the C-part of the fissile endonucleoprotein is referred to as the "C-endonucleoprotein." The fissile endonucleoprotein self-associates and catalyzes trans-protein splicing activity (referred to herein as "trans-splicing").

[0313] Other examples of the fission-type endonucleases of the present invention include: endonucleases (27, 28) of DnaE from *Nostoc punctata* (Npu), shown in Table 3 below as SEQ ID 1 encoded by the Npu-DnaE-n nucleotide sequence and SEQ ID 2 encoded by the Npu-DnaE-c nucleotide sequence; endonucleases (29) of DnaB from *Rhodophyton floccosum* (Rma), shown in Table 3 below as SEQ ID 4 encoded by the Rma-DnaB-n nucleotide sequence and SEQ ID 5 encoded by the Rma-DnaB-c nucleotide sequence; mutant N- and C-endonucleases, wherein the N-endonucleases are DnaE from Npu (SEQ ID 5) and the C-endonucleases are from *Syntrophus cytotoxicum* strain PCC6803 (Ssp (SEQ ID 6), (30)); the N-endonucleases and C-endonucleases of *Syntrophus cytotoxicum* strain PCC6803 are respectively represented as SEQ ID 6. 13 and 14 are included in the table below. Other endonucleoprotein systems may also be used. For example, synthetic rapid endonucleoproteins based on dnaE endonucleoprotein, Cfa-N, and Cfa-C endonucleoprotein pairs have been described (e.g., (31) and WO 2017 / 132580, which are incorporated herein by reference). Additional endonucleoproteins have been described in U.S. Patent No. 8,394,604, which include SspGyrB endonucleoprotein, SspDnaX endonucleoprotein, TerDnaE3 endonucleoprotein, TerThyX endonucleoprotein, and CnePrp8 endonucleoprotein. Further endonucleoproteins in this invention are those disclosed in WO2018071868, wherein the first pair of endonucleoproteins is listed in the table below and named SEQ ID 9 (N-endonucleoprotein) and SEQ ID 10 (C-endonucleoprotein); and the second pair of endonucleoproteins is listed, for example, SEQ ID 11 and SEQ ID 12.

[0314] Alternatively, the endonucleoprotein system can be a ligand-dependent endonucleoprotein that exhibits little or no protein splicing activity in the absence of a ligand (e.g., small molecules such as 4-hydroxytamoxifen, peptides, proteins, polynucleotides, amino acids, and nucleotides). Ligand-dependent endonucleoproteins include, for example, those described in US 2014 / 0065711 A1, which is incorporated herein by reference.

[0315] Table 3: Examples of the cleavage-type endogenous proteins of the present invention

[0316]

[0317]

[0318] As described herein, endogenous proteins derived from the same gene in different organisms and retaining trans-splicing activity are within the scope of this invention. As a non-limiting example, DNA-E fission-type endogenous proteins may be derived from fission-type endogenous proteins of the DnaE gene (e.g., DNA polymerase III subunit α) of cyanobacteria, including *Candida punctata* (Npu), *Syntropha cytosporum* PCC6803 (Ssp), *Ferula ferruginea* PCC 9605, *Bifidobacterium*, *Pseudobranchia monofibrillaris*, cyanobacteria SW_9_47_5, *Nostoc commune*, *Nostoc pubescens*, *Cyclocarya var. vulgaris* WH 8502, *Cyclocarya cacala* CCALA 043, and *Trichophyton rubrum*. As another example, DNA-B fission-type endogenous proteins may be derived from the DnaB gene of cyanobacteria, including, for example, *R. marineis* (Rma), *Syntropha cytosporum* PC6803 (Ssp), and *Porphyra yezoensis* chloroplasts (Ppu) as described in (59).

[0319] Therefore, the splitting endonucleoprotein of the present invention can be 100%, 98%, 80%, 75%, 70%, 65%, or 50% identical to naturally occurring endonucleoproteins, wherein the endonucleoproteins retain the ability to perform trans-splicing reactions. Fragments of naturally occurring or modified endonucleoproteins that retain trans-splicing activity are within the scope of the present invention.

[0320] For example, see the comparison between Npu (Nostoc punctata) DnaE and Synechocystis PCC6803 N-intrin:

[0321]

[0322] Furthermore, the comparison between Npu (Nostoc punctata) DnaE and Synechocystis PCC6803 C-intraprotein:

[0323]

[0324] Therefore, cleavage variants and fragments of the endoproteins of the present invention that retain trans-splicing activity are also within the scope of the present invention.

[0325] Interestingly, endoproteins have been reported to possess conserved functional features that ensure their splicing activity. Specifically, four endoprotein motifs have been identified (see below for their shared sequences): block AH (Pietrokovski 1994 and Perler 1997) and blocks N2 and N4 (Pietrokovski 1998). Intrin blocks A, N2, B, N4, F, and G are involved in protein splicing. Blocks C, D, E, and H are located in endonuclease domains, which are absent from cleavable endoproteins. Thus, cleavable endoproteins retain conserved motifs essential for trans-splicing activity. (Intrin Database, published in [Perler, FB (2002), InBase, the Intein Database, Nucleic Acids Res. 30, 383-384]).

[0326]

[0327] Although no single residue is invariant, serine (Ser) and cysteine ​​(Cys) in block A, histidine (His) in block B, histidine (His), asparagine (Asn), and serine (Ser) / cysteine ​​(Cys) / threonine (Thr) in block G are the most conserved residues in the splicing motif.

[0328] Comparison of the internal proteins of this invention:

[0329] CLUSTAL W alignment of all listed N-endoproteins:

[0330] CLUSTAL 2.1 multiple sequence alignment of all listed C-endoproteins

[0331]

[0332] In summary, the activity of endopeptides is background-dependent, and their junctions are surrounded by certain peptide sequences (called N- and C-exons) necessary for effective trans-splicing, the most important of which are the first residues in C-exons containing nucleophilic thiol or hydroxyl groups (e.g., cysteine, serine, or threonine).

[0333] The inventors have used endonuclein-mediated protein transsplicing to reconstruct large proteins in vivo. Split-type endonucleins, encoded by the endonuclein gene sequence, are produced as precursor peptides, and through their structural complementarity, they can be reassembled and catalyze protein transsplicing reactions.

[0334] In the context of protein trans-splicing, the N-endin gene fuses within the frame with the sequence encoding the N-terminal portion of the target protein; the C-endin gene fuses within the frame with the sequence encoding the C-terminal portion of the target sequence. After expression of the two precursor fusion proteins, the endin undergoes autocatalytic excision and forms a linked exon, such as the reconstructed target protein.

[0335] Therefore, the reconstruction of the target protein requires dividing the protein into two or three fragments, the coding sequences of which are cloned into AAV vectors, fused with N- or C-endoproteins, and under promoter control. The splitting point of each protein is selected considering the amino acid requirements at the binding site of the first residue in the C-exoprotein (e.g., the presence of an amino acid containing a nucleophilic thiol or hydroxyl group (i.e., cysteine, serine, or threonine)) and to maintain the integrity of key protein domains to facilitate proper protein folding and stability of each endoprotein-peptide precursor peptide and the resulting reconstructed protein.

[0336] Of particular note is that the inventors selected two types of linkage sites within the target proteins: protein ABCA4 splits at amino acids Cys1150, Ser1168, and Ser1090, and the cleavage-type intraprotein inserts at the splitting site. CEP290 protein splits at aa Cys1076, Ser1275, Cys929, and 1474; and at Ser 453 and Cys 1474.

[0337] Degradation signal

[0338] Regulated protein degradation protects cells from misfolded, aggregated, or otherwise abnormal proteins and also controls the levels of proteins that evolve into short-lived proteins in the body. It is primarily mediated by the ubiquitin (Ub)-proteasome system (UPS) and the autophagy-lysosome pathway, in which molecular chaperones are part of both systems.

[0339] Degradation signals are characteristic of proteins, making them targets of protein degradation pathways and thus reducing their half-life. Specifically, N-degradation determinants and C-degradation determinants are degradation signals, primarily determined by N-terminal and C-terminal residues of cellular proteins, respectively. N-degradation determinants and C-degradation determinants include, to varying degrees, adjacent sequence motifs, as well as internal lysine residues that function as polyubiquitination sites.

[0340] Within the meaning of this invention, an internal degradation determinant is defined as a degradation signal located within a protein sequence that is neither at the N-terminus nor the C-terminus, and whose functionally essential elements do not include N-terminal or C-terminal residues and mediate protein degradation.

[0341] The degradation determinant pathway includes multiple proteolytic systems that are characterized by their ability to recognize proteins containing N- or C- or internal degradation determinants, thereby causing these proteins to be degraded by the 26S proteasome or by autophagy.

[0342] Escherichia coli dihydrofolate reductase (ecDHFR) is a 159-residue enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate, a cofactor essential for several steps in prokaryotic primary metabolism. Many DHFR inhibitors have been developed as drugs, and one such inhibitor, trimethoprim (TMP), inhibits ecDHFR much more potently than mammalian DHFR. This large therapeutic window renders TMP “biosilenced” in mammalian cells. The specificity of the ecDHFR-TMP interaction, along with the commercial availability and attractive pharmacological properties of TMP, makes this protein-ligand pair ideal for development as a degradation system. (69) Thus, the presence of the DHFR amino acid sequence within the protein, preferably the ecDHFR amino acid sequence, functions as a target signal for the proteasome system, leading to protein degradation. In the presence of TMP, the protein is stabilized.

[0343] Conveniently, the ecDHFR-derived degradation determinant signals carrying point mutations developed by Iwamoto et al. include three amino acid mutations, R12Y, Y100I, and G67S (69), which confer functional activity only when placed in the N-terminus or internal position (e.g., degradation of fusion proteins).

[0344] The inventors, who identified the shortest active peptide, further improved the ecDHFR-derived degradation determinant. Advantageously, the shorter sequence allows for the installation of longer coding sequences within the same AAV vector.

[0345] In this invention, the ecDHFR-derived degradation determinant is fused to the N-terminus of the endonucleoprotein, where it is inactive. During protein transsplicing, the degradation determinant resides within the reconstructed endonucleoprotein and mediates its degradation.

[0346] The ecDHFR of this invention is WT ecDHFR, mutant DHFR, full-length ecDHFR, and shorter scDHFR.

[0347] The length of DHFR can range from 105 to 159 aa, with shortening occurring at the C-terminus.

[0348] ecDHFR Escherichia coli derivative, wild type

[0349] nucleotide sequence : (623 nt) SEQ ID No. 27

[0350]

[0351] amino acid sequence:

[0352] 159 aa- WT SEQ ID No. 28

[0353]

[0354] ecDHFR E. coli-derived, internal degradation determinant mutant (159 aa)

[0355] The mutation location is shown in bold - SEQ ID No. 29

[0356]

[0357] ecDHFR E. coli derivative, wild type, minimum active fragment

[0358] Nucleotide sequence: SEQ ID No. 30

[0359]

[0360] amino acid sequence SEQ ID No. 31

[0361]

[0362] ecDHFR E. coli-derived, internal degradation determinant mutant pe, minimum active fragment (104 aa)

[0363] (Mutation location is shown in bold) SEQ ID No. 32

[0364]

[0365] sequence

[0366] The coding sequence of the present invention can be operatively linked to a promoter sequence, optionally followed by an intron sequence, capable of regulating its expression in mammalian cells, preferably mammalian retinal cells, particularly photoreceptor cells, or hepatocytes, muscle cells, cardiac cells, neuronal cells, kidney cells, and endothelial cells. Exemplary promoters include, but are not limited to, ubiquitous, artificial, or tissue-specific promoters, including fragments and variants that retain transcription promoter activity, such as photoreceptor-specific promoters, including photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1), interreceptor photopigment-like binding protein promoter (IRBP), rhodopsin promoter (RHO), uveal dystrophy 2 promoter (VMD2), and rhodopsin kinase promoter (RK); muscle-specific promoters, including MCK and MYODI; liver-specific promoters, including thyroxine-binding globulin (TBG) and heterozygous liver-specific promoter (HLP) (67); neuron-specific promoters, including hSYN1 and CaMKIIa; and kidney-specific promoters, including Ksp-cadherin 16 and NKCC2. The ubiquitous promoters according to the invention are, for example, ubiquitous cytomegalovirus (CMV) (32) and short CMV (33) promoters.

[0367] Optionally, the promoter sequence includes an enhancer sequence, such as a globin IgG chimeric intron.

[0368] For the purposes of this invention, the coding sequences of EGFP (YP_009062989), ABCA4, and CEP290 are functionally linked to promoter sequences capable of regulating their expression in mammalian retinal cells, particularly photoreceptor cells, wherein the coding sequences are preferably selected from the sequences appended herein, or sequences that encode the same amino acid sequence due to the degeneracy of the genetic code.

[0369] Exemplary polyadenylation signals include, but are not limited to, bovine growth hormone polyadenylation signal (bGHpA), human β-globulin polyadenylation signal or short synthetic form (68), SV40 polyadenylation signal or other natural or artificial polyadenylation signals.

[0370] This invention provides the use of nucleotide sequences of degradation signals to reduce the stability of reconstructed endoproteins. Conveniently, one or more sequences can be repeated to maintain maximum effect.

[0371] Suitable degradation signals according to the invention include: (i) a short degradation determinant CL1, a C-terminal destabilized peptide that shares structural similarity with misfolded proteins and is therefore recognized by ubiquitination systems; (ii) ubiquitin, which mediates direct protein degradation or degradation via the N-terminal regular pathway at the N-terminus of the donor protein; and (iii) an N-terminal PB29 degradation determinant, which is a 9-amino acid peptide similar to the CL1 degradation determinant and is expected to fold into a structure recognized by enzymes of the ubiquitination pathway described herein and in (69), variant ecDHFR and its fragments, particularly ecDHFR-derived degradation determinant signals carrying point mutations, including three amino acid mutations R12Y, Y100I and G67S, which confer functional activity (e.g., degradation of fusion proteins) only when placed in an N-terminal or internal location.

[0372] Exemplary degradation signals are described in WO 201613932, which is incorporated herein by reference.

[0373] It will be readily understood by those skilled in the art that, in addition to variants that can be artificially created by skilled laboratory technicians, there are many variant sequences of proteins found in nature. The polynucleotides and polypeptides of this invention include the polynucleotides and polypeptides specifically illustrated herein, as well as any naturally occurring variants thereof, and any variants that can be artificially produced, provided that those variants retain the desired functional activity. Furthermore, polypeptides having the same amino acid sequence as the polypeptides illustrated herein, except for amino acid substitutions, additions, or deletions within the polypeptide sequence, are also within the scope of this invention, provided that these variant polypeptides retain substantially the same relevant functional activity as the polypeptides specifically illustrated herein. For example, conserved amino acid substitutions within a polypeptide that do not affect the polypeptide's function are within the scope of this invention. Therefore, the polypeptides disclosed herein should be understood to include variants and fragments of the specific example sequences discussed above. This invention also includes nucleotide sequences encoding the polypeptides disclosed herein. These nucleotide sequences can be readily constructed by those skilled in the art who possess knowledge of the protein and amino acid sequences provided herein. As those skilled in the art will understand, the degeneracy of the genetic code enables those skilled in the art to construct a variety of nucleotide sequences encoding a particular polypeptide or protein. The choice of a particular nucleotide sequence can depend, for example, on the codon selection of a particular expression system or host cell. Peptides having amino acid substitutions other than those specifically exemplified in the subject peptide are also included within the scope of this invention. For example, non-natural amino acids may substitute for amino acids in the peptides of this invention, provided that the peptide with the substituted amino acid retains substantially the same activity as the peptide without the substituted amino acid. Examples of non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, γ-aminobutyric acid, ε-aminohexanoic acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, 3-aminopropionic acid, leucine, valine, sarcosine, homocitrulline, sulfoalanine, τ-butylglycine, τ-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids, such as β-methyl amino acids, C-methyl amino acids, N-methyl amino acids, and general amino acid analogs. Non-natural amino acids also include amino acids having derivatized side groups. Furthermore, any amino acid in a protein can be in the D (dextral) or L (levorotatory) form. Amino acids are generally classified into the following categories: nonpolar, uncharged polar, basic, and acidic. Conservative substitutions fall within the scope of this invention, provided that the polypeptide with such substitution retains substantially the same biological activity as the polypeptide without such substitution. A conserved substitution refers to the replacement of a polypeptide with one class of amino acids by another amino acid of the same class. Table 4 provides a list of examples of amino acids belonging to each class.

[0374]

[0375] Polynucleotides having the same nucleotide sequence as the polynucleotides exemplified herein, except for nucleotide substitutions, additions, or deletions within the polynucleotide sequence, are also within the scope of this invention, provided that these variant polynucleotides retain substantially the same relevant functional activity as the polynucleotides specifically exemplified herein (e.g., they encode proteins with the same amino acid sequence or the same functional activity as the encoders of the exemplified polynucleotides). Therefore, the polynucleotides disclosed herein should be understood to include variants and fragments of the specific example sequences discussed above.

[0376] The present invention also contemplates polynucleotide molecules having a sequence sufficiently homologous to the polynucleotide sequence of the present invention to allow hybridization with that sequence under standard stringent conditions and standard methods (Maniatis, T. et al., 1982). The polynucleotides described herein may also be defined according to a more specific range of identity and / or similarity to the polynucleotides exemplified herein. Sequence identity is generally greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%. The identity and / or similarity of a sequence to the sequence in the examples herein can be 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. Unless otherwise stated, as used herein, the percentage of sequence identity and / or similarity between two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as described in Karlin and Altschul (1993). Such algorithms have been integrated into the NBLAST and XBLAST programs of Altschul et al. (1990). The NBLAST program can be used to perform a BLAST search with a score of 100 and a word length of 12 to obtain sequences with the desired percentage of sequence identity. For gap alignments used for comparison purposes, Gapped BLAST, as described by Altschul et al. (1997), can be used. When using the BLAST and gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See the NCBI / N1H website.

[0377] The plasmid of the present invention

[0378]

[0379]

[0380]

[0381] EGFP

[0382] p915_pAAV2.l-TBG-5' EGFP intraprotein (SEQ ID No. 33)

[0383] 5'ITR: Dashed underline (seq A starts at the 5th ' of the sequence)

[0384] TBG promoter: bold (seq B)

[0385] 5' EGFP: underlined (seq C)

[0386] N-endoprotein Npu DnaE: Double underline (seq D)

[0387] 3xflag: Italics (seq E)

[0388] WPRE: Italicized and underlined (seq F)

[0389] Bgh PolyA: Bold underline (seq G)

[0390] 3'ITR: Dashed underline (seq H starts at the 3' of the sequence)

[0391]

[0392]

[0393] P917_pAAV2.1-TBG-3' EGFP inner protein

[0394] 5' ITR (seq A)

[0395] TBG promoter (seq B)

[0396] C-intein Npu DnaE (seq I) SEQ ID No. 34

[0397]

[0398] 3' EGFP (seq L) SEQ ID No. 35

[0399]

[0400] 3xflag (seq E)

[0401] WPRE (seq F)

[0402] Bgh PolyA (seq G)

[0403] 3' ITR (seq H)

[0404] p914_pAAV2.1-CMV-5' EGFP inner protein

[0405] 5' ITR (seq A)

[0406] CMV promoter (seq M) SEQ. 36

[0407]

[0408] 5' EGFP (seq C)

[0409] N-endoprotein Npu DnaE (seq D)

[0410] 3xflag (seq E)

[0411] WPRE (seq F)

[0412] Bgh PolyA (seq G)

[0413] 3' ITR (seq H)

[0414] p916_pAAV2.1-CMV-3' EGFP inner protein

[0415] 5' ITR (seq A)

[0416] CMV promoter (seq M)

[0417] C-endoprotein Npu DnaE (seq I)

[0418] 3' EGFP (seq L)

[0419] 3xflag (seq E)

[0420] WPRE (seq F)

[0421] Bgh PolyA (seq G)

[0422] 3' ITR (seq H)

[0423] p932_pAAV2.1-GRKl-5' EGFP intraprotein

[0424] 5' ITR (seq A)

[0425] GRK1 promoter (seq N) SEQ. 37

[0426] 5' EGFP (seq C)

[0427] N-endoprotein Npu DnaE (seq D)

[0428] 3xflag (seq E)

[0429] WPRE (seq F)

[0430] Bgh PolyA (seq G)

[0431] 3' ITR (seq H)

[0432] p933_pAAV2.1-GRKl-3' EGFP intraprotein

[0433] 5' ITR (seq A)

[0434] GRK1 promoter (seq N)

[0435] C-endoprotein Npu DnaE (seq I)

[0436] 3' EGFP (seq L)

[0437] 3xflag (seq E)

[0438] WPRE (seq F)

[0439] Bgh PolyA (seq G)

[0440] 3' ITR (seq H)

[0441] p36 pAAV2.1-CMV-5' EGFP endogenous protein_ecDHFR

[0442] 5' ITR (seq A)

[0443] CMV promoter (seq M)

[0444] 5' EGFP (seq C)

[0445] N-endoprotein Npu DnaE (seq D)

[0446] 3xflag (seq E)

[0447] ecDHFR (seq O) SEQ ID No. 38

[0448]

[0449] WPRE (seq F)

[0450] Bgh PolyA (seq G)

[0451] 3' ITR (seq H)

[0452] p37 pAAV2.1-CMV-5' EGFP endogenous protein_small ecDHFR

[0453] 5' ITR (seq A)

[0454] CMV promoter (seq M)

[0455] 5' EGFP (seq C)

[0456] N-endoprotein Npu DnaE (seq D)

[0457] 3xflag (seq E)

[0458] small ecDHFR (seq P) SEQ ID No. 39

[0459]

[0460] WPRE (seqF)

[0461] Bgh PolyA (seq G)

[0462] 3' ITR (seq H)

[0463] p902_pAAV2.1-CMV-5' EGFP internal protein DnaB

[0464] 5' ITR (seq A)

[0465] CMV promoter (seq M)

[0466] 5' EGFP (seq C)

[0467] N-intein protein RmaDnaB (seq Q) SEQ. ID No. 40

[0468]

[0469] N-endoprotein Npu DnaE (seq D)

[0470] 3xflag (seq E)

[0471] WPRE (seq F)

[0472] Bgh PolyA (seq G)

[0473] 3' ITR (seq H)

[0474] p903_pAAV2.1-CMV-3' EGFP internal protein DnaB

[0475] 5' ITR (seq A)

[0476] CMV promoter (seq M)

[0477] C-endoprotein Rma DnaB (seq R) SEQ. ID No. 41

[0478]

[0479] 3' EGFP (seq L)

[0480] 3xflag (seq E)

[0481] WPRE (seq F)

[0482] Bgh PolyA (seq G)

[0483] 3' ITR (seq H)

[0484] p1256_pAAV2.1-CMV-5' EGFP intraprotein mDnaE

[0485] 5' ITR (seq A)

[0486] CMV promoter (seq M)

[0487] 5' EGFP (seq C)

[0488] N-endoprotein mDnaE (seq S) SEQ ID No. 42

[0489]

[0490] 3xflag (seq E)

[0491] WPRE (seq F)

[0492] Bgh PolyA (seq G)

[0493] 3' ITR (seq H)

[0494] p1257 pAAV2.1-CMV-3' EGFP endogenous protein mDnaE

[0495] 5' ITR (seq A)

[0496] CMV promoter (seq M)

[0497] C-endoprotein mDnaE (seq T) SEQ. 43

[0498]

[0499] 3' EGFP (seq L)

[0500] 3xflag (seq E)

[0501] WPRE (seq F)

[0502] Bgh PolyA (seq G)

[0503] 3' ITR (seq H)

[0504] CEP290

[0505] p1005 pAAV2.1-CMV260-5' CEP290 endogenous protein (Group 1)

[0506] 5' ITR (seq A)

[0507] CMV260 (seq U) SEQ ID No. 44

[0508]

[0509]

[0510] 5' CEP290: SEQ ID No. 45

[0511]

[0512]

[0513] N-endoprotein DnaE (seq D)

[0514] 3xflag (seq E)

[0515] shPolyA (seq V) SEQ ID No. 46

[0516]

[0517] 3'ITR (seq H)

[0518] p1093 pAAV2.1-CMV260-3' CEP290 endogenous protein (Group 1)

[0519] 5' ITR (seq A)

[0520] CMV260 (seq U)

[0521] 3' CEP290: SEQ ID No. 47

[0522]

[0523]

[0524]

[0525] C-endoprotein DnaE (seq I)

[0526] 3xflag (seq E)

[0527] shPolyA (seq V)

[0528] 3' ITR (seq H)

[0529] p1065 pAAV2.1-CMV260-5' CEP290 endogenous protein (Group 2)

[0530] 5' ITR (seq A)

[0531] CMV260 (seq U)

[0532] 5' CEP290: SEQ ID No. 48

[0533]

[0534]

[0535]

[0536] N-endoprotein DnaE (seq D)

[0537] 3xflag (seq E)

[0538] Bgh PolyA (seq G)

[0539] 3'ITR (seq H)

[0540] p1067 pAAV2.1-CMV260-3' CEP290 endogenous protein (Group 2)

[0541] 5' ITR (seq A)

[0542] CMV260 (seq U)

[0543] 3' CEP290: SEQ ID No. 49

[0544]

[0545]

[0546]

[0547] C-endoprotein DnaE (seq I)

[0548] 3xflag (seq E)

[0549] Bgh PolyA (seq G)

[0550] 3' ITR (seq H)

[0551] p1087 pAAV2.1-CMV260-5' CEP290 endogenous protein (Group 3)

[0552] 5' ITR (seq A)

[0553] CMV260 (seq U)

[0554] 5' CEP290: SEQ ID No. 50

[0555]

[0556]

[0557] N-endoprotein mDnaE (seq S)

[0558] 3xflag (seq E)

[0559] Bgh PolyA (seq G)

[0560] 3'ITR (seq H)

[0561] p1088 pAAV2.1-CMV260-3' CEP290 endogenous protein (Group 3)

[0562] 5' ITR (seq A)

[0563] CMV260 (seq U)

[0564] 3' CEP290: SEQ ID No. 51

[0565]

[0566]

[0567] C-endoprotein mDnaE (seq T)

[0568] 3xflag (seq E)

[0569] Bgh PolyA (seq G)

[0570] 3' ITR (seq H)

[0571] p1182 pAAV2.1-CMV260-5' CEP290 endogenous protein (Group 4)

[0572] 5' ITR (seq A)

[0573] CMV260 (seq U)

[0574] 5' CEP290: SEQ ID No. 52

[0575]

[0576]

[0577] N-endoprotein DnaE (seq D)

[0578] 3xflag (seq E)

[0579] WPRE (seq F)

[0580] Bgh PolyA (seq G)

[0581] 3'ITR (seq H)

[0582] p1183 pAAV2.1-CMV260-CEP290 in vivo proteins (group 4)

[0583] 5' ITR (seq A)

[0584] CMV260 (seq U)

[0585] C-endoprotein DnaE (seq I)

[0586] CEP290 body: SEQ ID No. 53

[0587]

[0588]

[0589] N-endoprotein Rma DnaB (seq Q)

[0590] 3xflag (seq E)

[0591] WPRE (seq F)

[0592] Bgh PolyA (seq G)

[0593] 3'ITR (seq H)

[0594] p1181 pAAV2.1-CMV260-3' CEP290 endogenous protein (Group 4 / Group 5)

[0595] 5' ITR (seq A)

[0596] CMV260 (seq U)

[0597] C-endoprotein Rma DnaB (seq R)

[0598] 3' CEP290: SEQ ID No. 54

[0599]

[0600]

[0601] 3xflag (seq E)

[0602] WPRE (seq F)

[0603] Bgh PolyA (seq G)

[0604] 3'ITR (seq H)

[0605] p1179 pAAV2.1-CMV260-5' CEP290 endogenous protein (Group 5)

[0606] 5' ITR (seq A)

[0607] CMV260 (seq U)

[0608] 5' CEP290: SEQ ID No. 55

[0609]

[0610] N-endoprotein mDnaE (seq S)

[0611] 3xflag (seq E)

[0612] WPRE (seq F)

[0613] Bgh PolyA (seq G)

[0614] 3'ITR (seq H)

[0615] p1180 pAAV2.1-CMV260-CEP290 in vivo proteins (group 5)

[0616] 5' ITR (seq A)

[0617] CMV260 (seq U)

[0618] C-endoprotein mDnaE (seq T)

[0619] CEP290 body: SEQ ID No. 56

[0620]

[0621]

[0622] N-endoprotein RmaDnaB (seq Q)

[0623] 3xflag (seq E)

[0624] WPRE (seq F)

[0625] Bgh PolyA (seq G)

[0626] 3'ITR (seq H)

[0627] p1152 pAAV2.1-GRKl-5' CEP290 endogenous protein (Group 5)

[0628] 5' ITR (seq A)

[0629] GRK1 promoter (seq N)

[0630] 5' CEP290: SEQ ID No. 57

[0631]

[0632]

[0633] N-endoprotein mDnaE (seq S)

[0634] 3xflag (seq E)

[0635] WPRE (seq F)

[0636] Bgh PolyA (seq G)

[0637] 3' ITR (seq H)

[0638] p1153 pAAV2.1-GRKl-CEP290 in vivo protein (group 5)

[0639] 5' ITR (seq A)

[0640] GRK1 promoter (seq N)

[0641] C-endoprotein mDnaE (seq T)

[0642] CEP290 body: SEQ ID No. 58

[0643]

[0644]

[0645] N-endoprotein RmaDnaB (seq Q)

[0646] 3xflag (seq E)

[0647] WPRE (seq F)

[0648] Bgh PolyA (seq G)

[0649] 3'ITR (seq H)

[0650] p1156 pAAV2.1-GRKl-3' CEP290 endogenous protein (Group 5)

[0651] 5' ITR (seq A)

[0652] GRK1 promoter (seq N)

[0653] C-endoprotein Rma DnaB (seq R)

[0654] 3' CEP290: SEQ ID No. 59

[0655]

[0656]

[0657] 3xflag (seq E)

[0658] WPRE (seq F)

[0659] Bgh PolyA (seq G)

[0660] 3' ITR (seq H)

[0661] pzac-GRKl-5' ABCA4 intraprotein (Group 1) SEQ ID No. 60

[0662] 5' ITR (seq A)

[0663] GRK1: Bold

[0664] 5' ABCA4: underline

[0665] N-endoprotein Npu DnaE: Double underscores

[0666] 3xflag: Italic

[0667] SV40: Bold underline

[0668] 3' ITR (seq H)

[0669]

[0670]

[0671] pzac-GRKl-3' ABCA4 intraprotein (Group 1) SEQ. No. 61

[0672] 5' ITR (seq A)

[0673] GRK1: Bold

[0674] 3' ABCA4: underline

[0675] C-endoprotein Npu DnaE: Double underscores

[0676] 3xflag: Italic

[0677] SV40: Bold underline

[0678] 3' ITR (seq H)

[0679]

[0680]

[0681]

[0682] pzac-CMV260-5' ABCA4 protein (Group 1) SEQ No. 62

[0683] 5' ITR (seq A)

[0684] CMV260: Bold

[0685] 5' ABCA4: underline

[0686] N-endoprotein Npu DnaE: Double underscores

[0687] 3xflag: Italic

[0688] SV40: Bold underline

[0689] 3' ITR (seq H)

[0690]

[0691]

[0692]

[0693] pzac-CMV260-3' ABCA4 intraprotein (Group 1) SEQ No. 63

[0694] 5' ITR (seq A)

[0695] CMV260: Bold

[0696] 3' ABCA4: underline

[0697] C-endoprotein Npu DnaE: Double underscores

[0698] 3xflag: Italic

[0699] SV40: Bold underline

[0700] 3' ITR (seq H)

[0701]

[0702]

[0703]

[0704] p38 pAAV2.1-CMV260- 5' ABCA4 endogenous protein_ecDHFR (Group 1)

[0705] 5' ITR (seq A)

[0706] CMV260 (seq U)

[0707] 5' ABCA4 (from group 1)

[0708] N-endoprotein Npu DnaE (seq D)

[0709] 3xflag (seq E)

[0710] ecDHFR (seq O)

[0711] WPRE (seq F)

[0712] SV40 PolyA (seq W)

[0713] 3' ITR (seq H)

[0714] p39 pAAV2.1-CMV260- 5' ABCA4 endogenous protein_small ecDHFR (Group 1)

[0715] 5' ITR (seq A)

[0716] CMV260 (seq U)

[0717] 5' ABCA4 (from group 1)

[0718] N-endoprotein Npu DnaE (seq D)

[0719] 3xflag (seq E)

[0720] Small ecDHFR (seq P)

[0721] WPRE (seq F)

[0722] SV40 PolyA (seq W)

[0723] 3' ITR (seq H)

[0724] p40 pAAV2.1-GRKl- 5' ABCA4 endogenous protein_ecDHFR (Group 1)

[0725] 5' ITR (seq A)

[0726] GRK1 (seq N)

[0727] 5' ABCA4 (from group 1)

[0728] N-endoprotein Npu DnaE (seq D)

[0729] 3xflag (seq E)

[0730] ecDHFR (seq O)

[0731] WPRE (seq F)

[0732] SV40 PolyA (seq W)

[0733] 3' ITR (seq H)

[0734] p41 pAAV2.1-GRKl-5'ABCA4 intraprotein_small ecDHFR (Group 1) SEQ ID No. 64

[0735] 5' ITR (seq A)

[0736] GRK1: Bold

[0737] 5' ABCA4: underline

[0738] N-endoprotein Npu DnaE: Double underscores

[0739] 3xflag: Italic

[0740] Small ecDHFR: bold underline

[0741] SV40: Bold underline

[0742] 3' ITR (seq H)

[0743]

[0744]

[0745] pzac-CMV260-5' ABCA4 intraprotein (Group 2) SEQ ID No. 65

[0746] 5' ITR (seq A)

[0747] CMV260: Bold

[0748] 5' ABCA4: underline

[0749] N-endoprotein Npu DnaE: Double underscores

[0750] 3xflag: Italic

[0751] SV40: Bold underline

[0752] 3' ITR (seq H)

[0753]

[0754]

[0755]

[0756] pzac-CMV260-3' ABCA4 intraprotein (Group 2) SEQ ID No. 66

[0757] 5' ITR (seq A)

[0758] CMV260: Bold

[0759] 3' ABCA4: underline

[0760] C-endoprotein Npu DnaE: Double underscores

[0761] 3xflag: Italic

[0762] SV40: Bold underline

[0763] 3' ITR (seq H)

[0764]

[0765]

[0766]

[0767] pzac-CMV260-5' ABCA4 protein (Group 3) SEQ No. 67

[0768] 5' ITR (seq A)

[0769] CMV260: Bold

[0770] 5' ABCA4: underline

[0771] N-endoprotein Npu DnaE: Double underscores

[0772] 3xflag: Italic

[0773] SV40: Bold underline

[0774] 3' ITR (seq H)

[0775]

[0776]

[0777]

[0778] pzac-CMV260-3' ABCA4 intraprotein (Group 3) SEQ ID No. 68

[0779] 5' ITR (seq A)

[0780] CMV260: Bold

[0781] 3' ABCA4: underline

[0782] C-endoprotein Npu DnaE: Double underscores

[0783] 3xflag: Italic

[0784] SV40: Bold underline

[0785] 3' ITR (seq H)

[0786]

[0787]

[0788]

[0789] p836 (IRBP_DsRed) SEQ ID No. 69

[0790] 5' ITR (seq A)

[0791] IRBP (bold)

[0792] WPRE: Italic underline DsRed: Underline BghpA: Bold underline

[0793] 3' ITR (seq H)

[0794]

[0795]

[0796] p1232 pAAV2.1_HLP_5' F8 endogenous protein (Group 1)

[0797] 5' ITR (seq A)

[0798] HLP promoter (seq J) SEQ ID No. 70

[0799]

[0800] F8 signal sequence (seq K) SEQ ID No. 71

[0801]

[0802] 5' F8: SEQ ID No. 72

[0803]

[0804]

[0805] N-endoprotein Npu DnaE (seq D)

[0806] 3xflag (seq E)

[0807] shPolyA (seq V)

[0808] 3' ITR (seq H)

[0809] p1389 pAAV2.1_HLP_3' F8 endogenous protein (Group 1)

[0810] 5' ITR (seq A)

[0811] HLP promoter (seq J)

[0812] F8 signal sequence (seq K)

[0813] C-endoprotein Npu DnaE (seq I)

[0814] 3' F8: SEQ ID No. 73

[0815]

[0816]

[0817]

[0818] 3xflag (seq E)

[0819] shPolyA (seq V)

[0820] 3' ITR (seq H)

[0821] p1207 pAAV2.1_HLP_5' F8 endogenous protein (Group 2)

[0822] 5' ITR (seq A)

[0823] HLP promoter (seq J)

[0824] F8 signal sequence (seq K)

[0825] 5' F8 (Group 2): SEQ ID No. 74

[0826]

[0827]

[0828] N-endoprotein Npu DnaE (seq D)

[0829] 3xflag (seq E)

[0830] shPolyA (seq V)

[0831] 3' ITR (seq H)

[0832] p1388 pAAV2.1_HLP_3' F8 endogenous protein (Group 2)

[0833] 5' ITR (seq A)

[0834] HLP promoter (seq J)

[0835] F8 signal sequence (seq K)

[0836] C-endoprotein Npu DnaE (seq I)

[0837] 3' F8: SEQ ID No. 75

[0838]

[0839]

[0840] 3xflag (seq E)

[0841] shPolyA (seq V)

[0842] 3' ITR (seq H)

[0843] The invention will now be described by way of non-limiting embodiments.

[0844] Example

[0845] Materials and methods

[0846] Generation of AAV vector plasmids

[0847] The plasmids used for AAV vector generation were derived from pAAV2.1(36) or pZac(37) plasmids containing the ITR of AAV serotype 2. For details on the design of AAV intraprotein plasmids, please refer to [link to relevant documentation]. Figure 1 A and Figure S5. The EGFP protein is split at amino acid (aa) C71. The ABCA4 protein is split at aa C1150 (group 1), aa S1168 (group 2), and aa C1090 (group 3) of the large cytoplasmic domain CD1 (34, 35). Although aa C1150 (group 1) and S1168 (group 2) fall within regions unrelated to known ABCA4 function, C1090 is included in the ABCA4 nucleotide-binding domain spanning aa929 to aa1148. All CEP290 splitting points fall within the coiled-coil domain (36): when CEP290 splits into two polypeptides, this occurs at aa C1076 (group 1) or S1275 (groups 2-3), and when it splits into three polypeptides, this occurs at aa C929 and C1474 (group 4) or aa S453 and C1474 (group 5).

[0848] The endogenous proteins contained in the plasmids were either endogenous proteins of DnaE from *Candida punctata* (27, 28), or endogenous proteins composed of mutant N- and C-endogenous proteins of DnaE from *Npu* and *Syntropha polycystica* PCC6803 (Ssp) (30), or endogenous proteins of DnaB from *Rhodophyton floccosum* (Rma) (29). The plasmids used in the study were controlled by the ubiquitous cytomegalovirus (CMV) (38) and short CMV (39) promoters or the photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1) 40 promoter. Plasmids encoding EGFP and CEP290 included bovine growth hormone polyadenylation signaling (bGHpA), while plasmids encoding ABCA4 included simian virus 40 (SV40) polyadenylation signaling.

[0849] AAV vector generation and characterization

[0850] AAV vectors were generated by triple transfection of HEK293 cells using TIGEM AAV Vector Core, as previously described (14, 41). No difference in vector yield was observed between AAV vectors that included or excluded the endonucleoprotein sequence.

[0851] Cell transfection and AAV infection

[0852] As described in (14), HEK293 cells were maintained and transfected using the calcium phosphate method (6-well plate format, 1 μg of each plasmid / well). For the experiments described in Figure S9, the amount of plasmid encoding the full-length gene was used, corresponding to the same number of molecules included in the protein grains within 1 μg of AAV. The total amount of DNA transfected in each well was kept constant by adding contaminant grains as needed.

[0853] Lipofectamine LTX (Invitrogen) transfection was used for Figure 2 HeLa cells used in experiments C and 2D (24-well plate format, 1 or 0.5 μg of each plasmid / well). AAV infection was performed as described (14).

[0854] iPSC and retinal differentiation culture

[0855] Human induced pluripotent stem cells (iPSCs) are derived from fibroblasts cultured from skin biopsies using the method described in (42). The STGD1 cell line carries the ABCA4 complex heterozygous variants c.4892T>C and c.4539+2001G>A, as also described in (43), or the complex heterozygous variants c.[2919-?_3328+?del; 4462T>C] and c.5196+1137G>A. c.[2919-?_3328+?del; 4462T>C] is an allele consisting of two variants. C.2919-?_3328+?del constitutes a deletion of exons 20, 21, and 22 and an unknown fragment of introns 19 and 22. This deletion is found in the cis configuration of c.4462T>C. iPSCs were maintained in 6-well plates coated with matrix gel (#354277, Corning® Matrigel® hESC-Qualified Matrix; Corning, NY) containing mTeSR™ medium (#85850; Stem cell technologies). Cells were passaged for 2–6 minutes at approximately 80% confluence using 0.5 mM EDTA (#AM9260G; Ambion). Retinal differentiation was based on a combination of previously described protocols (44, 45). Briefly, iPSCs were seeded in 96-well (9,000 cells / well) V-bottom plates containing RevitaCell Supplement (#A-2644501; Gibco, ThermoFisher) and 1% matrix gel to induce aggregate formation. Aggregates were then cultured to generate 3D retinal organoids as described in (46).

[0856] Western blot analysis and ELISA

[0857] Samples (HEK293 cells, retina, and retinal organoids) were lysed in RIPA buffer to extract EGFP, ABCA4, and CEP290 proteins. The lysis buffer was supplemented with protease inhibitors (Complete Protease Inhibitor Mixture Tablets; Roche, Basel, Switzerland) and 1 mM benzylsulfonyl. After lysis, ABCA4 samples were denatured at 37°C for 15 min in 1X Laemmli sample buffer supplemented with 2 M urea. EGFP and CEP290 samples were denatured at 99°C for 5 min in 1X Laemmli sample buffer. The lysates were separated by 12% (for EGFP samples) or 6% (for ABCA4 and CEP290 samples) SDS-polyacrylamide gel electrophoresis. The antibodies used for the immunoblotting method are as follows: anti-3xflag (1:1000, A8592; Sigma-Aldrich, St. Louis, Missouri, USA), for detecting EGFP, ABCA4, and CEP290 proteins; anti-ABCA4 (1:500, LS-C87292; LifeSpan BioSciences, Inc., Seattle, USA), for detecting ABCA4; anti-filamentin A (1:1000, #4762; Cell Signaling Technology, Danvers, Massachusetts, USA); anti-β-actin (1:1000, NB600-501; Novus Biological LLC, Littleton, Colorado, USA), for detecting filamentin A and β-actin, and used as loading controls in in vitro experiments; anti-Dysferlin (1:500, Dysferlin, clone Ham1 / 7B6, MONX10795); Tebu-bio (Le Perray-en-Yveline, France) was used to detect Dysferlin, which was used as a loading control in in vivo experiments. The EGFP, ABCA4, and CEP290 bands detected by Western blotting were quantified using ImageJ software (available for free download from http: / / rsbweb.nih.gov / ij / ).

[0858] for Figure 21 The experiment shown was performed on Abca4 cells injected with the AAV intraprotein carrier. - / - Abca4 mice and control littermates + / -Mouse retinal lysates were lysed in 30 μl of lysis buffer as described above, and 25 or 50 μl of the lysate were Western blotted with anti-ABCA4 antibody (LS-C87292; epitope conservation: 100% for human ABCA4; 86% for mouse ABCA4). The amount of ABCA4 in the retinal lysates (quantified by band intensity using ImageJ software) was then normalized relative to the volume of the retinal lysates loaded on an acrylamide gel. Figure 9 The experiment, as reported in the figure, involved treating HEK293 cells daily with increasing doses of trimethoprim (T7883, Sigma-Aldrich).

[0859] ELISA was performed on cells or mouse and porcine retinal lysates using the Max Discovery Green Fluorescent Protein ELISA Kit (Bioo Scientific Corporation, Austin, Texas, USA).

[0860] Southern blot analysis of rAAV vector DNA.

[0861] From 1.5 to 6x10 10 DNA was extracted from each viral particle (measured by GC). To digest the unpackaged genome, the vector solution was incubated with 30 μl of DNase (Roche) in a total volume of 300 μl at 37°C for 2 h, the total volume containing 50 mM Tris, pH 7.5, and 1 mM MgCl2. The DNase was then inactivated with 50 mM EDTA and incubated at 50°C for 1 h with proteinase K and 2.5% N-lauryl-sarcosil solution to lyse the capsid. The DNA was extracted twice with phenol-chloroform and precipitated with two volumes of 100% ethanol and 10% sodium acetate (3 M) and 1 μl of glycogen (20 μg). Alkaline agarose gel electrophoresis was performed as previously described (Sambrook, J., and Russell, DW 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York, USA. 999 pp). The biomarker was generated by double digestion of pF8-V3 with Small, producing a 5102 bp band. A probe specific to the HLP promoter was used.

[0862] Activated partial thrombin time (aPTT)

[0863] Nine blood samples were collected via retroorbital retraction into a buffered 0.109M trisodium citrate solution (BD, Franklin Lakes, NJ, USA). Plasma was separated by centrifugation at 13,000 rpm for 15 minutes.

[0864] Measure aPTT using the aPTT program according to the manufacturer's manual on a Coatron M4 (Teco, Bünde, Germany).

[0865] Immunoprecipitation and liquid chromatography / mass spectrometry analysis

[0866] Cells were seeded in 100 mm plates (1 x 10⁻⁶). 7 Transfected cells with AAV-EGFP or ABCA4 intracellular protein plasmids in suspension using the calcium phosphate method (20 μg each plasmid per plate). Cells were harvested 72 hours post-transfection, and EGFP and ABCA4 proteins were immunoprecipitated using anti-flag M2 magnetic beads (M8823; Sigma-Aldrich) according to the manufacturer's instructions. Proteins were eluted from the beads by incubation at 37°C for 15 min in sample buffer supplemented with 4 M urea. Proteins were then loaded onto 12% (for EGFP) or 6% (for ABCA4) SDS-polyacrylamide gel electrophoresis. Twenty-six and thirty-strip protein bands (from HEK293 cells independently transfected 2 and 3 times with AAV-EGFP and ABCA4 intracellular protein plasmids, respectively) after Coomassie blue staining were used for protein sequencing (Creative Proteomics, Shirley, NY). In summary, digestion was performed using three gel slides with each of the following enzymes: trypsin, chymotrypsin, Glu-C, Arg-C, Asp-N, and Lys-N. ABCA4 was also digested with pepsin. The resulting peptides were identified and quantified using nano-liquid chromatography-tandem mass spectrometry (nano LC-MS / MS). The obtained mass spectrometry data were analyzed using a PEAKS STUDIO 8.5. The inventors achieved 100% protein sequence coverage of both EGFP and ABCA4 proteins.

[0867] animal models

[0868] Animals were housed in the TIGEM animal facility (Naples) and kept under a 12-hour light / dark cycle. C57BL / 6J mice were purchased from Envigo (Italy).

[0869] Abca4 albino mice were produced through continuous hybridization and backcrossing with BALB / c mice (Rpe65 Leu450 homozygotes). - / -Mice were inbred. BXD24 / TyJ-Cep290 rd16 / J (referred to as rd16) mice were introduced from Jackson Laboratory (JAX original #000031). rd16 mice carry an 897 bp in-frame deletion, including exons 35–39 (46). Mice were maintained by crossing homozygous females with homozygous males. Hemophiliac mice B6;129S-F8 tm1Kaz The / J (referred to as F8tm1) mice were introduced from Jackson Laboratory (JAX original strain #004424). F8tm1 mice possess a neomycin resistance cassette, replacing a 293 bp sequence, including 7 bp at the 3' end of exon 16 and 286 bp at the 5' end of intron 16. Mouse colonies were maintained by crossing homozygous females with hemizygous males.

[0870] The Large White sows used in this study (Azienda Agricola Pasotti, Imola, Italy) were registered as purebred in the LWHerd manual of the Italian National Pig Breeders' Association and were kept in captivity at Centro di Biotecnologie AORN Antonio Cardarelli (Naples, Italy) and kept under a 12-hour light / dark cycle.

[0871] Subretinal injection of AAV vector in mice and pigs

[0872] This study was conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the Italian Ministry of Health's regulations on animal procedures. All procedures performed on mice were approved by the Italian Ministry of Health's Department of Public Health, Animal Health, Nutrition and Food Safety on March 6, 2015.

[0873] Subretinal injection in mice and pigs was performed as previously described (e.g., in 14). 1 μl or 0.5 μl (for rd16 pups) of the carrier solution was injected into the mouse eye. As described in the Results section, the AAV2 / 8 dose varied in different mouse experiments. Pig eyes were injected with 100 μl of the AAV2 / 8 carrier solution into two adjacent subretinal vesicles. The AAV2 / 8 dose was 2 × 10^11 GC per carrier / eye; therefore, co-injection of both AAV carriers resulted in a total dose of 4 × 10^11 GC / eye.

[0874] Histological, optical and fluorescence microscopy

[0875] To assess EGFP expression in tissue sections, retinal organoids and eyes from C57BL / 6J mice and Large White pigs were fixed and sectioned, as previously described. EGFP-positive frozen sections mounted with Vectashield and DAPI (Vector Lab Inc., Peterborough, UK) were analyzed under a confocal LSM-700 microscope (Carl Zeiss, Oberkochen, Germany) using appropriate excitation and detection settings, and were acquired at 40x magnification. Due to the prevalence of red-green color blindness, the colors of the original images were altered to avoid red and green appearing together. Figure 14 Modifications have been made.

[0876] To assess the thickness of the outer nuclear layer (ONL) in rd16 mice injected with the AAV CEP290 inner protein carrier, eyes were fixed overnight in 4% paraformaldehyde (PFA), then dehydrated in continuous ethanol, and then embedded in paraffin blocks. Continuous cross-sections (10 μm) from rd16 mice were cut along the horizontal meridian, gradually distributed on slides, and stained with hematoxylin and eosin (H&E). The sections were then analyzed under a microscope (Leica Microsystems GmbH; DM5000) and acquired at 20x magnification. For each eye, one image from the time-injection side of a section in the central region of the eye was analyzed. Three measurements of ONL thickness were taken in each image by an operator masked for genotype / treatment group using the “freehand line” tool of ImageJ software.

[0877] Immunofluorescence analysis

[0878] HeLa cells transfected with ABCA4 or CEP290 AAV protein plasmids were fixed in 4% PFA for 10 minutes 24 hours post-transfection. Cells were then blocked for 30 minutes in blocking buffer (PBS containing 0.05% saponin, 0.5% BSA, 50 mM NH4Cl, and 0.02% NaN3, pH 7.2) and incubated as follows:

[0879] - ABCA4 protein was detected by continuous staining with anti-FLAG M2 antibody (F1804, Sigma-Aldrich) for 1 hour; the endoplasmic reticulum was stained with anti-VAP-B antibody [Antonella De Matteis laboratory-produced ((47)]], and the trans-Golgi network was stained with TGN46 (AHP-499, Serotech). After washing in PBS, the cells were incubated with secondary antibodies for 30 minutes: goat anti-mouse Alexa Fluor 568; goat anti-rabbit Alexa Fluor 488; and donkey anti-goat Alexa Fluor 633, targeting anti-FLAG, -VAP-B, and -TGN46 antibodies, respectively.

[0880] - CEP290 protein was detected overnight using an anti-FLAG antibody (F7425, Sigma-Aldrich), and microtubules were stained using an anti-acetylated tubulin antibody (T6793, Sigma-Aldrich). After washing in PBS, cells were incubated for 1 hour with appropriate secondary antibodies: goat anti-rabbit Alexa Fluor 594 and donkey anti-mouse Alexa Fluor 488, targeting anti-FLAG and -Ac-tubulin antibodies, respectively.

[0881] Cell nuclei were stained with DAPI. Due to the prevalence of red-green color blindness, the original image's colors were modified to avoid red and green appearing together. Figure 2 CD and Figure 18 Modifications have been made.

[0882] The antibodies used for immunofluorescence of human retinal organoids are as follows:

[0883] Anti-human cone-inhibitor protein (CAR) (50, 51) (1:10000, hCAR, "Luminairefounders"; courtesy of Dr. Cheryl M. Craft, Doheny Eye Institute, Los Angeles, CA, USA); Anti-opsin, red / green (1:200, AB5405; Merck Millipore, Darmstadt, Germania); Anti-recovery protein (1:500, AB5585; Merck Millipore); Anti-CRX (A-9, 1:250, sc377138; Santa Cruz Biotechnology, Santa Cruz Biotechnology, Dallas, Texas, USA); Anti-rhodopsin (1D4, 1:200, ab5417, Abeam, Cambridge, MA, USA).

[0884] Transmission and scanning electron microscopy analysis

[0885] For electron microscopy (EM) analysis, Abca4 levels were measured 3 months after subretinal injection of AAV. - / - Mice were acclimatized overnight in darkness, and then their eyes were harvested. The eyes were fixed for 18 hours in 2% glutaraldehyde (GA) – 2% PFA in 0.1 M PHEM buffer at pH 6.9, followed by rinsing in 0.1 M PHEM buffer. The eyes were then dissected under an optical microscope to select the temporal injection area of ​​the optic cup. This portion of the optic cup was then embedded in 12% gelatin and injected with 2.3 M sucrose. Frozen sections (60 nm) were frozen in liquid nitrogen and cut using a Leica Ultramicrotome EM FC7 (Leica Microsystems). To avoid bias in attributing data to individual experimental groups, the area occupied by lipofuscin granules in the retinal pigment epithelium was measured by an operator masked for genotype / treatment groups using iTEM software (Olympus SYS, Hamburg, Germany). The area occupied by lipofuscin granules in at least 20 different images (25 μm) was measured using the “Free hand polygon” tool of iTEM software. 2 The area of ​​each lipofuscin granule in each field of view is measured in the area.

[0886] For scanning electron microscopy (SEM) analysis, retinal organoids were fixed in GA, stained with OsO4, dehydrated in ethanol, and dried using a critical point drying procedure. The dried samples were then mounted onto SEM columns and coated with a thin layer of gold. The surface tissue of the samples was analyzed for three-dimensional histology, and images were acquired using a JEOL 6700F scanning electron microscope (JEOL Ltd., Tokyo, Japan).

[0887] For ultrastructural analysis, retinal organoids were fixed overnight in a mixture of 2% PFA and 1% GA in 0.2 M PHEM buffer at pH 7.3. Following fixation, the samples were post-fixed as described previously. They were then dehydrated, embedded in epoxy resin, and polymerized at 60°C for 72 hours. Continuous 60 nm thin sections were cut using a Leica EM UC7 microtome.

[0888] EM images were acquired using a FEI Tecnai-12 electron microscope (FEI, Eindhoven, Netherlands) equipped with a VELETTA CCD digital camera.

[0889] Electrophysiological recordings and spectral-domain optical coherence tomography

[0890] As previously described (14), functional and morphological analyses were performed.

[0891] Pupil light response

[0892] Pupil light responses from rd16 mice were recorded under dark conditions using a TRC-50IX retinal camera (Topcon Biomedical Systems, Oakland, NJ) connected to a Nikon DLH digital camera via a charge-coupled device. Mice were exposed to 10 lux light stimulation for approximately 10 seconds, and one photograph of each eye was acquired using IMAGEnet software (Topcon Biomedical Systems). For each eye, the pupil diameter was normalized relative to the eye diameter (from the temporal to the nasal side).

[0893] Statistical analysis

[0894] One-way ANOVA (parametric test) or Kruskal-Wallis rank-sum test (non-parametric test) was performed to determine whether there were statistically significant differences between two or more groups of independent variables in terms of the dependent variable. P values ​​are as follows: ELISA assay for quantification of EGFP protein in vitro (p Kruskal-Wallis = 0.006036), in mouse retina (p ANOVA = 0.00585), and in porcine retina (p Kruskal-Wallis = 0.009005); Figure 5A(p ANOVA = 0.00585); Figure 5 B(pKruskal-Wallis = 5.547E-5); Figure 5 C (p ANOVA = 5.81E-10); ERG analysis (p ANOVA or p Kruskal-Wallis > 0.05 for all luminances analyzed for both a- and b-amplitude); OCT analysis in Figure S14 (p ANOVA = 0.52 for ABCA4, p ANOVA = 0.965 for CEP290). The statistically significant differences between groups, determined by multiple paired comparisons of group means, are as follows: ELISA assays for EGFP protein quantification performed in vitro (single AAV vs. double AAV = 0.012; AAV protein vs. double AAV = 0.012; single AAV vs. AAV protein = 0.222), in mouse retina (single AAV vs. double AAV = 0.0044; AAV protein vs. double AAV = 0.3754; single AAV vs. AAV protein = 0.0561), and in porcine retina (single AAV vs. double AAV = 0.012; AAV protein vs. double AAV = 0.012; single AAV vs. AAV protein = 0.841). Figure 5 A: + / + relative to - / - AAV protein = 0.4530; + / - relative to - / - = 0.0002; Figure 5 B: Wild-type protein content relative to rd16 AAV = 0.00131; Figure 5 C: Wild type relative to rd16 AAV protein 1E-07; wild type relative to rd16 neg < 1E-06.

[0895] Example

[0896] Example 1: In vitro reconstruction of full-length AAV-EGFP protein

[0897] The inventors tested the efficiency of protein trans-splicing mediated by intraretinal proteins in the retina; two AAV vectors were generated, each encoding Candida punctata. Figure 1 The reporter EGFP protein is a fusion of the N-terminal and C-terminal halves of the DnaE cleavage protein in A[Npu][A]. The EGFP protein cleaves at amino acid (aa) C71. Each AAV vector includes appropriate regulatory elements (i.e., a promoter and bovine growth hormone polyadenylation signal (bGHpA)) and a triple flag tag (3xflag) to allow detection of both halves as well as the full-length reconstructed EGFP protein. Figure 1 A).

[0898] Human embryonic kidney 293 (HEK293) cells were transfected with the AAV-EGFP DNA E intraprotein plasmid, and the production of single N-terminal and C-terminal EGFP protein, as well as the full-length EGFP protein, was evaluated. EGFP fluorescence was detected in cells co-transfected with the AAV-EGFP intraprotein plasmid, but not with the single N-terminal and C-terminal AAV-EGFP intraprotein plasmid, and was comparable to fluorescence observed in cells transfected with a single AAV plasmid encoding the full-length EGFP. Figure 12 As shown. Only after co-transfection with two AAV-EGFP intraprotein plasmids, Western blot (WB) analysis of HEK293 cell lysates confirmed the presence of the expected-size (approximately 28 kDa) trans-spliced ​​EGFP protein and the DnaE intraprotein (approximately 17 kDa) spliced ​​from the mature protein, as shown. Figure 1 As shown in B. Furthermore, quantification of band intensity indicated that the amount of EGFP protein from the AAV intrapeptide was 76 ± 37% of the amount observed with a single AAV plasmid (n = 3 independent experiments). To constrain the accuracy of protein remodeling, EGFP was immunopurified from HEK293 cells transfected with the AAV-EGFP intrapeptide and analyzed by liquid chromatography-mass spectrometry (LC-MS) to define its protein sequence. 3539 peptides were obtained from the proteolytic digestion of this sample, including 7 peptides containing cleavage sites (Table 5), covering the entire protein and confirming that the amino acid sequence of the EGFP remodeled from the AAV intrapeptide precisely corresponds to the amino acid sequence of wild-type EGFP.

[0899] Table 5: Peptides including EGFP cleavage points.

[0900] C = Cysteine ​​71

[0901]

[0902] Example 2: AAV-EGFP inner protein is more effective in vitro than dual AAV vectors.

[0903] To confirm EGFP protein remodeling from the AAV intraprotein vector, HEK293 cells were infected with AAV2 / 2-CMV-EGFP DnaE intraprotein or single and dual AAV vectors containing the same expression cassette. The multiplicity of infection (MOI) was 5 x 10^4 genomic copies (GC) / cell per vector, meaning that, assuming dual vectors underwent complete DNA or protein recombination, similar dosages were used across the three systems. To accurately quantify EGFP levels, cell lysates were harvested 72 hours post-infection. EGFP expression was assessed by both Western blotting and enzyme-linked immunosorbent assay (ELISA): EGFP expression obtained using the AAV intraprotein vector was approximately half that obtained using a single AAV vector (single AAV = 0.735 ± 0.2 ng EGFP / μg total lysate, n = 5 independent experiments; AAV intraprotein = 0.403 ± 0.04 ng EGFP / μg total lysate, n = 5 independent experiments) and 10-fold higher than that obtained using dual AAV vectors. Figure 1 As shown in C (double AAV = 0.046 ± 0.01 ng EGFP / μg total lysate, n=5 independent experiments). Furthermore, the intensity of full-length EGFP relative to the intensity of the excised endonucleoprotein was quantified by Western blotting; their relative abundance was found to be 1:0.2 (n=6 independent experiments). Figure 13 A).

[0904] Example 3: Subretinal administration of the AAV-EGFP intraretinal protein carrier resulted in efficient full-length protein remodeling in the retinas of both mice and pigs.

[0905] To investigate whether AAV intracellular protein-mediated transsplicing reconstructs full-length protein expression in the retina, 4-week-old C57BL / 6J mice were subretinally injected with AAV2 / 8-CMV-EGFP Dna E intracellular protein vectors (dose per eye: 5.8 x 10^9 GC). Eyes were harvested one month later and analyzed using microscopy. Most importantly, EGFP fluorescence was detected in the retinal pigment epithelium of all eyes, primarily in the photoreceptor. Figure 1 D). To compare transgene expression of AAV proteins in the photoreceptor with that of single and dual AAV transgenes, AAV2 / 8 vectors encoding EGFP encoded under the control of the photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1) promoter were subretinally injected into 4-week-old C57BL / 6J mice (dose per vector / eye: 5 x 10^9 GC). Eyes were harvested one month after injection and analyzed by fluorescence microscopy, ELISA, or Western blotting.

[0906] EGFP fluorescence was detected in the photoreceptor cell layer of the eyes of all injected vector groups, such as Figure 1 As seen in E, precise quantification of EGFP protein levels via ELISA confirmed that the efficiency of EGFP protein remodeling within AAV was lower than that of single AAV, but approximately 3 times higher than that of dual AAV (single AAV = 8.41 ± 2.48 ng EGFP / retina, n = 5 eyes; AAV inner protein = 3.72 ± 0.85 ng EGFP / retina, n = 7 eyes; dual AAV = 1.38 ± 0.43 ng EGFP / retina, n = 7 eyes). After Western blotting band intensity quantification, the relative amounts of full-length EGFP and excised inner protein were 1:3 (analyzed n = 14 eyes). Figure 13 B).

[0907] Subsequently, the inventors evaluated the efficiency of the AAV intraprotein vector in transducing photoreceptors in the porcine retina, which, due to its size and structure, served as an excellent preclinical model for evaluating viral vector transduction ((48)). Therefore, single, intraprotein, and dual AAV2 / 8-GRK1-EGFP vectors (dose per vector / eye: 2 x 10^11 GC, delivered via two adjacent subretinal vesicles) were injected subretinally into Large White pigs. Eyes were harvested one month post-injection and analyzed by fluorescence microscopy, ELISA, or WB. Notably, as assessed by EGFP fluorescence, the remodeling of EGFP protein mediated by the AAV intraprotein in the photoreceptor cell layer was higher than that mediated by dual AAV and indistinguishable from that mediated by the single AAV vector ( Figure 1 F). Precise quantification of EGFP in retinal lysates confirmed that the AAV intraprotein reconstructed the protein in amounts similar to those obtained using a single AAV vector, and approximately three times higher than those obtained using a dual AAV vector (single AAV = 247.5 ± 45.1 ng EGFP / retina, n = 5 eyes; AAV intraprotein = 227.0 ± 15.7 ng EGFP / retina, n = 5 eyes; dual AAV = 82.3 ± 9.6 ng EGFP / retina, n = 5 eyes). After Western blotting band intensity quantification, the relative amounts of full-length EGFP to excised intraprotein were 1:2 (n = 8 eyes). Figure 13 C).

[0908] Example 4: AAV-mediated protein trans-splicing reconstructing of full-length EGFP in 3D human retinal organoids.

[0909] As an additional preclinical model representing the human retina, the inventors generated 3D retinal organoids from human induced pluripotent stem cells (iPSCs) ((49, 50)). Six-month-old organoids ( Figure 14 A) Includes cells stained with markers of mature photoreceptors, such as Figure 14 As shown in B; organoids were successfully transduced using AAV2 vectors with photoreceptor-specific promoters, namely AAV2 / 2 CMV EGFP and AAV2 / 2IRBP DsRed vectors, as shown in Figure B. Figure 14 C is shown by fluorescence analysis. (Light) Figure 14 D) and electrons ( Figure 14 EF microscopy revealed the presence of buds on the outer segments of the photoreceptor. Nine-month-old 3D human retinal organoids incubated for 30 days with the AAV-GRK1-EGFP internal protein vector (dose per vector / organoid: 1 × 10^12 GC) showed EGFP fluorescence. Figure 1 G). Western blot analysis of retinal organoid lysates (G). Figure 15 After band intensity quantification, full-length EGFP expression (n=4 organoids) was confirmed to be approximately 5 times richer than the excised endonuclein.

[0910] Example 5: Intraprotein-mediated transsplicing of large proteins (effective AAV intraprotein-mediated transsplicing requires identification of the optimal ABCA4 and CEP90 cleavage sites).

[0911] To test whether protein trans-splicing can be developed into a mechanism for reconstructing large therapeutic proteins, the inventors developed AAV-ABCA4 and -CEP290 internal protein vectors.

[0912] ABCA4 and CEP290 split into two (AAV I, AAV II) or three (AAV I, AAV II, AAV III) fragments, and their coding sequences are cloned into single AAV vectors, which are then fused with the coding sequences of the N-terminus and C-terminus of the splitting endoprotein, as shown below. Figure 16 As shown. The AAV intraprotein vector includes ubiquitous short CMV [(shCMV), for all groups] or the GRK1 promoter (for group 1 of ABCA4 and group 5 of CEP290).

[0913] The splitting point of each protein was chosen considering both the amino acid residue requirements at the junction for effective protein trans-splicing (18, 51) and the maintenance of the integrity of key protein domains, which should benefit each individual polypeptide and thus the correct folding and stability of the ultimately reconstructed protein. Additional splitting intraproteins were also considered. CEP290 groups (groups 4 and 5) were generated where the protein was split into 3 polypeptides. Figure 16B) to allow the inclusion of posttranscriptional regulatory elements of marmot hepatitis virus [WPRE, (52)] to increase transgene expression. To prevent unwanted trans-splicing between AAV I and AAV III, which could reduce the amount of full-length protein produced, groups 4 and 5 include two different cleavage-type endogenous proteins at the two cleavage junctions, specifically, a DnaB endogenous protein from *Halomycinus rubrum* and a wild-type or mutant DnaE endogenous protein, which the inventors show do not cross-react ( Figure 17 ).

[0914] The inventors compared the ability of each group of AAV intraprotein granules to reconstruct ABCA4 and CEP290 after transfection into HEK293 cells. Western blot analysis of cell lysates 72 hours post-transfection showed that the expected full-length ABCA4 and CEP290 proteins (approximately 250 kDa and 290 kDa, respectively) were reconstructed by each group of AAV intraprotein granules, although with varying efficiencies. Figure 2 (AB). Groups 1 and 5 were found to be the most effective for the remodeling of ABCA4 and CEP290 proteins, respectively, and were therefore used in all subsequent experiments.

[0915] To ensure the accuracy of protein reconstruction, the inventors immunopurified ABCA4 from HEK293 cells transfected with Group 1 and performed LC-MS analysis to define its protein sequence. The proteolysis of this sample yielded 3108 peptides (22 of which included cleavage sites (Table 6)) covering the entire protein, and it was confirmed that the amino acid sequence of ABCA4 reconstructed from AAV protein particles precisely corresponds to the amino acid sequence of wild-type ABCA4. The amino acid sequence of ABCA4 reconstructed from AAV protein particles matched the amino acid sequence of wild-type ABCA4. Alignment was performed between the wild-type ABCA4 sequence and the peptides identified in the LC-MS analysis of ABCA4 reconstructed from AAV protein particles.

[0916] Table 6: Peptides including the ABCA4 cleavage point.

[0917] NB: C Cysteine ​​1150

[0918]

[0919]

[0920] The inventors then evaluated the intracellular localization of protein products from different plasmids containing the endoprotein, comparing it to the localization of the full-length protein. Full-length ABCA4 is known to be localized in the endoplasmic reticulum (ER) when expressed in cultured cell lines (53, 54). Two ABCA4 peptides from group 1 were found to co-localize in the ER, while no co-localization was observed at the transport Golgi network. Figure 2 C). Similar localization was observed in cells co-transfected with these two AAV protein plasmids and in cells transfected with a plasmid encoding the full-length ABCA4 protein, thus confirming that exogenously expressed ABCA4 is primarily localized in the ER in cell lines.

[0921] As for CEP290, the full-length protein has been reported to exhibit a mixed distribution pattern, with a predominantly punctate and a minor filamentous pattern (55). Profiling of the domains responsible for CEP290 subcellular targeting revealed that the N-terminal domain (aa 1-362) targets the protein to vesicular structures due to its membrane-interacting ability, while the region near the C-terminus of CEP290 (encompassing the myosin tail homology domain of most of the protein) mediates microtubule binding (aa 580-2479) and, when expressed in a truncated form, exhibits a prominent filamentous distribution consistent with acetylated tubulin (Ac-Tub). Consistent with Drivas et al., immunofluorescence analysis of HeLa cells transfected alone with AAV I, II, or III intraprotein plasmids or co-transfected with AAV I+II, AAV I+III, and AAV II+III showed that products from AAV I and AAV II exhibited a predominantly punctate pattern, while products from AAV III (encompassing the myosin tail homology domain) displayed a fibrous pattern and were the only products completely co-localized to the Ac-tub (Fig. 2D). Therefore, products from AAV I+II exhibited a predominantly punctate pattern, while products from AAV I+III and AAV II+III showed a combination of microtubule-filamentous and punctate patterns. Cells co-transfected with the three AAV CEP290 intraprotein plasmids showed a predominantly punctate signal, partially aligned along microtubules, comparable to the signal observed in cells transfected with plasmids encoding the full-length CEP290 protein. Figure 2 D and Figure 18 ).

[0922] The inventors then compared the protein yield obtained using the optimal AAV-ABCA4 and -CEP290 intraparticle protein groups with the protein yield obtained from a single AAV plasmid encoding the corresponding full-length protein. For this purpose, HEK293 cells were transfected with the same equimolar amounts of single or AAV intraparticle protein groups, and cell lysates were analyzed by Western blotting 72 hours post-transfection. Figure 19Quantification of band intensity indicated that the expression of ABCA4 and CEP290 from the AAV intraplasm was 61 ± 4% (n = 3 independent experiments) and 58 ± 4% (n = 3 independent experiments), respectively, as observed with the corresponding single AAV plasmids.

[0923] Example 6: AAV intracellular protein carrier mediates the expression of large therapeutic proteins in vitro and in the retina.

[0924] The inventors compared the efficiency of AAV intraprotein-mediated large protein remodeling with that of dual AAV vectors in vitro and in mouse and porcine retinas. HEK293 cells were infected with dual or intraprotein AAV2 / 2 vectors encoding ABCA4 (group 1) or CEP290 (group 5) (moi: 5 × 10^4 GC / cell for each vector), and cell lysates were analyzed by Western blotting after 72 hours. Figure 3 As shown in A and 3B, both the AAV-ABCA4 and -CEP290 intraprotein carriers mediate large protein remodeling more effectively than dual AAV carriers. As expected, in addition to full-length proteins, shorter peptides derived from a single AAV intraprotein carrier (in the case of ABCA4 and CEP290) or from trans-splicing between AAV II and AAV III (in the case of CEP290) were observed. Figure 3 (A and 3B).

[0925] Furthermore, compared to dual-carrier therapy (dose per ABCA4 carrier / eye: 3.3 x 10^9 GC, dose per CEP290 carrier / eye: 1.1 x 10^9 GC), subretinal injection of AAV-GRK1-ABCA4 or -CEP290 intraocular protein (groups 1 and 5, respectively) into 4-week-old wild-type mice was significantly improved. Animals were sacrificed 4–7 weeks post-injection, and protein expression in retinal lesions was assessed by Western blotting. In 10 / 11 (91%) eyes injected with AAV-ABCA4 intraocular protein (…),… Figure 4 Full-length protein was detected in eyes A and 20) and 5 / 10 (50%) injected with AAV-CEP290 intraocular protein (Fig. 4B). Conversely, full-length protein expression was evident in eyes 5 / 9 (56%) and 0 / 5 injected with dual AAV vectors, ABCA4 and CEP290, respectively. Similar to what was observed in vitro, peptides derived from a single AAV intraocular protein vector (in the case of both ABCA4 and CEP290) and from trans-splicing occurring between AAV II and AAV III (in the case of CEP290) were detected. Figure 4 (A and 4B).

[0926] To investigate the efficiency of protein remodeling mediated by AAV endogenous proteins relative to endogenous proteins, 1-4 month old Abca4 cells were subjected to... - / - Mice were subretinally injected with AAV-GRK1-ABCA4 intracellular protein carriers (Group 1) (dose per ABCA4 carrier / eye: 5.5 x 10^9 GC). One month later, Abca4 from unaffected and AAV-injected mice was analyzed by Western blotting using antibodies that recognize mouse and human ABCA4. - / - ABCA4 expression in mouse retinal lesions ( Figure 21 The expression of the AAV protein ABCA4 was found to be 8.6 ± 1.3% of the endogenous ABCA4.

[0927] To confirm the effective remodeling of large proteins in clinically relevant porcine retina, AAV2 / 8-GRK1-ABCA4 intracellular protein (Group 1) or a dual-carrier (dose per carrier / eye: 2 x 10^11 GC, delivered via two adjacent subretinal vesicles) was injected subretinally into Large White pigs, and protein expression was analyzed by Western blotting one month post-injection. Notably, the AAV intracellular protein was found to remodel the full-length ABCA4 protein more effectively than the dual AAV carrier. Figure 4 C).

[0928] Finally, human retinal organoids from iPSCs of healthy individuals or STGD1 patients were infected with AAV2 / 2-GRK1-ABCA4 protein carriers (Group 1) after 121 days of culture [when photoreceptor maturation began (20)] (dose per carrier / organoid: 1 x 10^12 GC). Organoids were lysed between 20 and 40 days post-infection and analyzed by Western blotting. ABCA4 of the expected size was detected in all infected organoids. Figure 4 D and Figure 22 (n=3 and n=4 were from normal controls and STGD1 organoids, respectively).

[0929] Example 7: Subretinal administration of AAV intracellular protein carrier improves retinal phenotype in STGD1 and LCA10 mouse models

[0930] To determine whether photoreceptor transduction obtained using AAV intracellular protein carriers is therapeutically relevant, STGD1(Abca4) was used. - / - The assay was tested in the retina of LCA10 (rd16) mouse models.

[0931] To 1-month-old Abca4 - / -Mice were subretinally injected with AAV2 / 8-GRK1-ABCA4 protein carriers (Group 1) (dose per carrier / eye: 4.3–4.8 x 10^9 GC). Eyes were harvested three months later, and ultrathin retinal sections were analyzed by transmission electron microscopy to measure the amount of lipofuscin, which accumulates in Abca4. - / - In the retinal pigment epithelium (RPE) of mice (56, 57). Notably, lipofuscin in the RPE accumulates in Abca4, a protein carrier injected with AAV. - / - Significant reduction was observed in the eyes, but no reduction was observed in the eyes injected with the negative control (p = 0.0163). Figure 5 A and Figure 23 ).

[0932] In parallel, AAV2 / 8-GRK1-CEP290 intraretinal protein carriers (group 5) were injected subretinally into 4–6 day old rd16 mice (dose per carrier / eye: 5.5 x 10^8 GC). Microscopic analysis of retinal sections performed 1 month after injection showed that, as a result of progressive retinal degeneration (55), the thickness of the outer nuclear layer (ONL), including the photoreceptor nucleus, was significantly reduced in rd16 mice compared with wild-type mice (p = 0.00048). Figure 5 B). Notably, the ONL thickness of the rd16 retina injected with the AAV intracellular protein carrier was significantly higher (approximately 60%, p = 0.00281) than that of the rd16 retina injected with the negative control. Figure 5 B). Therefore, retinal function tests based on pupillary light response (PLR) showed that rd16 mice injected with the AAV protein carrier exhibited significantly higher pupillary constriction than rd16 mice injected with the negative control (approximately 20%, p = 0.00073). Figure 5 C).

[0933] Furthermore, the inventors investigated the safety of the AAV intracellular protein carriers in the retina. For this purpose, AAV2 / 8-GRK1-ABCA4 or -CEP290 intracellular protein carriers (groups 1 and 5, respectively) were injected subretinally into wild-type C57BL / 6J mice (dose per ABCA4 carrier: 4.3 x 10^9 GC; dose per CEP290 carrier / eye: 1.1 x 10^9 GC), and retinal electrical activity was measured by Ganzfeld electroretinography (ERG) at 6 and 4.5 months post-injection, respectively. In both studies, the a-wavelength and b-wavelength amplitudes were similar between the eyes of mice injected with the AAV intracellular protein carriers (n=14-15 for ABCA4 and n=11 for CEP290) and the eyes injected with negative control AAV carriers (n=8 for ABCA4 and n=5 for CEP290) or PBS (n=6-7 for ABCA4 and n=6 for CEP290). Similarly, ONL thickness, measured by optical coherence tomography, was similar in eyes injected with AAV intracellular protein, negative control, and PBS. Figure 24 ).

[0934] Example 8: Safe AAV Intraprotein-Mediated Large Gene Delivery

[0935] Although no obvious signs of toxicity were observed in wild-type mice injected with AAV endonuclease, the inventors have evaluated the inclusion of a degradation determinant in the trans-splicing system, which, once embedded in the excised endonuclease, leads to rapid ubiquitination of the fusion protein, subsequently triggering proteasome disruption. Figure 6 Most of the degradation determinants described are functional at the N-terminal or C-terminal positions (i.e., CL1, SMN, CIITA, ODC), and these degradation determinants cannot fuse with N-terminal or C-terminal intramolecular proteins because they would lead to the degradation of a single host protein, thus eliminating polypeptides that need to participate in protein trans-splicing (PTS) reactions. Therefore, the inventors selected mutant forms of dihydrofolate reductase (ecDHFR) from Escherichia coli, which include three amino acid mutations that confer functional activity only at the N-terminal or intramolecular positions: R12Y, Y100I, and G67S (69).

[0936] To test the efficiency of ecDHFR in reducing the amount of excised endonuclease, the inventors generated an AAV vector encoding the N-terminal half of EGFP fused with the N-terminal half of Npu DnaE and ecDHFR (pAAV2.1-CMV-5'EGFP endonuclease_ecDHFR). Therefore, the degradation determinant would be located at its C-terminus, where it should be inactive. HEK293 cells were transfected using a combination of the AAV-EGFP-ecDHFR endonuclease and vector II (encoding the C-terminal half of EGFP fused with the C-terminal half of Npu DnaE (pAAV2.1-CMV-3'EGFP endonuclease)) and the production of full-length EGFP protein and excised endonuclease were evaluated. Trans-spliced ​​EGFP protein, with similar protein levels to AAV endonuclease, was detected by Western blotting analysis. Furthermore, after co-transfection with the AAV-EGFP-ecDHFR endonuclease, the amount of excised endonuclease in HEK293 cell lysates was significantly reduced. Figure 7 Then, the inventors decided to apply the same strategy to the large ABCA4 protein (pAAV2.1-CMV260-5' ABCA4 inner protein_ecDHFR). With regard to EGFP, the inventors found that the amount of similarity of the full-length ABCA4 from the AAV-ABCA4-ecDHFR inner protein plasmid compared to the AAV-ABCA4-inner protein was (…). Figure 8 A). Importantly, complete elimination of the excised endonucleoprotein was observed (Figure 8B).

[0937] To demonstrate the inventors' observation of ecDHFR-mediated DnaE degradation, cells were treated with trimethoprim (TMP). TMP is an antibiotic that binds to ecDHFR, thereby preventing protein degradation and allowing the fusion protein to escape degradation (69). HEK293 cells co-transfected with AAV-ABCA4-ecDHFR protein particles were treated with increased doses of TMP, and it was found that the DnaE protein no longer degraded, and TMP stabilized ecDHFR in a dose-dependent manner, implying that the reduction in DnaE protein was mediated by ecDHFR. Figure 9 ).

[0938] One limitation of including degradation determinants in vectors (beyond the endoprotein) is a further reduction in AAV cloning ability, resulting in AAV vectors being too large for some applications. In fact, the length of ecDHFR is 159 aa. Therefore, the inventors designed a shorter 105 aa ecDHFR variant that retains amino acids allegedly crucial for its activity at the N-terminus or internal position. The inventors tested this small ecDHFR in both EGFP and ABCA4 endoproteins (pAAV2.1-CMV-5' EGFP endoprotein_small ecDHFR; pAAV2.1-CMV260-5' ABCA4 endoprotein_small cDHFR). After co-transfection of AAV-EGFP- or ABCA4-small ecDHFR endoproteins, they found similar full-length protein expression compared to AAV endoproteins. Figure 10 and 11 A) and a significant reduction in DnaE proteins ( Figure 10 and 11 B).

[0939] These results indicate that the PTS system contains ecDHFR or small ecDHFR to mediate selective intraprotein degradation without significantly affecting the efficacy of protein transsplicing and therapeutic protein production.

[0940] Example 9: Intraprotein-mediated protein transsplicing in the liver

[0941] To test the efficiency of endoprotein-mediated protein trans-splicing in the liver, two AAV vectors were generated, each encoding the N-terminal or C-terminal half of a reporter protein EGFP protein fused to the N-terminal and C-terminal halves of the DnaE fission-type endoprotein from Candida punctata.

[0942] Five-week-old C57 / BL6 mice were injected retroorbitally with AAV2 / 8 vectors carrying the liver-specific human thyroxine-binding globulin (TBG) promoter (dose per vector / kg: 5 × 10⁻⁶). 11 (GC). Liver samples were harvested and lysed 4 weeks after injection for analysis using anti-3xflag antibody via Western blotting to detect EGFP-3xflag and endonucleoprotein-3xflag. Quantification of EGFP band intensity indicated that AAV endonucleoprotein transduced the liver more efficiently than dual AAV, with a protein content approximately 6-7 times higher.

[0943] Example 10: AAV intraprotein vector can be used to deliver the affected large F8 gene in hemophilia A.

[0944] The F8 gene, mutated in hemophilia A, is too large (approximately 7 kb) to be delivered by a single AAV in its wild-type conformation. For this reason, the B-domain deletion (BDD) conformation of the gene alone is suitable for AAV gene therapy. Recently, a 5 kb expression cassette containing BDD-F8, a short liver-specific promoter, and a polyA signal has been packaged into AAV5 and shown to produce therapeutic levels of FVIII (71) in mice and cynomolgus monkeys (70) as well as in HemA patients. However, the genome of this vector is slightly too large, and when packaged as a heterogeneous truncated genome library into the AAV capsid, it induces efficient transduction after remodeling in target cells. The ultra-large AAV vector is less efficient compared to normal-sized vectors, and the quality of the product with the heterogeneous truncated genome may hinder its further commercial development.

[0945] To overcome the limited cargo capacity of AAVs, a protein trans-splicing strategy was designed, which involves two independent AAV vectors with regular-sized genomes, each encoding one of the two halves of a large FVIII protein flanked by a split Npu DnaE protein.

[0946] The wild-type F8 gene splits into two distinct sites within its B domain: group 1 and group 2. This results in a liver-specific heterozygous liver promoter (HLP) and a short synthetic polyA-based F8 intraprotein vector (…). Figure 25 A). As shown by Southern blotting, the vector genome, unlike its ultra-large AAV BDD-F8 control, was appropriately packaged into the AAV capsid (A). Figure 25 B).

[0947] To determine the therapeutic relevance of this strategy, AAV2 / 8 F8 intraorbital protein carriers were administered via retroorbital infusion (dose per carrier / animal: 4-5 x 10⁻⁶). 11 GC was administered systemically to 7-8 week old hemophilia A knockout mice. Plasma aPTT (activated partial thromboplastin kinase time) analysis at 8 weeks post-injection showed slight correction for the bleeding phenotype, although not at the same level as the ultra-large single AAV BDD-F8 control. Figure 25 C).

[0948] References

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Claims

1. A vector system for expressing a coding sequence in cells, said coding sequence comprising a first part (CDS1), a second part (CDS2), and optionally a third part (CDS3), said vector system comprising: a) A first carrier, the first carrier comprising: -The first portion (CDS1) of the encoded sequence, - The first nucleotide sequence encoding the N-endoprotein, said sequence being located at the 3' end of CDS1; and b) A second carrier, the second carrier comprising: -The second part (CDS2) of the encoded sequence, - A second endonuclein nucleotide sequence encoding C-endon, said sequence being located at the 5' end of CDS2; When the first and second vectors are inserted into the cell, the protein product encoding the sequence is generated by protein splicing. Or the carrier system may include: a') A first carrier, the first carrier comprising: -The first portion (CDS1) of the encoded sequence, - The first intraprotein nucleotide sequence encoding the first N-endoprotein, said sequence being located at the 3' end of CDS1; and b') A second carrier, the second carrier comprising: -The second part (CDS2) of the encoded sequence, - A second endonuclein nucleotide sequence encoding the first C-endon, said sequence being located at the 5' end of CDS2; - A third endonuclein nucleotide sequence encoding the second N-endon, said sequence being located at the 3' end of CDS2; and c') A third carrier, the third carrier comprising: - The third part (CDS3) of the encoded sequence - The fourth endonuclein nucleotide sequence encoding the second C-endon, said sequence being located at the 5' end of CDS3. The first endonucleoprotein nucleotide sequence is different from the third endonucleoprotein nucleotide sequence, and the second endonucleoprotein sequence is different from the fourth endonucleoprotein nucleotide sequence. When the first vector, the second vector, and the third vector are inserted into the cell, the protein product encoding the sequence is generated by protein splicing.

2. The vector system according to claim 1, wherein the first endonucleoprotein, the second endonucleoprotein, the third endonucleoprotein, and the fourth endonucleoprotein encode a splitting endonucleoprotein, preferably, the maximum length of the splitting endonucleoprotein is 150 amino acids, more preferably, the splitting endonucleoprotein is a DnaE or DnaB endonucleoprotein.

3. The carrier system according to claim 1 or 2, wherein - The first endonucleoprotein nucleotide sequence encodes an endonucleoprotein selected from the group consisting of: SEQ ID No. 1, 3, 5, 7, 9, 11, 13 or variants thereof, fragments thereof or homologues thereof; - The second endonucleoprotein nucleotide sequence encodes an endonucleoprotein selected from the group consisting of: SEQ ID No. 2, 4, 6, 8, 10, 12, 14 or variants thereof, fragments thereof or homologues thereof; - The third endonuclein nucleotide sequence encodes an endonuclein selected from the group consisting of: SEQ ID No. 1, 3, 5, 7, 9, 11, 13 or variants thereof, fragments thereof or homologues thereof; - The fourth endonucleoprotein nucleotide sequence encodes an endonucleoprotein selected from the group consisting of: SEQ ID No. 2, 4, 6, 8, 10, 12, 14 or variants thereof, fragments thereof or homologues thereof.

4. The carrier system according to any of the preceding claims, wherein the first carrier, the second carrier, and the third carrier further include a promoter sequence operatively connected to the 5' end portion of the first portion (CDS1) of the coding sequence, the second portion (CDS2) of the coding sequence, or the third portion (CDS3) of the coding sequence.

5. The vector system according to any of the preceding claims, wherein the first vector, the second vector, and the third vector further comprise a 5'-terminal repeat (5'-TR) nucleotide sequence and a 3'-terminal repeat (3'-TR) nucleotide sequence, preferably the 5'-TR is a 5'-inverted terminal repeat (5'-ITR) nucleotide sequence, and the 3'-TR is a 3'-inverted terminal repeat (3'-ITR) nucleotide sequence.

6. The vector system according to any of the preceding claims, wherein the first vector, the second vector, and the third vector further comprise a polyadenylation signal nucleotide sequence, and / or wherein at least one of the first vector, the second vector, or the third vector further comprises a nucleotide sequence encoding a degradation signal.

7. The carrier system according to claim 6, wherein the degradation signal is selected from the group consisting of CL1, PB29, SMN, CIITA, ODC, ecDHFR or fragments thereof.

8. The vector system according to any of the preceding claims, wherein the coding sequence is split into a first portion, a second portion and optionally a third portion at positions consisting of nucleophilic amino acids that do not fall into the structural or functional domains of the encoded protein product, wherein the nucleophilic amino acids are selected from serine, threonine or cysteine.

9. The vector system according to any of the preceding claims, wherein at least one of the first vector, the second vector, and the third vector further comprises at least one enhancer or regulatory nucleotide sequence operatively linked to the coding sequence.

10. The carrier system according to any of the preceding claims, wherein the coding sequence encodes a protein capable of correcting a pathological state or symptom, preferably, the symptom being retinal degeneration, metabolic disease, hematologic disease, neurodegenerative disease, hearing loss, channel disease, lung disease, myopathy, or heart disease.

11. The vector system according to any of the preceding claims, wherein the coding sequence encodes a protein capable of correcting a pathological state or symptom, preferably, the symptom is retinal degeneration, preferably, the retinal degeneration is hereditary, preferably, the pathology or disease is selected from the group consisting of: retinitis pigmentosa (RP), Lieber's congenital amaurosis (LCA), Stargardt disease (STGD), Usher syndrome (USH), Alstrom syndrome, congenital stationary night blindness (CSNB), macular dystrophy, latent macular dystrophy, and diseases caused by mutations in the ABCA4 gene.

12. The vector system according to any one of claims 1 to 10, wherein the coding sequence encodes a protein capable of correcting Duchenne muscular dystrophy, cystic fibrosis, hemophilia A, Wilson's disease, phenylketonuria, dysferlinopathies, Rett's syndrome, polycystic kidney disease, Niemann-Pick type C, and Huntington's disease.

13. The vector system according to any one of claims 1 to 11, wherein the coding sequence is the coding sequence of a gene selected from the group consisting of: ABCA4, MYO7A, CEP290, CDH23, EYS, PCDH15, CACNA1, SNRNP200, RP1, PRPF8, RP1L1, ALMS1, USH2A, GPR98, HMCN1.

14. The vector system according to any one of claims 1 to 12, wherein the coding sequence is the coding sequence of a gene selected from the group consisting of: DMD, CFTR, F8, ATP7B, PAH, DYSF, MECP2, PKD, NPC1HTT.

15. The carrier system according to any one of the preceding claims, comprising: a) A first carrier, the first carrier comprising, in the 5'-3' direction: -5'-inverted terminal repeat (5'-ITR) sequence; -Initiation subsequence; - The 5' end portion (CDS1) of the encoded sequence, which is operatively connected to and under the control of the promoter; -The first endonuclein nucleotide sequence encoding the N-endoprotein; and -3'-inverted terminal repeat (3'-ITR) sequence; and b) A second carrier, the second carrier comprising, in the 5'-3' direction: -5'-inverted terminal repeat (5'-ITR) sequence; -Initiation subsequence; -The second endonuclein nucleotide sequence encoding C-endoprotein; - The 3' end portion (CDS2) of the encoded sequence; and -3'-inverted terminal repeat (3'-ITR) sequence; Or include: a') A first carrier, the first carrier comprising, in the 5'-3' direction: -5'-inverted terminal repeat (5'-ITR) sequence; -Initiation subsequence; - The 5' end portion of the encoded sequence (CDS1'), said 5' end portion being operatively connected to and under the control of the promoter; -The first endonuclein nucleotide sequence encoding the first N-endonuclein; and -3'-inverted terminal repeat (3'-ITR) sequence; and b') A second carrier, the second carrier comprising, in the 5'-3' direction: -5'-inverted terminal repeat (5'-ITR) sequence; -Initiation subsequence; -The nucleotide sequence of the second endonuclein encoding the first C-endonuclein; - The second part (CDS2') of the encoded sequence; and -The nucleotide sequence of the third endonuclein encoding the second N-endonuclein; -3'-inverted terminal repeat (3'-ITR) sequence; and c') A third carrier, the third carrier comprising, in the 5'-3' direction: -5'-inverted terminal repeat (5'-ITR) sequence; -Initiation subsequence; - The nucleotide sequence of the fourth inner protein encoding the second C-inner protein; - The third part (CDS3') of the encoded sequence; and -3'-inverted terminal repeat (3'-ITR) sequence.

16. The vector system according to any of the preceding claims, wherein the coding sequence encodes the ABCA4 gene, preferably, the coding sequence splits at nucleotides corresponding to aa Cys1150, Ser1168, and Ser1090 of the ABCA4 protein, and a splitting endoprotein is inserted at the splitting point; or the coding sequence encodes the CEP290 gene, preferably, the coding sequence splits at nucleotides corresponding to aa Cys1076 and Ser1275 of the CEP290 protein, preferably, the coding sequence encoding the CEP290 gene splits at nucleotide sequences corresponding to aaCys 929 and 1474, Ser 453, and Cys 1474 of the CEP290 protein, and two splitting endoproteins are inserted at the splitting point.

17. The vector system according to any of the preceding claims, wherein the first vector, the second vector, and the third vector are independently viral vectors, preferably adenovirus vectors or adeno-associated virus (AAV) vectors, preferably, the first, second, and third AAV vectors are selected from the same or different AAV serotypes, preferably the serotypes are selected from serotype 2, serotype 8, serotype 5, serotype 7 or serotype 9, serotype 7m8, serotype sh10; serotype 2 (quad YF).

18. A host cell, said host cell being transformed with a vector system according to any of the preceding claims.

19. The carrier system according to any one of claims 1 to 17 or the host cell according to claim 18, for medical applications.

20. The vector system according to any one of claims 1 to 19 or the host cell according to claim 18, for use in gene therapy, preferably for the treatment and / or prevention of pathologies or diseases characterized by retinal degeneration, metabolic disorders, hematologic disorders, neurodegenerative diseases, hearing loss, channel diseases, lung diseases, myopathy, or heart diseases.

21. The vector system or host cell used according to claim 20, wherein the retinal degeneration is hereditary, preferably, the pathology or disease is selected from the group consisting of: retinitis pigmentosa (RP), Lieber congenital amaurosis (LCA), Staggart disease (STGD), Usher syndrome (USH), Alstrém syndrome, congenital stationary night blindness (CSNB), macular dystrophy, latent macular dystrophy, and diseases caused by mutations in the ABCA4 gene.

22. The vector system or host cell used according to claim 20, for the prevention and / or treatment of Dishene muscular dystrophy, cystic fibrosis, hemophilia A, Wilson's disease, phenylketonuria, dysferlin lesions, Rett syndrome, polycystic kidney disease, Niemann-Pick type C disease, and Huntington's disease.

23. A pharmaceutical composition comprising a carrier system according to any one of claims 1 to 17 or a host cell according to claim 18, and a pharmaceutically acceptable carrier.

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