Intracellular protein and its use

Abstract

The present invention relates to constructs, vectors, related host cells and pharmaceutical compositions that allow for efficient gene therapy, particularly of genes larger than 5 Kb.

Description

Technical Field

[0001] The present invention relates to constructs, vectors, related host cells and pharmaceutical compositions that allow for effective gene therapy, in particular for diseases caused by mutations in genes with coding sequences (CDS) greater than 5 Kb. Background Art

[0002] 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 inherited metabolic and blinding diseases, and clinical trials of AAV-based gene therapy approaches for diseases in different therapeutic areas are also increasing. The diseases range from ophthalmology to blood diseases to musculoskeletal and metabolic diseases.

[0003] However, limitations in AAV vector cargo capacity have prevented the development of AAV-based therapies for diseases caused by mutations in genes with coding sequences (CDS) larger than 5 kb, also referred to herein as large genes.

[0004] Genetic diseases caused by large gene mutations (listed in Table 1 below) include Duchenne muscular dystrophy caused by DMD gene mutations, cystic fibrosis caused by CFTR gene mutations, hemophilia A caused by F8 gene mutations, dysferlinopathies caused by DYSF gene mutations, polycystic kidney disease caused by PKD gene mutations, Wilson disease caused by ATP7B gene mutations, Huntington's disease caused by HTT gene mutations, and Niemann-Pick disease type C caused by NPC1 gene mutations.

[0005] Table 1: Genetic diseases caused by large gene mutations

[0006]

[0007]

[0008] In addition, several inherited retinal degenerations (IRDs) are due to mutations in the large genes listed in Table 2 below. In Europe and the United States, IRDs affect approximately 1 in 3000 people (58).

[0009] The most common and severe IRDs are retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and Stargardt disease (STGD), which are usually inherited as monogenic conditions with an overall global prevalence of 1 in 2,000 (1) and are the leading causes of blindness worldwide. Most mutations that cause IRDs occur in genes expressed in the neuronal photoreceptors (PRs), rods, and / or cones, in the retina (2).

[0010] Gene therapy holds great promise for the treatment of IRDs. The first adeno-associated virus (AAV)-based gene therapy product for an inherited form of blindness was approved in December 2017 (3). In addition, numerous 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 retinal gene therapy vector to date (4, 5), its limited cargo capacity hinders its use for the treatment of conditions requiring the delivery of DNA sequences larger than 5 kb (6), which include not only the transgene but also the cis-regulatory elements necessary for its expression.

[0011] Examples of disease genes larger than 5 kb are summarized in Table 2 below.

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

[0013]

[0014]

[0015] Stargardt disease (STGD; MIM#248200) is the most common form of inherited macular degeneration caused by mutations in the ABCA4 gene (CDS: 6822 bp), which encodes the all-trans retinal transporter located in the PR outer segments (7); Usher syndrome type IB (USH1B; MIM#276900) is the most severe form of RP and deafness caused by mutations in the MYO7A gene (CDS: 6648 bp), which encodes an unconventional MYO7A (8), an actin-based motor expressed in both the PR and RPE within the retina (9-11).

[0016] Cone-rod dystrophy type 3, fundus flavimaculatus, age-related macular degeneration type 2, early-onset severe retinal dystrophy, and retinitis pigmentosa type 19 are also associated with ABCA4 mutations (referred to herein as ABCA4-related diseases).

[0017] The present inventors and others have shown 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 exploit concatemerization and recombination of the AAV genome to reconstitute the full-length genome in cells co-infected with multiple AAV vectors. However, for photoreceptors, which are the primary therapeutic target for most inherited retinal diseases, the efficiency of transgene expression 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 concatemer formation, stability of heterogeneous mRNA, and splicing efficiency across vector junctions.

[0018] The present inventors showed in WO2014 / 170480 and Colella et al. (15) that dual AAV vectors can reconstruct large genes by splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybridization), and found that dual trans-splicing and hybrid vectors are particularly effective for treating inherited retinal degenerations. In addition, Maddalena et al. (14) demonstrated a triple AAV vector approach for genes up to 14 kb. However, the efficiency of transgene expression achieved using dual or triple AAV vectors is lower than that achieved using single AAV vectors (6, 13, 14). This may be due to various limiting steps required for efficient transduction, including: correct DNA concatemer formation, stability of heterogeneous mRNA, and splicing efficiency across vector junctions. In addition, the triple AAV vector strategy produces gene expression levels below the threshold required for therapeutic approaches.

[0019] Thus, there remains a need for constructs and vectors that can be used to reconstitute the expression of large genes for effective gene therapy.

[0020] The inventors have now discovered that delivering multiple AAV vectors, each encoding a reporter or one of the fragments of a large therapeutic protein, flanked by short split inteins, can achieve protein trans-splicing and full-length protein reconstitution both in vitro and in vivo.

[0021] Inteins are genetic elements that are transcribed and translated within host proteins and that self-excise in the absence of energy, exogenous host-specific proteases, or cofactors in a manner similar to inteins, without leaving amino acid modifications in the final protein product (16, 17, 27, 28). The activity of inteins is context-dependent, with certain peptide sequences (called N- and C-exteins) surrounding their junctions being necessary for efficient trans-splicing to occur, the most important of which are sulfhydryl- or hydroxyl-containing amino acids (e.g., cysteine, serine, or threonine) as the first residue in the C-extein (18). Split inteins are a subset of inteins that are expressed as two separate polypeptides at the termini of two host proteins and catalyze their trans-splicing to produce a single larger polypeptide (19). Introns, including split inteins, are widely used in biotechnology applications, including protein purification and labeling procedures (19, 20), and in reconstitution of the widely used CRISPR / Cas9 genome editing nuclease (21, 22).

[0022] Several attempts have been made to utilize intein-based protein splicing to reconstitute the expression of therapeutic genes, including the factor VIII gene, where it was demonstrated that fusion of the heavy and light chain genes of the factor VIII Synechocystis sp. (Ssp) DnaB intein could induce factor VIII reconstitution in cell culture and in animal models (23, 24). Similarly, a highly functional form of the dystrophin gene was expressed in vitro and in vivo by splitting the 6.3-kb Becker-type dystrophin gene onto two AAV vectors and fusing each half to split inteins obtained from either the Synechocystis sp. PCC 6803 (Ssp) DnaB intein or the Rhodothermus marinus (Rma) DnaB intein (25). Furthermore, a protein trans-splicing strategy mediated by a split intein (i.e., the N. punctiforme DnaE split intein) was reported to reconstitute the large pore-forming subunit of the L-type calcium channel from two separate fragments in cardiac cells (26). US 6,544,786 further reports the use of split-intrin to deliver the dystrophin minigene.

[0023] The present inventors exploited the intrinsic ability of split-intrin to mediate protein trans-splicing to fragment a large full-length protein into two or three split-intrin-flanked polypeptides, and then reconstitute the large full-length protein, the coding sequence of which is suitable for a single AAV vector.

[0024] Therefore, the present invention achieves reconstitution of cellular large proteins by providing target cells with two or more fragments of the large protein, which are fused to split-type intein to promote intein-mediated trans-splicing and reconstitute functional proteins. Summary of the Invention

[0025] The present invention provides gene therapy using AAV vectors for diseases caused by gene mutations, particularly mutations in genes with coding regions exceeding 5 kb.

[0026] Based on the discovery that unicellular organisms use split-intrin-mediated protein trans-splicing for protein remodeling, the inventors constructed multiple AAV vectors, each encoding one of the reporter or large therapeutic protein fragments flanked by short split-intrins, thereby triggering protein trans-splicing and full-length protein remodeling in vivo and in vitro.

[0027] Advantageously, the AAV-based protein trans-splicing-mediated remodeling of disease proteins achieved by the present invention provides higher target protein expression levels compared to AAV-based large protein methods known in the art. This is likely due to overcoming various limiting steps required for efficient transduction of dual-vector-based systems, including: formation of correct DNA concatemers, stability of heterologous mRNAs, and splicing efficiency across vector junctions.

[0028] The present invention provides a vector system for expressing a coding sequence in a cell, wherein the coding sequence consists of a first part (CDS1), a second part (CDS2) and an optional third part (CDS3), and the vector system comprises:

[0029] a) a first carrier, the first carrier comprising:

[0030] - said first part of said coding sequence (CDS1),

[0031] - a first intein nucleotide sequence encoding an N-intein, said sequence being located at the 3′ end of CDS1; and

[0032] b) a second carrier, the second carrier comprising:

[0033] - said second part of the coding sequence (CDS2),

[0034] - a second intein nucleotide sequence encoding C-intein, said sequence being located at the 5' end of CDS2;

[0035] wherein when said first vector and said second vector are inserted into a cell, said protein product of said coding sequence is produced by protein splicing;

[0036] Alternatively, the vector system comprises:

[0037] a′) a first carrier, the first carrier comprising:

[0038] - said first part of said coding sequence (CDS1),

[0039] - a first intein nucleotide sequence encoding a first N-intein, said sequence being located at the 3' end of CDS1; and

[0040] b′) a second carrier, the second carrier comprising:

[0041] - said second part of the coding sequence (CDS2),

[0042] - a second intein nucleotide sequence encoding the first C-intein, said sequence being located at the 5' end of CDS2;

[0043] - a third intein nucleotide sequence encoding a second N-intein, said sequence being located at the 3' end of CDS2; and

[0044] c′) a third carrier, the third carrier comprising:

[0045] - the third part of the coding sequence (CDS3)

[0046] - a fourth intein nucleotide sequence encoding a second C-intein, said sequence being located at the 5' end of CDS3

[0047] wherein the first intein nucleotide sequence is different from the third intein nucleotide sequence, and the second intein sequence is different from the fourth intein nucleotide sequence, wherein when the first vector, the second vector, and the third vector are inserted into a cell, the protein product of the coding sequence is produced by protein trans-splicing.

[0048] Preferably, in the vector system, the first intein, the second intein, the third intein and the fourth intein encode split-type inteins, preferably, the maximum length of the split-type intein is 150 amino acids, more preferably, the split-type intein is DnaE or DnaB intein.

[0049] According to the present invention, an intein is a fragment of a protein that is able to cut off itself and join the remaining parts (exteins) with peptide bonds in a process called protein splicing. The fragments are called "intrins" and represent the internal protein sequence, and "exteins" represent the external protein sequence, wherein the upstream extein is called "N-extein" and the downstream extein is called "C-extein", and the upstream intein is called "N-intrin" and the downstream intein is called "C-intrin".

[0050] Thus, in the context of the present invention, an N-intrin is an intein fragment located at the N-terminus of a first polypeptide (and fused to the first polypeptide), and a C-intrin is an intein fragment located at the C-terminus of a second polypeptide (and fused to the second polypeptide), wherein following expression of the two polypeptides, the two intein fragments undergo protein trans-splicing and are ligated to form the complete intein, and the two polypeptides are ligated together, wherein the full-length protein is reconstituted when the two polypeptides form the full-length protein.

[0051] According to the present invention, the first intein sequence is an N-intein sequence and the second intein sequence is a C-intein sequence, wherein the N-intein and the C-intein are preferably derived from the same intein or split-type intein gene. Alternatively, the N-intein and the C-intein are derived from two different intein genes, which are capable of naturally undergoing a trans-splicing reaction or are modified to undergo the trans-splicing reaction. Accordingly, the same gene can come from the same organism or different organisms. For example, the widely used split-type intein is derived 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-intein coding sequence is fused in frame with the sequence encoding the N-terminal portion of the target protein; the C-intein coding sequence is fused in frame with the sequence encoding the C-terminal portion of the target sequence. After expressing the two precursor fusion proteins, the intein undergoes autocatalytic excision and forms a connected exogenous protein, such as a reconstructed target protein.

[0052] According to the present invention, the coding sequence of target protein can be split into three parts. Accordingly, the first protein sequence is an N-protein sequence and the second protein sequence is a C-protein sequence, wherein the first protein coding sequence is fused in frame with the sequence of the N-part of the coding target protein at the C-terminus, and the second protein coding sequence is fused in frame with the N-terminus of the sequence of the coding target protein mid-part. Accordingly, the N-protein and the C-protein are preferably derived from identical protein or split-type protein genes. Alternatively, the N-protein and the C-protein are derived from two different protein genes, and the protein genes can naturally carry out trans-splicing reaction, or are modified to carry out the trans-splicing reaction. Accordingly, the same gene can be from the same organism or different organisms. In the configuration of the present invention, the third protein is an N-protein coding sequence fused in frame with the sequence of the C-terminus of the coding target protein mid-part, and the fourth protein is a C-protein coding sequence fused in frame with the sequence of the N-terminus of the C part of the coding target protein. Accordingly, the third and fourth intein are preferably derived from the same intein or split-type intein gene. Alternatively, the N-intein and the C-intein are derived from two different intein genes, which are naturally capable of undergoing a trans-splicing reaction or are modified to undergo the trans-splicing reaction. Accordingly, the same gene may be from the same organism or from different organisms. Within the scope of the present invention, the first and second intein and the third and fourth intein are derived from different intein genes, and the first intein selectively binds to the second intein, while the third intein selectively binds to the fourth intein.

[0053] In the present invention, when the first vector, the second vector, and the optional third vector are inserted into a cell, at least two fusion proteins or three fusion proteins are formed, and when the two or three fusion proteins are contacted, the protein product of the coding sequence is produced. The contacting step is carried out under conditions that allow binding of the N-intrin to the C-intrin.

[0054] In the present invention, when the first, second, and third vectors are inserted into cells, three independent polypeptides are produced, and full-length proteins are generated through trans-splicing. The key to the formation of the three AAV intraprotein vectors is the use of different intraproteins, namely DnaE and DnaB, which do not cross-react, thereby preventing inappropriate trans-splicing between the polypeptides produced by the first and third vectors.

[0055] According to a preferred embodiment of the present invention, the vector system for expressing the coding sequence of the target gene in a cell comprises two vectors, each vector comprising a portion of the coding sequence flanked by an intein sequence, wherein the 5′ end of the coding sequence is flanked by an N-intein sequence at the 3′ end, and the 3′ end of the coding sequence of the target gene is flanked by a C-intein sequence, such that when the two vectors are expressed in a cell, two fusion proteins are produced and the full-length protein of interest is generated due to a spontaneous trans-splicing reaction.

[0056] According to another preferred embodiment of the present invention, the vector system for expressing the coding sequence of the target gene in the cell comprises three vectors, each vector comprising a portion of the coding sequence flanked by an intein sequence, wherein the coding sequence is divided into three parts, such that the 5′ end of the coding sequence is flanked by the sequence of the first N-intein at the 3′ end; the middle portion of the coding sequence is flanked by the first C-intein at the 5′ end and by the second N-intein at the 3′ end; the 3′ portion of the coding sequence is flanked by the second C-intein at the 5′ end, such that when all three vectors are expressed in the cell, three fusion proteins are produced and the target full-length protein is generated due to spontaneous trans-splicing reactions, wherein the first N-intein reacts with the first C-intein and the second N-intein reacts with the second C-intein.

[0057] The split inteins of the present invention can be encoded by a single gene that is then engineered to encode two separate intein fragments, such as a split intein; alternatively, naturally occurring split inteins 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 inteins of the present invention are inteins derived from intein proteins (e.g., small inteins) or split inteins formed by trans-splicing reactions, and are 150 aa or less in length.

[0058] The split-type inteins of the present invention may be 100%, 98%, 80%, 75%, 70%, 65%, 60%, 55%, 50% identical to the naturally occurring inteins or SEQ ID Nos. 1 to 14 (homologs), wherein the inteins retain the ability to undergo trans-splicing reactions. Fragments or variants of naturally occurring or modified inteins that retain trans-splicing activity are within the scope of the present invention.

[0059] Conveniently, the split inteins of the present invention may be derived from the same gene isolated from different organisms.Preferred intein genes are Dna B and DnaE.

[0060] In a preferred embodiment, the protein of the present invention is a split protein derived from the DnaE gene (e.g., DNA polymerase III subunit α) from 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, Trichodesmium erythraeum); preferably, the protein of the present invention is derived from the Dna E gene isolated from Nostoc punctata or Synechocystis PCC6803.

[0061] In another preferred embodiment, the intein of the present invention is a split-type intein derived from the DnaB gene from cyanobacteria, including, for example, R. marinus (Rma), Synechocystis sp. PC6803 (Ssp), and Porphyra purpurea chloroplast (Ppu) as described in (59).

[0062] Preferably,

[0063] - the first intein nucleotide sequence encodes an intein selected from the group consisting of SEQ.ID.No.1, 3, 5, 7, 9, 11, 13 or variants thereof, or fragments thereof or homologs thereof;

[0064] - the second intein nucleotide sequence encodes an intein selected from the group consisting of SEQ.ID.No. 2, 4, 6, 8, 10, 12, 14 or variants thereof, or fragments thereof or homologs thereof;

[0065] - the third intein nucleotide sequence encodes an intein selected from the group consisting of SEQ.ID.No.1, 3, 5, 7, 9, 11, 13 or a variant thereof, or a fragment thereof or a homolog thereof;

[0066] - the fourth intein nucleotide sequence encodes an intein selected from the group consisting of SEQ.ID.No. 2, 4, 6, 8, 10, 12, 14 or a variant thereof, or a fragment thereof or a homolog thereof;

[0067] Preferably, when the first protein or the third protein is SEQ.ID.No.1, the second protein or the fourth protein is SEQ.ID 2; or when the first protein or the third protein is SEQ ID 3, the second protein or the fourth protein is SEQ ID 4; or when the first protein or the third protein is SEQ ID5, the second protein or the fourth protein is SEQ ID6; or when the first protein or the third protein is SEQ ID 7, the second protein or the fourth protein is SEQ ID 8; or when the first protein or the third protein is SEQ ID 9, the second protein or the fourth protein is SEQ ID 10; or when the first protein or the third protein is SEQ ID11, the second protein or the fourth protein is SEQ ID 12.

[0068] Preferably, when the first protein is SEQ ID 1 and the second protein is SEQ ID 2, the third protein is not SEQ ID 1 and the fourth protein is not SEQ ID 2; preferably, when the first protein is SEQ ID 3 and the second protein is SEQ ID 4, the third protein is not SEQ ID 3 and the fourth protein is not SEQ ID 4; preferably, when the first protein is SEQ ID 5 and the second protein is SEQ ID 6, the third protein is not SEQ ID 5 and the fourth protein is not SEQ ID 6; preferably, when the first protein is SEQ ID 7 and the second protein is SEQ ID 8, the third protein is not SEQ ID 7 and the fourth protein is not SEQ ID 8; preferably, when the first protein is SEQ ID 9 and the second protein is SEQ ID 10, the third protein is not SEQ ID 9 and the fourth protein is not SEQ ID 10; preferably, when the first protein is SEQ ID 11 and the second protein is SEQ ID 12, the third protein is not SEQ ID 11 and the fourth protein is not SEQ ID 12.

[0069] In a specific embodiment, the first protein is SEQ ID 1, the second protein is SEQ ID 2, the third protein is SEQ ID 3, and the fourth protein is SEQ ID 4; or, the first protein is SEQ ID 5, the second protein is SEQ ID 6, the third protein is SEQ ID 3, and the fourth protein is SEQ ID 4.

[0070] In a preferred embodiment, the first vector, the second vector and the third vector further comprise a promoter sequence, which is operably linked to the 5′ terminal portion of the first portion (CDS1) of the coding sequence, or the 5′ terminal portion of the second portion (CDS2) of the coding sequence, or the 5′ terminal portion of the third portion (CDS3) of the coding sequence.

[0071] Preferred promoters are ubiquitous, artificial or tissue-specific promoters, including fragments and variants thereof that retain transcriptional promoter activity. Particularly preferred promoters are photoreceptor-specific promoters, including photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1), interphotoreceptor retinoid binding protein promoter (IRBP), rhodopsin promoter (RHO), vitelliform macular dystrophy 2 promoter (VMD2), rhodopsin kinase promoter (RK); further particularly preferred promoters are muscle-specific promoters, including MCK, MYODI; liver-specific promoters, including thyroxine-binding globulin (TBG), hybrid liver-specific promoter (HLP) (67); neuron-specific promoters, including hSYN1, CaMKIIa; kidney-specific promoters, including Ksp-cadherin 16, NKCC2. Ubiquitous promoters according to the present invention are, for example, ubiquitous cytomegalovirus (CMV) (32) and short CMV (33) promoters. More preferred promoters within the scope of the present invention are GRK1, TBG, CaMKIIa, Ksp-Cadherin 16.

[0072] In a more preferred embodiment, 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.

[0073] In a more preferred embodiment, the first vector, the second vector and the third vector further comprise a polyadenylation signal nucleotide sequence.

[0074] In a more preferred embodiment, the coding sequence is split into the first part, the second part and optionally the third part at a position consisting of a nucleophilic amino acid that does not fall within the structural or functional domain of the encoded protein product, wherein the nucleophilic amino acid is selected from serine, threonine or cysteine.

[0075] Preferably, at least one of the first vector, the second vector and the third vector further comprises at least one enhancer or regulatory nucleotide sequence operably linked to the coding sequence.

[0076] Preferred enhancer or regulatory nucleotide sequences are the globin IgG chimeric intron, the woodchuck hepatitis virus posttranscriptional regulatory element.

[0077] Optionally, at least one of the first vector, the second vector and the third vector further comprises at least one degradation signal to reduce the stability of the reconstituted protein.

[0078] Preferably, the degradation signal is the CL1 degron or the PB29 degron. 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 degron as described herein. Most preferably, the fragment retains the degradation properties of ecDHFR, preferably, the properties of a variant DHFR that functions as an internal degron, preferably, the fragment is mini-ecDHFR, wherein the mini-ecDHFR is a variant that functions as an internal degron.

[0079] Preferably, the coding sequence encodes a protein capable of correcting a pathological condition or disorder, preferably, the disorder is retinal degeneration, a metabolic disorder, a blood disorder, a neurodegenerative disorder, hearing loss, a channelopathy, a lung disease, a myopathy, a heart disease, a muscular dystrophy.

[0080] Further preferably, the coding sequence encodes a protein capable of correcting a pathological condition or disorder, preferably, the disorder is retinal degeneration, preferably, the retinal degeneration is hereditary, preferably, the pathology or disease is selected from the group consisting of: retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt disease (STGD), Ussher syndrome (USH), Alstrom syndrome, congenital stationary night blindness (CSNB), macular dystrophy, recessive macular dystrophy, 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 a fragment thereof or a direct homolog thereof or a minigene thereof with a coding sequence length of more than 5 kb, that is, a minimal gene fragment including one or more exons and the regulatory elements required for this gene to express itself in the same manner as a wild-type gene fragment.

[0081] More preferably, the coding sequence encodes a protein capable of correcting muscular dystrophy, such as Duchenne muscular dystrophy, cystic fibrosis, hemophilia A, Wilson's disease,

[0082] Phenylketonuria, dysferlinopathies, Rett's syndrome, polycystic kidney disease, Niemann-Pick type C, Huntington's disease.

[0083] 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 a fragment thereof or a direct homolog thereof or a minigene thereof with a coding sequence length of more than 5 kb, i.e., the minimum gene fragment including one or more control regions necessary for this gene to express itself in the same manner as a wild-type gene fragment.

[0084] Further 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 a fragment thereof or a direct homolog thereof, or a minigene thereof with a coding sequence length exceeding 5 kb, i.e., a minimal gene fragment comprising one or more regulatory elements necessary for the gene to express itself in the same manner as a wild-type gene fragment.

[0085] In a particularly preferred embodiment of the present invention, the coding sequence encodes the ABCA4 gene. Preferably, the coding sequence is cleaved at nucleotides corresponding to aa Cys1150, Ser1168, and Ser1090 of the ABCA4 protein, and the split-in protein is inserted at the cleavage point.

[0086] In another preferred embodiment, the coding sequence encodes the CEP290 gene. Preferably, the coding sequence is split at the nucleotides corresponding to aa Cys 1076 and Ser 1275. More preferably, the coding sequence is split at the nucleotide sequences corresponding to aa Cys 929 and 1474, Ser 453, and Cys 1474 of the CEP290 protein, with two split-proteins inserted at the split point.

[0087] EGFP SEQ ID No. 15

[0088] The first amino acid of C-exonin is highlighted in the sequence. Splitting Cys.71 (bold)

[0089]

[0090] ABCA4 SEQ ID No. 16

[0091] The first amino acid of C-exonin is highlighted in the sequence.

[0092] Splitting group 1 Cys.1150 (bold)

[0093] Split group 2Ser.1168 (underlined)

[0094] Split Group 3Ser.1090 (italics)

[0095]

[0096] CEP290 SEQ ID No. 17

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

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

[0099] Split Group 2-3Ser.1275 (underline)

[0100] Split group 4 Cys.929 and Cys.1474 (italics)

[0101] (double underscore)

[0102]

[0103] F8 SEQ ID No.18

[0104] The first amino acid of c-exonin is highlighted in the sequence. Split Group 1 Cys.1312 (Underlined) Group Split 2 Ser.984 (Bold)

[0105]

[0106] ecDHFRSEQ ID No.19

[0107]

[0108] mini ecDHFRSEQ ID No.20

[0109]

[0110] In a preferred embodiment, the vector system of the present invention comprises:

[0111] a) a first vector, wherein the first vector comprises, in the 5′-3′ direction:

[0112] -5′-inverted terminal repeat (5′-ITR) sequence;

[0113] - promoter sequence;

[0114] - a 5' terminal portion of the coding sequence (CDS1), said 5' terminal portion being operably linked to and under the control of said promoter;

[0115] - a first intein nucleotide sequence encoding an N-intein; and

[0116] -3′-inverted terminal repeat (3′-ITR) sequence; and

[0117] b) a second vector, the second vector comprising in the 5′-3′ direction:

[0118] -5′-inverted terminal repeat (5′-ITR) sequence;

[0119] - promoter sequence;

[0120] - a second intein nucleotide sequence encoding C-intein;

[0121] - the 3' terminal part of the coding sequence (CDS2); and

[0122] -3′-inverted terminal repeat (3′-ITR) sequence;

[0123] Or include:

[0124] a′) a first vector, the first vector comprising in the 5′-3′ direction:

[0125] -5′-inverted terminal repeat (5′-ITR) sequence;

[0126] - promoter sequence;

[0127] - the 5′-terminal portion of the coding sequence (CDS1′), said 5′-terminal portion being operably linked to and under the control of said promoter;

[0128] - a first intein nucleotide sequence encoding a first N-intein; and

[0129] -3′-inverted terminal repeat (3′-ITR) sequence; and

[0130] b′) a second vector, the second vector comprising in the 5′-3′ direction:

[0131] -5′-inverted terminal repeat (5′-ITR) sequence;

[0132] - promoter sequence;

[0133] - a second intein nucleotide sequence encoding the first C-intein;

[0134] - the second part of the coding sequence (CDS2'); and

[0135] - a third intein nucleotide sequence encoding a second N-intein;

[0136] -3′-inverted terminal repeat (3′-ITR) sequence; and

[0137] c′) a third vector, said third vector comprising in the 5′-3′ direction:

[0138] -5′-inverted terminal repeat (5′-ITR) sequence;

[0139] - promoter sequence;

[0140] - a fourth intein nucleotide sequence encoding a second C-intein;

[0141] - said third part of said coding sequence (CDS3'); and

[0142] -3' inverted terminal repeat (3'-ITR) sequence.

[0143] Preferably, the first vector, the second vector and the third vector are independently viral vectors, preferably adenoviral vectors or adeno-associated virus (AAV) vectors, preferably, the first vector, the second vector and the third adeno-associated virus (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 (quadY-F).

[0144] The present invention also provides a host cell transformed with the vector system defined above.

[0145] Preferably, the vector system or the host cell is for medical use, preferably for use in gene therapy, preferably for the treatment and / or prevention of pathologies or diseases characterized by retinal degeneration, metabolic disorders, blood disorders, neurodegenerative diseases, hearing loss, channelopathies, lung diseases, myopathies, heart diseases, muscular dystrophy.

[0146] Preferably, the retinal degeneration is hereditary, preferably, the pathology or disease is selected from the group consisting of: retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt disease (STGD), Ussher syndrome (USH), Alström syndrome, congenital stationary night blindness (CSNB), macular dystrophy, recessive macular dystrophy, diseases caused by mutations in the ABCA4 gene.

[0147] Preferably, the vector system or the host cell is used to prevent and / or treat Duchenne muscular dystrophy, cystic fibrosis, hemophilia A, Wilson's disease, phenylketonuria, dysferlinosis, Rett syndrome, polycystic kidney disease, Niemann-Pick disease type C, or Huntington's disease.

[0148] The present invention also provides a pharmaceutical composition comprising the vector system or host cell of the present invention and a pharmaceutically acceptable carrier. Detailed Description of the Invention BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1: AAV intracellular proteins reconstitute EGFP in vitro and in mouse and pig retinas at levels higher than dual AAV and up to levels achieved with single AAV.

[0150] (A) Schematic diagram of AAV intron-mediated protein trans-splicing. ITR: AAV2 inverted terminal repeat; CDS: coding sequence; ■: 3xflag tag; PolyA: polyadenylation signal.

[0151] (B) Western blot (WB) analysis of lysates from HEK293 cells transfected with full-length or AAV intron CMV-EGFP plasmids. pEGFP: full-length EGFP plasmid; pAAV I+II: AAV-EGFP I+II intron plasmid; pAAV I: single AAV-EGFP I intron plasmid; pAAV II: single AAV-EGFP II intron plasmid; Neg: untransfected cells. Arrows indicate full-length EGFP protein (EGFP), the N-terminal and C-terminal halves of the EGFP protein (B and A, respectively), and the reconstituted intron excised from the full-length EGFP protein (C). WB represents n = 3 independent experiments.

[0152] (C) Western blot analysis of lysates from HEK293 cells infected with single, intron, or dual AAV2 / 2-CMV-EGFP vectors. Western blot analysis represents n=5 independent experiments.

[0153] (D) Retinal cryosections from C57BL / 6J mice injected subretinally with an AAV2 / 8-CMV-EGFP endonuclease vector. Scale bar: 50 μm. RPE: retinal pigment epithelium; OS: outer segment; ONL: outer nuclear layer.

[0154] (EF) Retinal cryosections from C57BL / 6J mice (E) or Large White pigs (F) injected subretinally with single, intronic, or dual AAV2 / 8-GRK1-EGFP vectors. Scale bars: 50 μm (E); 200 μm (F). OS: outer segment; ONL: outer nuclear layer.

[0155] (G) Fluorescence analysis of retinal organoids infected with AAV2 / 2-GRK1-EGFP-intrin vector at day 293 of culture. Scale bar: 100 μm.

[0156] Figure 2 : Optimization of AAV intraproteins allows correct reconstitution of large ABCA4 and CEP290 proteins.

[0157] (AB) Western blot (WB) analysis of lysates from HEK293 transfected with different sets of AAV-shCMV-ABCA4 or -CEP290 protein plasmids (Groups 1 and 5, respectively). Figure 16 Schematic representation of the various groups used is shown. WB represents n=3 independent experiments.

[0158] (CD) Representative images of immunofluorescence analysis of HeLa cells transfected with AAV-shCMV-ABCA4 (C) or AAV-shCMV-CEP290 (D) endonuclease plasmids. pABCA4 (C) or pCEP290 (D): plasmids containing the full-length expression cassette; pAAV-endonuclease: AAV-endonuclease plasmid (group 1 in C or group 5 in D); I+II+III: AAV I+II+III endonuclease plasmid; I+II: AAV I+II endonuclease plasmid; I+III: AAV I+III endonuclease plasmid; II+III: AAV II+III endonuclease plasmid; I: single AAV I endonuclease plasmid; II: single AAV II endonuclease plasmid; III: single AAV III endonuclease plasmid; Neg: untransfected cells.

[0159] Cells were stained for 3xFLAG and VAP-B (endoplasmic reticulum markers) and TGN46 (trans-Golgi network marker) in C, or for acetylated tubulin (microtubule marker) in D. White arrows point to Figure 18 Cells are shown at higher magnification.

[0160] Figure 3 : AAV intracellular vectors reconstitute large ABCA4 and CEP290 proteins more efficiently than dual AAV vectors.

[0161] Western blot (WB) analysis of lysates of HEK293 cells infected with dual or intron AAV2 / 2-shCMV-ABCA4 (A) or -CEP290 (B) vectors.

[0162] AAV endonuclease: AAV-ABCA4 (Group 1, A) or -CEP290 (Group 5, B) endonuclease vector; I+II+III: AAV I+II+III endonuclease vector; I+II: AAV I+II endonuclease vector; I+III: AAV I+III endonuclease vector; II+III: AAVII+III endonuclease vector; I: single AAV I endonuclease vector; II: single AAV II endonuclease vector; III: single AAVIII endonuclease vector; Dual AAV: dual AAV vector; Neg: AAV-EGFP vector.

[0163] (A) Arrows indicate 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.

[0164] (B) Arrows indicate 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 represents n=3 independent experiments.

[0165] Figure 4 : AAV intracellular proteins reconstitute large proteins in mouse, porcine, and human photoreceptors at therapeutic levels.

[0166] (AC) Western blot (WB) analysis of retinal lysates from wild-type mice (A, B) or Large White pigs (C) injected with dual or intron AAV2 / 8-GRK1-ABCA4 (A, C) or -CEP290 (B) vectors (Groups 1 and 5, respectively). AAV intron: AAV intron vector; dual AAV: dual AAV vector; Neg: AAV-EGFP vector or PBS.

[0167] (D) Western blot analysis of lysates from human iPSC-derived 3D retinal organoids infected with an AAV2 / 2-GRK1-ABCA4 endogenous vector. AAV endogenous: AAV-ABCA4 endogenous vector; Neg: uninfected organoids; - / -: organoids derived from STGD1 patients.

[0168] (A, C, D) Arrows indicate full-length ABCA4 protein (ABCA4), and A: protein product derived from AAV I; B: protein product derived from AAV II. *Protein products with potentially different post-translational modifications.

[0169] (B) Arrows indicate full-length CEP290 protein (CEP290); A: protein product derived from AAV II+III; and D: protein product derived from AAV III.

[0170] Figure 5 : Subretinal administration of AAV intracellular proteins improves retinal phenotypes in a mouse model of inherited retinal degeneration.

[0171] (A) Abca4 treated with AAV protein - / -Quantification of the average area occupied by lipofuscin in the RPE of mice. Each point represents the average value measured for each eye. The average value of lipofuscin area for each group is shown in the figure. + / + or + / -: Abca4 injected with control + / + or + / - Eye (PBS); - / -: Abca injected with negative control - / - Eyes (AAV I ABCA4 or AAV IIABCA4 or PBS); - / - AAV intracellular protein: Abca4 injected with AAV intracellular protein vector (Group 1) - / - *ANOVA p value < 0.05; ***ANOVA p value < 0.001.

[0172] (B) Representative images of retinal sections from wild-type, uninjected mice and rd16 mice injected subretinally with an AAV2 / 8-GRK1-CEP290 intron vector (AAV intron, group 5) or a negative control (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.

[0173] (C) Representative images of eyes from wild-type uninjected mice and rd16 mice injected subretinal with AAV2 / 8-GRK1-CEP290 intracellular protein vector (AAV intracellular protein, group 5) or negative control (Neg; i.e., AAV I+II or AAV II+III or PBS). The white circle defines the pupil.

[0174] Figure 6 : Schematic diagram of large protein remodeling mediated by protein trans-splicing.

[0175] The coding sequence (CDS) of a large gene is split into two halves (5′ and 3′), flanked by inverted terminal repeats (ITRs), and packaged separately in two AAV capsids. Following co-transduction of the same cells, different mechanisms are explored to reconstitute full-length protein expression by linking these two halves at the protein level. The 5′-vector comprises the 5′ CDS, 5′ intein (n-intein), and degron, while the 3′-vector comprises the 3′ CDS and 3′ intein (c-intein); both vectors include a promoter and polyA. Pairing of the two hemipolypeptides is mediated by intein self-recognition; subsequent self-excision of the intein from the host protein results in reconstitution of the full-length protein. The degron, now embedded within the excised intein, is rapidly ubiquitinated and degraded by the proteasome.

[0176] Figure 7: In vitro EGFP expression from AAV intein vectors with and without degradation signals.

[0177] Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intein plasmids containing ecDHFR (+) or without ecDHFR (-). Arrows indicate full-length EGFP protein (EGFP), cleaved intein containing the degron (DnaE+ecDHFR) or without this degron (DnaE).

[0178] Figure 8 : In vitro expression of ABCA4 from AAV intracellular protein vectors with and without degradation signals.

[0179] Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intein plasmids containing ecDHFR (+) or without ecDHFR (-). Arrows indicate full-length ABCA4 protein (ABCA4), cleaved intein containing the degron (DnaE+ecDHFR) or without this degron (DnaE).

[0180] Figure 9 :The expression of endogenous protein DnaE-ecDHFR is dependent on TMP.

[0181] Western blot analysis of lysates from HEK293 cells transfected with AAV_ABCA4 intein plasmids containing ecDHFR (pAAV intein+ecDHFR) or without ecDHFR (pAAV intein) and treated with increasing doses of Trimetrophin (1 to 50 mM). Arrows indicate excised intein with or without degron (DnaE+ecDHFR) or without degron (DnaE).

[0182] Figure 10 : In vitro EGFP expression from AAV intein vectors with and without degradation signals.

[0183] Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intein plasmids containing mini-ecDHFR (+) or without mini-ecDHFR (-). Arrows indicate full-length EGFP protein (EGFP), cleaved intein containing the degron (DnaE+mini-ecDHFR) or without this degron (DnaE).

[0184] Figure 11 : In vitro expression of ABCA4 from AAV intracellular protein vectors with and without degradation signals.

[0185] Western blot (WB) analysis of lysates from HEK293 cells transfected with AAV intein plasmids containing mini-ecDHFR (+) or without mini-ecDHFR (-). Arrows indicate full-length ABCA4 protein (ABCA4), cleaved intein containing the degron (DnaE+mini-ecDHFR) or without this degron (DnaE).

[0186] Figure 12 : Analysis of EGFP fluorescence in HEK293 cells transfected with AAV I+II, but not single AAV I or AAV II protein plasmids.

[0187] Fluorescence analysis of HEK293 cells transfected with full-length or intron CMV-EGFP plasmids. pEGFP: plasmid containing the full-length EGFP expression cassette; pAAV I+II: AAV I+II intron plasmid; pAAV I: single AAV I intron plasmid; pAAVII: single AAV II intron plasmid; Neg: untransfected cells. Scale bar: 100 μm.

[0188] Figure 13 : The intein relative to the full-length protein varies between species.

[0189] 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 intein vectors (B-C). AAV intein: cells infected with AAV intein vectors (A) or injected eyes (B, C); Neg: uninfected cells (A) or PBS-injected eyes (B, C). Arrows indicate full-length EGFP protein (EGFP) and excised intein (DnaE).

[0190] Figure 14 : Characterization of human iPSC-derived 3D retinal organoids.

[0191] (A) Light microscopy analysis of retinal organoids at 183 days in culture.

[0192] (B) Immunofluorescence analysis using antibodies against mature photoreceptor markers. Scale bar: 100 μm.

[0193] (C) Fluorescence analysis of retinal organoids infected with AAV2 / 2-CMV-EGFP and AAV2 / 2-IRBP-DsRed vectors. Scale bar: 100 μm.

[0194] (D) Outer segment-like structures protruding from the surface of retinal organoids were observed at day 230 of culture. The inset shows the presence of outer segment (OS)-like structures with radial organization. NR: neural retina; RPE: retinal pigment epithelium.

[0195] (E) Scanning electron microscopy analysis showed the presence of inner segment (IS), connecting cilium (CC), and outer segment (OS)-like structures. Scale bar: 4 μm.

[0196] (F) Electron microscopy analysis shows the presence of the external limiting membrane (*), centriole (C), basal body (BB), connecting cilium (CC), and sketch of the outer segment (OS).

[0197] Inset shows the presence of disordered membrane disks in OS. Scale bar: 500 nm.

[0198] D: Number of days of cultivation.

[0199] Figure 15 : Low intrin relative to full-length protein in human 3D retinal organoids.

[0200] Western blot analysis of lysates from human iPSC-derived 3D retinal organoids infected with an AAV2 / 2-GRK1-EGFP intron vector. AAV intron: AAV intron vector; Neg: uninfected organoids. Arrows indicate full-length EGFP protein (EGFP) and excised intron (DnaE).

[0201] Figure 16 : Schematic diagram of the proteins within AAV-ABCA4 and -CEP290 in each group.

[0202] (A) AAV-ABCA4-intrin constructs. (Groups 1-2 are exemplified by constructs) n-DnaE: n-intrin from DnaE of Npu; c-DnaE: c-intrin from DnaE of Npu; (Group 3) n-mDnaE: n-intrin from mutant DnaE of Npu (mNpu); c-mDnaE: c-intrin from DnaE of mNpu.

[0203] (B) AAV-CEP290-intrin construct. (Group 1) n-DnaE: n-intrin of DnaE from Npu; c-DnaE: c-intrin of DnaE from Npu; shPolyA: short synthetic polyA; (Group 2) n-DnaE: n-intrin of DnaE from mNpu; c-DnaE: c-intrin of DnaE from mNpu; (Group 3) n-mDnaE: n-intrin of DnaE from mNpu; c-mDnaE: c-intrin of DnaE from mNpu; (Group 4) n-DnaE: n-intrin of DnaE from Npu; c-DnaE: c-intrin of DnaE from Npu between AAV I and AAV II; n-DnaB: n-intrin of DnaB from Rhodothermus marinus (Rma); c-DnaB: n-intrin of DnaB from AAV II and AAV c-inner protein of DnaE from Rma between III; wpre: woodchuck hepatitis virus post-transcriptional regulatory element. (Group 5) n-mDnaE: n-inner protein of DnaE from mNpu; c-mDnaE: c-intrin of DnaE from mNpu between AAV II and AAV III; n-DnaB: n-intrin of DnaB from Rhodothermus marinus (Rma); c-DnaB: c-intrin of DnaE from Rma between AAV II and AAV III; wpre: woodchuck hepatitis virus posttranscriptional regulatory element. (AB) ITR: AAV2 inverted terminal repeat; : 3xflag tag; Promoter: short CMV promoter for in vitro experiments and human G protein-coupled receptor (GRK1) promoter for in vivo experiments; PolyA: simian virus 40 polyadenylation signal (for ABCA4, A) and bovine growth hormone polyadenylation signal (for CEP290, B). The amino acids at the split point for each group are indicated in the figure. The predicted protein molecular weight is shown below each AAV vector.

[0204] Figure 17 : The combination of heterologous N- and C-intrins does not result in detectable reconstitution of EGFP protein in vitro.

[0205] Fluorescence analysis of HEK293 cells transfected with full-length or intronic AAV-CMV-EGFP plasmids. N+C-DnaE: AAV I+II fused to the inner protein from DnaE; N+C-DnaB: AAV I+II fused to the inner protein from DnaB; N+C-mDnaE: AAV I+II fused to the split inner protein from mDnaE; N-DnaE+C-DnaB: AAV I fused to the n-inner protein from DnaE and AAV II fused to the c-inner protein from DnaB; N-DnaB+C-DnaE: AAV I fused to the n-inner protein from DnaB and AAV II fused to the c-inner protein from DnaE; N-mDnaE+C-DnaB: AAV I fused to the n-inner protein from mDnaE and AAV II fused to the c-inner protein from DnaB; N-DnaB+C-mDnaE: AAV I fused to the n-inner protein from DnaB I and AAV II fused with c-intrin from mDnaE; pEGFP: plasmid containing the full-length EGFP expression cassette; Neg: untransfected cells. Scale bar: 100 μm.

[0206] Figure 18 : CEP290 is arranged along microtubules.

[0207] Figure 2 Magnification of a single cell in D. Immunofluorescence analysis of HeLa cells transfected with a plasmid containing the full-length CEP290 expression cassette (pCEP290) or with a CEP290 protein plasmid (Group 5, pAAV I+II+III). Cells were stained for 3xFLAG and acetylated tubulin (a microtubule marker). Scale bar: 50 μm.

[0208] Western blot (WB) analysis of lysates from HEK293 cells transfected with full-length or AAV intein plasmids encoding short CMV-ABCA4 (Group 1, A) or -CEP290 (Group 5, B).

[0209] (A) pABCA4: full-length ABCA4 expression cassette; Group 1: ABCA4 (Cys. 1150)-intrin plasmid.

[0210] (B) pCEP290: full-length CEP290 expression cassette; Group 5: CEP290 (Ser.453 and Cys.1474)-intrin plasmid.

[0211] Neg: AAV EGFP plasmid. Western blot represents n=3 independent experiments.

[0212] Figure 19Compared with transfection with a single plasmid carrying the full-length expression cassette, transfection with an AAV protein plasmid resulted in reconstitution of ABCA4 and CEP290 proteins at lower amounts.

[0213] Western blot (WB) analysis of lysates from HEK293 cells transfected with full-length or AAV intrapeptide plasmids encoding short CMV-ABCA4 (A) or -CEP290 (B). (A) pABCA4: full-length ABCA4 expression cassette; Group 1: ABCA4 (Cys. 1150)-interpeptide plasmid. (B) pCEP290: full-length CEP290 expression cassette; Group 5: CEP290 (Ser. 453 and Cys. 1474)-interpeptide plasmid. Neg: AAV EGFP plasmid. WB represents n = 3 independent experiments.

[0214] Figure 20 : Subretinal delivery of AAV intracellular protein vectors results in ABCA4 expression in the mouse retina.

[0215] Western blot analysis of retinal lysates from wild-type mice injected with dual or Neg AAV2 / 8-GRK1-ABCA4 vectors (Group 1). AAV Neg: AAV Neg vector; Dual AAV: dual AAV vector; Neg: AAV-EGFP vector.

[0216] Figure 21 : AAV proteins can reconstitute about 10% of endogenous Abca4.

[0217] Abca4 injected with AAV2 / 8-GRK1-ABCA4 protein vector (Group 1) + / - or Abca4 - / - Western blot (WB) analysis of mouse retinal lysates. mAbca4: Abca4 + / - Retinas; AAV endonuclease: AAV endonuclease-injected retinas; Neg: uninjected retinas. The same retinal lysates from Abca4+ / - were loaded on gels #2 and #3. The percentage of AAV endonuclease ABCA4 expression relative to endogenous expression is shown below each lane.

[0218] Figure 22 : AAV intracellular protein reconstitutes full-length ABCA4 protein in human retinal organoids.

[0219] Western blot analysis of lysates from human iPSC-derived 3D retinal organoids infected with an AAV2 / 2-GRK1-ABCA4 intron vector (Group 1). AAV intron: AAV intron vector; Neg: uninfected organoids. - / -: organoids derived from STGD1 patients; + / -: organoids derived from healthy donors.

[0220] Figure 23 Subretinal administration of AAV intracellular protein vectors results in lipofuscin in Abca4 - / - Accumulation was reduced in mice.

[0221] Representative images of transmission electron microscopy analysis showing wild type and Abca4 injected with negative control (Neg) or AAV intein vector (Group 1) - / - Lipofuscin granules in mouse RPE. White arrows indicate lipofuscin granules; M: mitochondria.

[0222] Figure 24 : Subretinal delivery of AAV intracellular protein vectors in mice does not modify ONL thickness.

[0223] Spectral domain optical coherence tomography analysis of C57BL / 6J mouse eyes injected subretinal with AAV intraprotein vectors, irrelevant AAV vectors (AAV neg), or PBS. Black bars represent eyes 6 months after injection with AAV-ABCA4 intraprotein vectors (Group 1) and their corresponding controls; white bars represent eyes 4.5 months after injection with AAV-CEP290 intraprotein vectors (Group 5) and their corresponding controls. Data are presented as mean ± SE. Mean values ​​are shown above the corresponding bars.

[0224] Figure 25 : AAV protein vectors can deliver full-length wild-type F8.

[0225] A) Schematic diagram of a single AAV B domain deleted variant 3 Factor VIII (F8-V3) and AAV F8 intein vector.

[0226] The coding sequence of the F8 gene is divided into two halves (5' and 3' F8), flanked by inverted terminal repeats (ITRFs), which are packaged in two AAV capsids. The 5' vector includes 5' F8 and the 5' inner protein (n-DnaE), while the 3' vector includes 3' F8 and the 3' inner protein (c-DnaE). Both vectors contain the HLP promoter and synthetic polyA. V3: variant 3; SS: signal sequence.

[0227] B) Unlike the single, extra-large AAV F8-V3, the F8 inner proteins are correctly packaged into AAV capsids with defined vector genomes.

[0228] Southern blot analysis of vector genome integrity using an HLP promoter-specific probe showed that the truncation products in the super-large AAV F8-V3 were absent in the AAV F8 intein vector. Neg: negative control.

[0229] AAV F8 intein vectors showed mild correction of the bleeding phenotype in hemophilia A knockout mice at 8 weeks post-injection.

[0230] aPTT analysis of plasma samples from hemophilia A knockout mice 8 weeks after injection of AAV F8 protein (two split points) showed a slight correction of the phenotype compared to controls injected with PBS. aPTT: activated partial thromboplastin time.

[0231] Gene therapy

[0232] Over the past decade, gene therapy has been applied to treat disease 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, are currently one of the most popular gene delivery tools. Most systems consist of a vector capable of accommodating the gene of interest and helper cells capable of providing viral structural proteins and enzymes to allow production of infectious viral particles containing the vector. Adeno-associated viruses are a family of viruses that differ in nucleotide and amino acid sequence, genome structure, pathogenicity, and host range. This diversity provides opportunities for developing diverse therapeutic applications using viruses with diverse biological properties. As with any delivery vehicle, efficiency, the ability to target specific tissues or cell types, expression of the gene of interest, and the safety of adeno-associated virus-based systems are crucial for the successful application of gene therapy. In recent years, numerous efforts have been made in these research areas. Various modifications have been made to adeno-associated virus-based vectors and helper cells to alter gene expression, target delivery, increase viral titer, and enhance safety. The present invention represents an improvement on this design process, as it serves as a tool for the efficient delivery of target genes whose size exceeds the cargo size limitations of a single adeno-associated virus-based vector. Viruses are a logical tool for gene delivery. They replicate in cells and therefore evolve mechanisms to enter cells and use cellular machinery to express their own genes. The concept of viral-based gene delivery is to engineer viruses so that they can 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, slow viruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, picornaviruses, and alpha viruses. The present invention preferably uses adeno-associated viruses. Therefore, viral-based vectors for gene delivery include but are not limited to adenoviral vectors, adeno-associated virus (AAV) vectors, pseudotyped AAV vectors, herpes virus vectors, retroviral vectors, slow virus vectors, and baculovirus vectors.

[0233] Ideal adeno-associated virus-based vectors for gene delivery must be efficient, cell-specific, regulated and safe. Delivery efficiency is crucial because it can determine the efficacy of the treatment. Current efforts are aimed at achieving cell type-specific infection and gene expression through adeno-associated virus vectors. In addition, adeno-associated virus vectors are being developed to regulate the expression of target genes because long-term or regulated expression may be required for treatment. Safety is a major issue with viral gene delivery because most viruses are pathogens or have pathogenic potential.

[0234] Adeno-associated virus (AAV) is a small virus that infects humans and some other primates. AAV is not known to cause disease, and therefore the virus elicits a very mild immune response. Gene therapy vectors using AAV can infect both dividing and dormant cells and persist in an extrachromosomal state without integrating into the host cell genome. These properties make AAV an attractive candidate for creating viral vectors for gene therapy and for establishing isogenic human disease models.

[0235] Wild-type AAV has garnered significant interest among gene therapy researchers due to its many characteristics. Chief among these is its apparent lack of pathogenicity. It can also infect non-dividing cells and 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 carry the threat of random insertions and mutagenesis, which sometimes contribute to the development of cancer. The AAV genome most frequently integrates into the aforementioned sites, while random integration into the genome is negligible. However, in the development of AAV as a gene therapy vector, this integration ability has been eliminated by removing the rep and cap sequences from the vector DNA. The desired gene, along with a promoter driving gene transcription, is inserted between inverted terminal repeats (ITRs), which facilitate concatemer formation in the cell nucleus after the single-stranded vector DNA is converted to double-stranded DNA by the host cell DNA polymerase complex. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact for the life of the host cell. In dividing cells, AAV DNA is lost through cell division because episomal DNA is not replicated along with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at a very low frequency. AAVs also exhibit very low immunogenicity, which appears to be limited to the production of neutralizing antibodies, while they do not induce a well-defined cytotoxic response. This feature, along with the ability to infect dormant cells, suggests that AAV is superior to adenovirus as a human gene therapy vector.

[0236] AAV genome, transcriptome, and proteome

[0237] 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) located at either end of the DNA strand and two open reading frames (ORFs): rep and cap. The former consists of four overlapping genes encoding the Rep proteins required for the AAV life cycle, while the latter includes overlapping nucleotide sequences for the capsid proteins: VP1, VP2, and VP3, which interact to form the icosahedral capsid.

[0238] ITR sequence

[0239] Inverted terminal repeat (ITR) sequences each comprise 145 bases. They are so named because of their symmetry, which has been shown to be necessary for efficient amplification of the AAV genome. Another characteristic of these sequences is their ability to form hairpins, which contributes to so-called self-priming, which allows for the synthesis of a second DNA strand that is independent of the primer enzyme. It has also been shown that ITRs are necessary for the integration of AAV DNA into the host cell genome (human chromosome 19) and for the rescue thereof, as well as for the efficient encapsidation of AAV DNA and the generation of fully assembled, deoxyribonuclease-resistant AAV particles.

[0240] For gene therapy, the ITR appears to be the only sequence required in cis near the therapeutic gene: structural (cap) and packaging (rep) genes can be delivered in trans. Based on this hypothesis, many methods have been established to efficiently generate recombinant AAV (rAAV) vectors containing reporter or therapeutic genes. However, it has also been shown that the ITR is not the only element required in cis for efficient replication and encapsidation. Several research groups have identified sequences within the coding sequence of the rep gene, termed cis-acting Rep-dependent elements (CAREs). When present in cis, CAREs have been shown to enhance replication and encapsidation.

[0241] AAV serotypes

[0242] To date, dozens of different AAV variants (serotypes) have been identified and classified (60). All known serotypes can infect cells from a variety of different 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 genome of an AAV serotype in the capsid of a second AAV serotype; for example, the AAV2 / 8 vector contains the AAV8 capsid and the AAV 2 genome (61). Such vectors are also called chimeric vectors.

[0243] Serotype 2

[0244] To date, serotype 2 (AAV2) has been the most extensively examined. AAV2 has a natural tropism for skeletal muscle, neurons, vascular smooth muscle cells, and hepatocytes. Three cellular receptors for AAV2 have been described: heparan sulfate proteoglycans (HSPGs), avβ5 integrin, and fibroblast growth factor receptor 1 (FGFR-1). The first functions as a primary receptor, while the latter two have co-receptor activity, enabling AAV entry into cells via receptor-mediated endocytosis. These findings have been challenged by Qiu, Handa, and others. HSPGs function as the primary receptor, although their abundance in the extracellular matrix can clear AAV particles and impair infection efficiency.

[0245] The study shows that adeno-associated virus type 2 (AAV-2) apparently kills cancer cells without harming healthy cells. "Our results show that adeno-associated virus type 2, which infects most populations without known adverse effects, kills multiple cancer cell types but has no effect on healthy cells," said Craig Meyers, professor of immunology and microbiology at the Penn State College of Medicine. This could lead to a new class of anti-cancer agents.

[0246] Other serotypes

[0247] Although AAV2 is the most commonly used serotype in various AAV-based studies, other serotypes have been shown to be more effective as gene delivery vectors. For example, AAV6 performs better at infecting airway epithelial cells; AAV7 exhibits very high transduction rates of murine skeletal muscle cells (similar to AAV1 and AAV5); AAV8 excels at transducing hepatocytes and photoreceptors; and AAV1 and 5 have been shown to be very effective at delivering genes to vascular endothelial cells. In the brain, most AAV serotypes display neuronal tropism, while AAV5 also transduces astrocytes. AAV6 (a hybrid of AAV1 and AAV2) displays lower immunogenicity than AAV2.

[0248] Serotypes can differ in the receptors to which they bind. For example, transduction by AAV4 and AAV5 can be inhibited by soluble sialic acid (a different form of each of these serotypes), and AAV5 has been shown to enter cells through platelet-derived growth factor receptors. Novel AAV variants, such as the quadruple tyrosine mutant or AAV 2 / 7m8, have been shown to transduce the outer retina from the vitreous in small animal models (62, 63). Another AAV mutant, named ShH1O, is an AAV6 variant with improved glial tropism after intravitreal administration (64). Another AAV mutant with a particularly favorable tropism for the retina is AAV2 (quad YF) (65).

[0249] The gene delivery vector of the present invention can be administered to a patient. The administration can be "in vivo" administration or "ex vivo" administration. A skilled person can determine the appropriate dosage rate. The term "administering" includes delivery by viral or non-viral techniques. Viral delivery mechanisms include, but are not limited to, the above-mentioned adenoviral vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.

[0250] Non-viral delivery systems include DNA transfection such as electroporation, lipid-mediated transfection, compacted DNA-mediated transfection; liposomes, immunoliposomes, lipofectin, cationic surface amphiphiles (CFAs), and combinations thereof.

[0251] Delivery of one or more therapeutic genes by the vector system according to the present invention can be used alone or in combination with other treatments or components of a treatment.

[0252] Pharmaceutical composition

[0253] The present invention also provides for treating individual pharmaceutical compositions by gene therapy, wherein the composition includes a therapeutically effective amount of the vector / construct or host cell of the present invention, and the vector / construct or host cell includes one or more deliverable therapeutic and / or diagnostic transgenes or viral particles produced or obtained therefrom. The pharmaceutical composition can be used for human or animal use. Usually, the physician will determine the actual dosage that is most suitable for the individual subject, and it varies according to the age, body weight and reaction of the specific individual. The composition can optionally include a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The selection of pharmaceutical carriers, excipients or diluents can be selected according to the intended route of administration and standard pharmaceutical practice. As a carrier, excipient or diluent or outside a carrier, excipient or diluent, the pharmaceutical composition can be any suitable adhesive, lubricant, suspending agent, coating agent, solubilizing agent and other carrier agents (such as lipid delivery systems) that can help or increase the virus to enter the target site. Where appropriate, the pharmaceutical compositions may be administered by any one or more of the following means: by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder; by use of a skin patch, orally, in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules, alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavorings or colorings; preferably, they may be injected parenterally, for example intracavernous, intravenous, intramuscular or subcutaneously. For parenteral administration, the compositions are preferably administered in the form of a sterile aqueous solution which may contain other substances, such as sufficient salts or simple sugars to render the solution isotonic with the blood. For buccal or sublingual administration, the compositions may be administered in the form of tablets or lozenges formulated in conventional manner.

[0254] The preferred formulation is in the case of topical administration of the carrier system in the conjunctival sac or under the conjunctiva, preferably 1 to 10 times per day, preferably for 1 day to 6 months, preferably for 1 day to 30 days.

[0255] Preferred administrations are intracameral administration, intravitreal injection, subretinal injection, parabulbar and / or retrobulbar injection, intrastromal corneal injection.

[0256] Preferably, the pharmaceutical composition of the present invention is for topical ophthalmic use and is therefore an ophthalmic composition.

[0257] The carrier system according to the present invention may be administered by any convenient route, but a preferred route of administration is topical administration to the ocular surface, in particular topical administration to the cornea. An even more preferred route is instillation into the conjunctival sac.

[0258] A particular object of the present invention is the use of the carrier system for the production of ophthalmic compositions for topical administration to the eye for medical use.

[0259] More generally, a preferred embodiment of the present invention is a composition formulated for topical application to a local, superficial or restricted area of ​​the eye and / or ocular adnexa, comprising a carrier system, optionally together with one or more pharmaceutically acceptable additives (e.g., diluents or carriers).

[0260] As used herein, the terms "vehicle," "diluent," "carrier," and "additive" are interchangeable.

[0261] The ophthalmic compositions of the present invention may be in the form of solutions, emulsions or suspensions (eyewashes), ointments, gels, aerosols, mists or liniments together with an ophthalmic carrier that is pharmaceutically acceptable, ophthalmically tolerated and compatible with the active ingredient.

[0262] Specific ocular routes of administration for delayed release are also within the scope of the invention, for example, as an ocular erodible insert or polymeric film "reservoir" system to be positioned in the conjunctival sac or contact lens.

[0263] The ophthalmic compositions of the present invention can be administered topically, eg, by delivering the composition into direct contact with the eye and / or ocular adnexa.

[0264] Pharmaceutical compositions containing at least the carrier system of the present invention may be prepared by any conventional technique, such as described in Remington: The Science and Practice of Pharmacy, Ed.: EW Martin, Mack Publishing Company, 19th ed., Easton, Pa.

[0265] In one embodiment, the composition is formulated as a liquid, wherein the carrier system can be a solution or suspension. The composition can be formulated in 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).

[0266] Preferably, the liquid is an aqueous liquid. Further preferably, the liquid is sterile. Sterility can be imparted by any conventional method, such as filtering, irradiation or heating, or by carrying out the manufacturing process under aseptic conditions.

[0267] The liquid may include one or more lipophilic vehicles.

[0268] In one embodiment of the present invention, the composition is formulated as an ointment. Preferably, one of the carriers in the ointment can be a petrolatum carrier.

[0269] The pharmaceutically acceptable vehicle can generally be any conventionally used pharmaceutically acceptable vehicle, which should be selected according to the specific formulation, intended route of administration, etc. In addition, the pharmaceutically acceptable vehicle can be any acceptable additive listed in the FDA's "Inactive Ingredients List", which can be obtained, for example, through the Internet address http: / / www.fda.gov / cder / drug / iig / default.htm.

[0270] At least one pharmaceutically acceptable diluent or carrier may be a buffer. For some purposes, it is generally desirable that the composition include a buffer capable of buffering the solution to a pH in the range of 5 to 9, such as pH 5 to 6, pH 6 to 8, or pH 7 to 7.5.

[0271] However, in other embodiments of the present invention, the pharmaceutical composition may not include a buffer at all, or may include only a micromolar amount of a buffer. The buffer may, for example, be selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, borate, carbonate, glycinate, histidine, glycine, succinate, and triethanolamine buffers. Thus, the buffer may be K2HPO4, Na2HPO4, or sodium citrate.

[0272] In a preferred embodiment, the buffer is TRIS buffer. TRIS buffer is 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.

[0273] In addition, the buffer can be selected, for example, from a USP compatible buffer for parenteral use, particularly when the pharmaceutical formulation is 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.

[0274] The compositions may include preservatives such as thimerosal, chlorobutanol, benzalkonium chloride, or chlorhexidine; buffers such as phosphates, borates, carbonates, and citrates; and thickening agents such as high molecular weight carboxyvinyl polymers, such as those sold under the trademark Carbopol by BF Goodrich Chemical Company, hydroxymethylcellulose, and polyvinyl alcohol, all of which are in accordance with the prior art.

[0275] In some embodiments of the present invention, pharmaceutically acceptable additives include stabilizers. Stabilizers can be, for example, detergents, amino acids, fatty acids, polymers, polyols, metal ions, reducing agents, chelating agents, or antioxidants, but any other suitable stabilizer can also be used with the present invention. For example, stabilizers can 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.

[0276] In addition, the stabilizer 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 polyvinyl pyrrolidone; polyols such as sorbitol, mannitol, glycerol, sucrose, glucose, propylene glycol, ethylene glycol, lactose, trehalose; antioxidants such as ascorbic acid, cysteine ​​hydrochloride, thioglycerol, thioglycolic acid, thiosorbitol and glutathione; reducing agents such as several thiols; chelating agents such as EDTA salts, glutamic acid and aspartic acid.

[0277] The pharmaceutically acceptable additives may include one or more selected from the group consisting of isotonic salt, hypertonic salt, hypotonic salt, buffer, and stabilizer.

[0278] 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.

[0279] In some embodiments of the present invention, pharmaceutically acceptable additives include mucolytics (eg, N-acetylcysteine), hyaluronic acid, cyclodextrin, petroleum.

[0280] Exemplary compounds that can be included in the pharmaceutical compositions of the present invention to promote and accelerate transdermal delivery of the topical compositions to the eye or adnexal tissues include, but are not limited to, alcohols (ethanol, propanol, and nonanol), fatty alcohols (lauryl alcohol), fatty acids (valeric acid, caproic acid, and capric acid), fatty acid esters (isopropyl myristate and isopropyl n-hexanoate), alkyl esters (ethyl acetate and butyl acetate), polyols (propylene glycol, propylene glycol, and hexanetriol), sulfoxides (dimethyl sulfoxide and decyl methyl sulfoxide), amides (urea, dimethylacetamide, and pyrrolidone derivatives), surfactants (sodium lauryl sulfate, cetyltrimethylammonium bromide, poloxamers, spans, tweens, bile salts, and lecithin), terpenes (d-limonene, alpha-terpeneol, 1,8-cineole, and menthone), and alkanones (n-heptane and n-nonane). Additionally, topically administered compositions may include surface adhesion molecule modulators, including but not limited to cadherin antagonists, selectin antagonists, and integrin antagonists.

[0281] In addition, the ophthalmic solution may contain a thickening agent, such as hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose, polyvinylpyrrolidone, etc., to improve the retention of the drug in the conjunctival sac.

[0282] In one embodiment, the carrier system used according to the present invention can be combined with an ophthalmically acceptable preservative, surfactant, viscosity enhancer, penetration enhancer, buffer, sodium chloride and water to form an aqueous, sterile, ophthalmic suspension or solution. The ophthalmic solution can also include an ophthalmically acceptable surfactant to help dissolve the carrier system. The ophthalmic solution formulation can be prepared by dissolving the carrier system in a physiologically acceptable isotonic aqueous buffer.

[0283] To prepare sterile ophthalmic ointment formulations, the carrier system can be combined with a preservative in an appropriate vehicle, such as mineral oil, liquid lanolin, or white petrolatum. Sterile ophthalmic gel formulations can be prepared by suspending the carrier system in a hydrophilic base prepared from a combination of, for example, carbopol-940, according to published formulations similar to ophthalmic formulations; preservatives and tonicity agents may be added.

[0284] Preferably, the formulation of the present invention is an aqueous, non-irritating ophthalmic composition for topical administration to the eye, comprising: a therapeutically effective amount of a carrier system for topical treatment; a xanthine derivative present in an amount ranging from the amount of a water-soluble derivative of the composition to 0.05% weight / volume of the composition, which is effective to reduce 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 buffering agent to provide an isotonic, aqueous, non-irritating ophthalmic composition.

[0285] Drug delivery devices

[0286] In one embodiment, the present invention includes a drug delivery device composed of at least a carrier system and a pharmaceutically compatible polymer. For example, the composition is incorporated into or coated onto the polymer. The composition is chemically bound or physically entrapped by the polymer. The polymer can be hydrophobic or hydrophilic. The polymeric device includes a variety of physical arrangements. Exemplary physical forms of the polymeric device include, but are not limited to, a membrane, a stent, a chamber, a sphere, a microsphere, a scaffold, or other structure. The polymeric device has an interior surface and an exterior surface. The device has one or more internal chambers. These chambers contain one or more compositions. The device contains a polymer of one or more chemically distinguishable monomers. The subunits or monomers of the device are polymerized in vitro or in vivo.

[0287] In a preferred embodiment, the invention comprises a device comprising a polymer and a biologically active composition incorporated into or onto the polymer, wherein the composition comprises a carrier system, and wherein the device is implanted or injected into ocular surface tissue, adnexal tissue in contact with ocular surface tissue, a fluid-filled ocular or adnexal cavity, or an ocular or adnexal cavity.

[0288] Exemplary mucoadhesive polyanionic natural or semisynthetic polymers that can form the device include, but are not limited to, polygalacturonic acid, hyaluronic acid, carboxymethyl amylose, carboxymethyl chitosan, chondroitin sulfate, heparin sulfate, and mesoglycan. In one embodiment, the device comprises a biocompatible polymer matrix that can optionally be fully or partially biodegradable. A hydrogel is an example of a suitable polymer matrix material. Examples of materials that can form hydrogels include polylactic acid, polyglycolic acid, PLGA polymers, alginate and alginate derivatives, gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids such as polypeptides, particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon-caprolactone, polyanhydrides; polyphosphazenes, polyvinyl alcohol, poly(oxyalkylenes), particularly poly(ethylene oxide), poly(allylamine) (PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamer, poly(uronic acid), poly(vinyl pyrrolidone), and copolymers thereof, 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, fibrin, hyaluronic acid, agarose, and laminin-rich gels.

[0289] A preferred hydrogel material is alginate or a modified alginate material. Alginate molecules are composed 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., Ca+2, Mg+2, Ba+2) and form stable hydrogels when exposed to these molecules.

[0290] The device can be administered topically, subconjunctivally or in the episcleral space, subcutaneously, or intraductally. Specifically, the device is placed on the surface of or just below the ocular tissue. Alternatively, the device is placed inside the tear duct or gland. The composition incorporated into or onto the polymer is released or diffused from the device.

[0291] 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 the contact lens dissolves over time while releasing the composition into closely juxtaposed tissue. Similarly, in various embodiments, the drug delivery device is optionally biodegradable or permanent.

[0292] For example, the composition is incorporated into or applied to the lenses. The composition is chemically bound or physically entrapped by the contact lens polymer. Alternatively, the color additive is chemically bound or physically entrapped 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 dose of the therapeutic composition that remains bound or entrapped in the polymer. Alternatively, or in addition, an ultraviolet (UV) absorber is chemically bound or physically entrapped within the contact lens polymer. The contact lens polymer is hydrophobic or hydrophilic.

[0293] Exemplary materials for making hydrophobic lenses having a means for 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, crifocon A, crifocon B, dimefocon A, enflufocon A, enflofocon B, erifocon A, flurofocon A, flusilfocon A, flusilfocon B, flusilfocon C, flusilfocon D, flusilfocon E, 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 CC), 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, sulfocon A, sulfocon B, telafocon A, tisilfocon A, tolofocon A), trifocon A, unifocon A, vinafocon A and wilofocon A. Exemplary materials for making hydrophilic lenses having a means for 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, droxfilcon A, and A), elastofilcon A, epsilonfilcon A, esterifilcon A, etafilcon A, focofilcon AA), Galyfilcon A, Genfilcon A, Govafilcon 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, Lotrafilcon B, Mafilcon A, Mesafilcon A), methafilcon B, mipafilcon A, nelfilcon A, netrafilcon A, ocufilcon A, ocufilcon B, C, ocufilcon D, ocufilcon E, ofilcon A, omafilcon A, oxyfilcon A, pentafilcon A, perfllcon A, pevafilcon A, phemfilcon A, polymacon, senofilcon A, silafilcon A, siloxyfilcon A, surfilcon A, tefilcon A A), tetrafilcon A, trilfilcon A, vmcon A, viflcon B and xylofilcon A.

[0294] Compositions formulated as gels or gel-like substances, emulsions, or viscous emulsions are within the scope of the present 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 that does not adversely affect the area being treated and is suitable for formulating compositions and pharmaceutical compositions for topical administration to the skin, eye, or mucous membranes may be used. For example, the gelling component can be selected from the group consisting of acrylic acid, carbomer, carboxypolymethylene, such materials sold under the trademark Carbopol (e.g., Carbopol 940) by BF Goodrich Company, polyethylene-polypropylene glycol, such materials sold under the trademark Poloxamer by BASF (e.g., Poloxamer 188), cellulose derivatives, such as hydroxypropylcellulose, hydroxyethylcellulose, hydroxyethylenecellulose, methylcellulose, carboxymethylcellulose, propylene glycol alginate, polyvinylpyrrolidone, veegum (magnesium aluminum silicate), Pemulen, Simulgel (e.g., Simulgel 600, Simulgel EG and Simulgel NS), Capigel, Colafax, plasdones, etc., and mixtures thereof.

[0295] The gel or gel-like substance according to the present 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.

[0296] The gel-like substances of the present invention include hydrogels, colloidal gels formed as dispersions in water or other aqueous media. Thus, a hydrogel forms after colloid formation, where the dispersed phase (colloid) combines with the continuous phase (i.e., water) to produce a viscous, jelly-like product; for example, coagulated silicic acid. A hydrogel is a three-dimensional network of hydrophilic polymer chains cross-linked by chemical or physical bonds. Due to the hydrophilic nature of the polymer chains, the hydrogel absorbs water and swells. The swelling process is similar to the dissolution of non-crosslinked hydrophilic polymers. By definition, water constitutes at least 10% of the total weight (or volume) of the hydrogel.

[0297] Examples of hydrogels include synthetic polymers such as polyhydroxyethylmethacrylate, as well as chemically or physically cross-linked polyvinyl alcohol, polyacrylamide, poly(N-vinylpyrrolidone), polyethylene oxide, and hydrolyzed polyacrylonitrile. Examples of hydrogels that are organic polymers include covalently or ionically cross-linked polysaccharide-based hydrogels such as alginates, pectins, carboxymethylcellulose, heparin, multivalent metal salts of hyaluronate, and hydrogels derived from chitin, chitosan, pullulan, gellan, and xanthan gum. The specific hydrogels used in our experiments were cellulosic compounds (i.e., hydroxypropyl methylcellulose [HPMC]) and high molecular weight hyaluronic acid (HA).

[0298] Hyaluronic acid is a polysaccharide produced by various body tissues. U.S. Patent No. 5,166,331 discusses different fractions of purified hyaluronic acid for use as a substitute for intraocular fluid and as a carrier for topical ophthalmic drugs. Other U.S. patent applications discussing ophthalmic uses of hyaluronic acid include Serial Nos. 11 / 859,627; 11 / 952,927; 10 / 966,764; 11 / 741,366; and 11 / 039,192. Macromolecular 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; 60 / 567,423; and 11 / 695,527. The use of various active agents is known with high-viscosity hyaluronic acid. 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.

[0299] Sustained release formulations as described in WO2010048086 are within the scope of the present invention.

[0300] Those skilled in the art are familiar with standard methods for incorporating polynucleotides or vectors into host cells, such as transfection, lipofection, electroporation, microinjection, viral infection, heat shock, chemical permeabilization of membranes followed by transformation, or cell fusion.

[0301] As used herein, the term "host cell or genetically engineered host cell" refers to a host cell that has been transduced, transformed or transfected with the aforementioned constructs or vectors.

[0302] As representative examples of suitable host cells, bacterial cells, such as Escherichia coli, Streptomyces (Streptomyces), Salmonella typhimurium (Salmonella typhimurium), fungal cells such as yeast, insect cells such as Sf9, animal cells such as CHO or COS, plant cells, etc. can be enumerated. According to the teachings of this article, the selection of suitable hosts is considered to be within the scope of those skilled in the art. Preferably, the host cell is an animal cell, most preferably, a human cell. The present 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 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.

[0303] In the case of ex vivo gene therapy, the host cell may be a cell isolated from a patient, such as a hematopoietic stem cell, which, after introduction of a transgene, is reintroduced into the patient in need thereof.

[0304] AAV-based viral delivery system

[0305] Construction of AAV vectors can be performed according to procedures and using techniques known to those skilled in the art. The theory and practice of adeno-associated virus vector construction and its use in therapy are described in several scientific and patent publications (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.2006Mar;13(3):463-83).

[0306] Suitable administration forms of pharmaceutical compositions containing AAV vectors include, but are not limited to, injectable solutions or suspensions, eye washes, and eye ointments. In a preferred embodiment, the AAV vector is administered by intrathecal injection. In a particularly preferred embodiment, the AAV vector is administered in the anterior chamber or the retrobulbar space and in the vitreous body as a subretinal injection. Preferably, the viral vector is delivered by 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).

[0307] The dose of virus used for treatment should be determined on a case-by-case basis, depending on the route of administration, severity of the disease, general condition of the patient, and other clinical parameters. In general, an appropriate dose range is 10 8 to 10 13 vg (vector genome) / eye.

[0308] Intrinsic protein

[0309] Inteins are protein fragments that can cleave themselves and join the remaining parts (exteins) with peptide bonds in a process called protein splicing. This fragment is called "intrin," representing the internal protein sequence, and "extein," representing the external protein sequence, with the upstream extein being called "N-extein" and the downstream extein being called "C-extein." The products of the protein splicing process are two stable proteins: the mature protein and the intein.

[0310] An intein may also exist as two fragments encoded by two separately transcribed and translated genes, referred to herein as a "split intein."

[0311] The inteins of the present invention include, but are not limited to, the split-type inteins listed in the New England Biolabs intein database disclosed in (66).

[0312] Split inteins can be generated starting from inteins by first removing the homing endonuclease domain sequence to generate a small intein. The small intein can then be cleaved at one or more sites that are aligned with the protein sequence of inteins of known crystal structure to generate split intein designs and assayed for trans-splicing activity according to the protocols included in this disclosure.

[0313] The desired properties of split-type inteins, including activity, efficiency, versatility, and stability, can be further improved by site-directed mutagenesis or modification of intein sequences based on rational design, and / or by directed evolution using methods such as functional selection, phage display, and ribosome display.

[0314] An example of a split-type intein is an intein derived from DnaE, ​​which is the catalytic subunit a of DNA polymerase III in cyanobacteria and is encoded by two independent genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene is referred to herein as the "N-intein." The intein encoded by the dnaE-c gene is referred to herein as the "C-intein." Typically, the N-part of a split-type intein is referred to as the "N-intein," and the C-part of a split-type intein is referred to as the "C-intein." Split-type inteins self-associate and catalyze trans protein splicing activity (referred to herein as "trans-splicing").

[0315] Other examples of split-type endonucleases of the present invention include: endonucleases of DnaE from Nostoc punctata (Npu) (27, 28), represented 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 of DnaB from Rhodothermus marinus (Rma) (29), represented in the table 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 DnaE from Synechocystis sp. strain PCC6803 (Ssp (SEQ ID 6), (30); the N-endonucleases and C-endonucleases of Synechocystis sp. strain PCC6803 are represented as SEQ.ID 4, respectively. 13 and 14 are included in the table below. Other endonuclease systems may also be used. For example, synthetic rapid endonucleases based on the dnaE endonuclease, Cfa-N and Cfa-C endonuclease pairs have been described (e.g., (31) and WO 2017 / 132580, which are incorporated herein by reference). Additional endonucleases have been described in U.S. Patent No. 8,394,604, including the SspGyrB endonuclease, the SspDnaX endonuclease, the TerDnaE3 endonuclease, the TerThyX endonuclease and the CnePrp8 endonuclease. Further endonucleases in the present invention are those disclosed in WO2018071868, wherein the first pair of endonucleases are listed in the table below and are designated as SEQ ID 9 (N-endonuclease) and SEQ ID 10 (C-endonuclease); the second pair of endonucleases are listed, e.g., SEQ ID 11 and SEQ ID 12.

[0316] Alternatively, the intein system can be a ligand-dependent intein that exhibits no or minimal protein splicing activity in the absence of a ligand (e.g., a small molecule, such as 4-hydroxytamoxifen, a peptide, a protein, a polynucleotide, an amino acid, and a nucleotide). Ligand-dependent inteins include, for example, those described in US2014 / 0065711 A1, which is incorporated herein by reference.

[0317] Table 3: Examples of split-type proteins of the present invention

[0318]

[0319]

[0320]

[0321] As described herein, proteins derived from the same gene of different organisms that retain trans-splicing activity are within the scope of the present invention. As a non-limiting example, the DNA-E split-type protein can be derived from the split-type protein of the DnaE gene (e.g., DNA polymerase III subunit α) of cyanobacteria, including Candida punctata (Npu), Synechocystis PCC6803 (Ssp), Feldspar PCC 9605, Bifidobacterium, Pseudocladus monofidum, Cyanobacterium SW_9_47_5, Nodularia foamycis, Nodularia hair-like, Varroa crocodile algae WH 8502, Cuban chromococcus CCALA 043, and Trichoderma rubrum. As another example, the DNA-B split-type protein can be derived from the DnaB gene from cyanobacteria, including, for example, Rhabdothermosphaerella marineensis (Rma), Synechocystis PC6803 (Ssp), and Porphyra purpurogenum (Ppu), as described in (59).

[0322] Thus, the split-inteins of the present invention may be 100%, 98%, 80%, 75%, 70%, 65%, 50% identical to a naturally occurring intein, wherein the intein retains the ability to undergo a trans-splicing reaction. Fragments of naturally occurring or modified inteins that retain trans-splicing activity are within the scope of the present invention.

[0323] See, for example, the alignment between Npu (Nostoc punctata) DnaE and Synechocystis sp. PCC6803 N-intrin:

[0324]

[0325]

[0326]

[0327] Also, alignment between Npu (Nostoc punctata) DnaE and Synechocystis sp. PCC6803 C-intrin:

[0328]

[0329]

[0330] Therefore, split-form intein variants and fragments of the intein of the present invention that retain trans-splicing activity are also within the scope of the present invention.

[0331] Interestingly, inteins are reported to have conserved functional features that ensure their splicing activity. Specifically, four intein motifs have been identified (for their consensus sequences, see below): blocks AH (Pietrokovski 1994 and Perler 1997) and blocks N2 and N4 (Pietrokovski 1998). Intein blocks A, N2, B, N4, F and G are involved in protein splicing. Blocks C, D, E, H are located in the nuclease domain, which is absent from the split-type intein. Therefore, the split-type intein retains the conserved motifs that are essential for trans-splicing activity. (Intein database, disclosed in [Perler, FB (2002), InBase, the Intein Database, Nucleic Acids Res. 30, 383-384]).

[0332]

[0333] 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.

[0334] Comparison of proteins within the present invention:

[0335] CLUSTAL W alignment of all listed N-intrins:

[0336]

[0337]

[0338] CLUSTAL 2.1 multiple sequence alignment of all C-intrins listed

[0339]

[0340] In summary, intein activity is context-dependent, with certain peptide sequences surrounding the junction (termed N- and C-exonins) being necessary for efficient trans-splicing to occur. The most important of these is an amino acid containing a nucleophilic thiol or hydroxyl group (e.g., cysteine, serine, or threonine) as the first residue in the C-exonin.

[0341] The present inventors have used intein-mediated protein trans-splicing to reconstitute large proteins in vivo. Split intein, encoded by intein gene sequences, is produced as a precursor polypeptide that, through their structural complementation, can reassemble and catalyze protein trans-splicing reactions.

[0342] In the context of protein trans-splicing, the N-intrin gene is fused in frame to the sequence encoding the N-terminal portion of the target protein; the C-intrin gene is fused in frame to the sequence encoding the C-terminal portion of the target sequence. After expression of the two precursor fusion proteins, the intrin undergoes autocatalytic cleavage and forms a linked exon, such as the reconstituted target protein.

[0343] Therefore, the reconstruction of the protein of interest requires that the protein be divided into two or three fragments, the coding sequences of which are cloned into AAV vectors, fused to the N- or C-intrin and under the control of a promoter. The split point of each protein is selected taking into account the amino acid requirements at the junction as the first residue in the C-exon protein (e.g., the presence of an amino acid containing a nucleophilic thiol or hydroxyl group (i.e., cysteine, serine, or threonine)) and taking into account the maintenance of the integrity of key protein domains to facilitate the correct protein folding and stability of each intrin-polypeptide precursor polypeptide and the resulting reconstructed protein.

[0344] Of particular note, the inventors selected two attachment points within the proteins of interest: the ABCA4 protein is cleaved at amino acids Cys1150, Ser1168, and Ser1090, and the split-type protein is inserted at the cleavage point. The CEP290 protein is cleaved at aa Cys1076, Ser1275, Cys929, and 1474; Ser 453, and Cys 1474.

[0345] Degradation signal

[0346] Regulated protein degradation protects cells from misfolded, aggregated, or otherwise abnormal proteins and also controls the levels of proteins that have evolved to be short-lived in vivo and is primarily mediated by the ubiquitin (Ub)-proteasome system (UPS) and the autophagy-lysosome pathway, of which molecular chaperones are part.

[0347] Degradation signals are characteristics of proteins that make them targets for protein degradation pathways, thereby reducing their half-life. Specifically, N-degrons and C-degrons are degradation signals whose primary determinants are the N-terminal and C-terminal residues of cellular proteins, respectively. N-degrons and C-degrons include, to varying degrees, adjacent sequence motifs and also include internal lysine residues that function as polyubiquitination sites.

[0348] Within the meaning of the present invention, an internal degron is defined as a degradation signal located within a protein sequence that is neither at the N-terminus nor at the C-terminus and whose functionally essential elements do not include the N-terminal residue or the C-terminal residue and that mediates protein degradation.

[0349] Degron pathways encompass multiple groups of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-, C-, or internal-degrons, leading to their degradation by the 26S proteasome or autophagy.

[0350] Escherichia coli dihydrofolate reductase (ecDHFR) is a 159-residue enzyme that catalyzes the reduction of dihydrofolate to tetrahydrofolate, an essential cofactor 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 it inhibits mammalian DHFR. This large therapeutic window renders TMP “biosilent” in mammalian cells. The specificity of the ecDHFR-TMP interaction, as well as the commercial availability and attractive pharmacological properties of TMP, make this protein-ligand pair ideal for development as a degradation system. (69) Therefore, the presence of a DHFR amino acid sequence within a protein, preferably an ecDHFR amino acid sequence, functions as a targeting signal for the proteasome system, leading to protein degradation. In the presence of TMP, the protein is stabilized.

[0351] Conveniently, the ecDHFR-derived degron signal carrying point mutations developed by Iwamoto et al. includes three amino acid mutations, R12Y, Y100I, and G67S (69), which confer functional activity (e.g., degradation of fusion proteins) only when placed within the N-terminus or internal positions.

[0352] The inventors who identified the shortest active peptide have further improved the ecDHFR-derived degron. Advantageously, the shorter sequence allows for the installation of longer coding sequences in the same AAV vector.

[0353] In the present invention, the ecDHFR-derived degron is fused to the N-terminus of the intein, where it is inactive. Upon protein trans-splicing, the degron is localized within the reconstructed intein and mediates its degradation.

[0354] The ecDHFR of the present invention is WT ecDHFR, mutant DHFR, full-length ecDHFR, or shorter scDHFR.

[0355] DHFR can range from 105 to 159 aa in length, with shortening occurring at the C-terminus.

[0356] ecDHFR E. coli-derived, wild-type

[0357] Nucleotide sequence :(623nt)SEQ ID No.27

[0358]

[0359] Amino acid sequence:

[0360] 159aa-WT SEQ ID No. 28

[0361]

[0362] ecDHFR E. coli-derived, internal degron mutant (159aa)

[0363] The mutation position is shown in bold - SEQ ID No. 29

[0364]

[0365] ecDHFR E. coli-derived, wild-type, minimal active fragment

[0366] Nucleotide sequence: SEQ ID No. 30

[0367]

[0368] Amino acid sequence SEQ ID No.31

[0369]

[0370] ecDHFR E. coli derived, internal degradation determinant mutant pe, minimal active fragment (104aa)

[0371] (The mutation position is shown in bold) SEQ ID No. 32

[0372]

[0373] sequence

[0374] The coding sequence of the present invention can be operably 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, heart cells, neuronal cells, kidney cells, endothelial cells. Exemplary promoters include, but are not limited to, ubiquitous, artificial or tissue-specific promoters, including fragments and variants thereof that retain transcriptional promoter activity, such as photoreceptor-specific promoters, including photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1), interphotoreceptor retinoid binding protein promoter (IRBP), rhodopsin promoter (RHO), vitelliform macular dystrophy 2 promoter (VMD2), rhodopsin kinase promoter (RK); muscle-specific promoters, including MCK, MYODI; liver-specific promoters, including thyroxine binding globulin (TBG), hybrid liver-specific promoter (HLP) (67); neuron-specific promoters, including hSYN1, CaMKIIa; kidney-specific promoters, including Ksp-cadherin 16, NKCC2. Ubiquitous promoters according to the invention are for example the ubiquitous cytomegalovirus (CMV) (32) and short CMV (33) promoters.

[0375] Optionally, the promoter sequence includes an enhancer sequence, such as the globin IgG chimeric intron.

[0376] For the purposes of the present 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, in particular in photoreceptor cells, and the coding sequences are preferably selected from the sequences attached herein, or sequences encoding the same amino acid sequence due to the degeneracy of the genetic code.

[0377] Exemplary polyadenylation signals include, but are not limited to, the bovine growth hormone polyadenylation signal (bGHpA), the human beta globin polyadenylation signal or a short synthetic form (68), the SV40 polyadenylation signal, or other natural or artificial polyadenylation signals.

[0378] The present invention provides the use of a nucleotide sequence of a degradation signal to reduce the stability of a reconstituted intein. Conveniently, one or more sequences may be repeated to maintain maximum effect.

[0379] Suitable degradation signals according to the present invention include: (i) the short degron CL1, a C-terminal destabilizing peptide that shares structural similarity with misfolded proteins and is therefore recognized by the ubiquitination system, (ii) ubiquitin, which, when fused at the N-terminus of the donor protein, mediates direct protein degradation or degradation via the N-end rule pathway, and (iii) the N-terminal PB29 degron, which is a 9-amino acid peptide that, like the CL1 degron, is predicted to fold into a structure recognized by enzymes of the ubiquitination pathway, variant ecDHFR and fragments thereof described herein and (69), particularly ecDHFR-derived degron signals carrying point mutations, including the three amino acid mutations R12Y, Y100I, and G67S, which confer functional activity (e.g., degradation of the fusion protein) only when placed at the N-terminus or within internal positions.

[0380] Exemplary degradation signals are described in WO 201613932, which is incorporated herein by reference.

[0381] Those skilled in the art will readily appreciate that, in addition to variants that can be artificially created by a skilled laboratory technician, there may be many variant sequences of proteins found in nature. The polynucleotides and polypeptides of the present invention include the polynucleotides and polypeptides specifically exemplified herein, as well as any natural variants thereof, and any variants that can be artificially created, as long as those variants retain the desired functional activity. In addition, polypeptides having the same amino acid sequence as the polypeptides exemplified herein, except for amino acid substitutions, additions, or deletions within the polypeptide sequence, are also within the scope of the present invention, as long as these variant polypeptides retain substantially the same relevant functional activity as the polypeptides specifically exemplified herein. For example, conservative amino acid substitutions within a polypeptide that do not affect the function of the polypeptide are within the scope of the present invention. Therefore, the polypeptides disclosed herein should be understood to include variants and fragments of the specific example sequences discussed above. The present invention also includes nucleotide sequences encoding the polypeptides disclosed herein. These nucleotide sequences can be easily constructed by those skilled in the art with knowledge of the protein and amino acid sequences provided herein. As understood by those skilled in the art, the degeneracy of the genetic code enables technicians to construct a variety of nucleotide sequences encoding a particular polypeptide or protein. The selection of a particular nucleotide sequence may depend, for example, on the codon usage of a particular expression system or host cell. Polypeptides having amino acid substitutions other than those specifically exemplified in the subject polypeptides are also included within the scope of the present invention. For example, non-natural amino acids can replace the amino acids of the polypeptides of the present invention as long as the polypeptide with the substituted amino acids retains the same activity as the polypeptide with the unsubstituted amino acids. 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-aminocaproic acid, 6-aminocaproic acid, 2-aminoisobutyric acid, 3-aminopropionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, τ-butylglycine, τ-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino 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 with derivatized side groups. In addition, any amino acid in a protein can be in either the D (dextrorotatory) or L (levorotatory) form. Amino acids can generally be classified into the following categories: non-polar, uncharged polar, basic, and acidic. Conservative substitutions, which refer to replacing a polypeptide with one class of amino acids with another amino acid of the same class, fall within the scope of the present invention as long as the polypeptide with the substitution retains substantially the same biological activity as the polypeptide without the substitution. Table 4 provides a list of examples of amino acids belonging to each class.

[0382]

[0383] 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 the present invention, as long as these variant polynucleotides retain substantially the same relevant functional activity as the polynucleotides specifically exemplified herein (e.g., they encode proteins having the same amino acid sequence or the same functional activity as those encoded by the exemplified polynucleotides). Thus, the polynucleotides disclosed herein should be understood to include variants and fragments of the specific exemplified sequences discussed above.

[0384] The present invention also contemplates polynucleotide molecules having sequences sufficiently homologous to the polynucleotide sequences of the present invention to allow hybridization thereto under standard stringent conditions and standard methods (Maniatis, T. et al., 1982). The polynucleotides described herein may also be defined in terms of more specific identity and / or similarity ranges to the polynucleotides exemplified herein. Sequence identity is typically 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 identities and / or similarities of the sequences compared to the sequences exemplified 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 indicated, as used herein, percent sequence identity and / or similarity between two sequences can be determined using the algorithm of Karlin and Altschul (1990), as modified as described in Karlin and Altschul (1993). Such an algorithm has been incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used, as described in Altschul et al. (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See the NCBI / N1H website.

[0385] Plasmid of the present invention

[0386]

[0387]

[0388]

[0389] EGFP

[0390] p915_pAAV2.1-TBG-5′EGFP inner protein (SEQ ID No. 33)

[0391] (seqA is at the 5′ beginning of the sequence)

[0392] TBG promoter: bold (seq B)

[0393] 5′EGFP: underlined (seq C)

[0394] (seq D)

[0395] 3xflag: italic (seq E)

[0396] WPRE: italic underline (seq F)

[0397] Bgh PolyA: bold underline (seq G)

[0398] (seq H is at the 3′ beginning of the sequence)

[0399]

[0400]

[0401] P917_pAAV2.1-TBG-3'EGFP protein

[0402] 5'ITR(seqA)

[0403] TBG promoter (seq B)

[0404] C-intein Npu DnaE (seq I) SEQ ID No.34

[0405]

[0406] 3′EGFP (seq L) SEQ ID No. 35

[0407]

[0408] 3xflag(seq E)

[0409] WPRE(seq F)

[0410] Bgh PolyA(seq G)

[0411] 3′ITR (seq H)

[0412] p914_pAAV2.1-CMV-5′EGFP inner protein

[0413] 5′ITR(seqA)

[0414] CMV promoter (seq M) SEQ.36

[0415]

[0416] 5′EGFP (seq C)

[0417] N-intrin Npu DnaE (seq D)

[0418] 3xflag(seq E)

[0419] WPRE(seq F)

[0420] Bgh PolyA(seq G)

[0421] 3′ITR (seq H)

[0422] p916_pAAV2.1-CMV-3′EGFP protein

[0423] 5′ITR(seqA)

[0424] CMV promoter (seq M)

[0425] C-intrin Npu DnaE (seq I)

[0426] 3′EGFP(seq L)

[0427] 3xflag(seq E)

[0428] WPRE(seq F)

[0429] Bgh PolyA(seq G)

[0430] 3′ITR (seq H)

[0431] p932_pAAV2.1-GRK1-5′EGFP protein

[0432] 5′ITR(seqA)

[0433] GRK1 promoter (seq N) SEQ.37

[0434]

[0435] 5′EGFP (seq C)

[0436] N-intrin Npu DnaE (seq D)

[0437] 3xflag(seq E)

[0438] WPRE(seq F)

[0439] Bgh PolyA(seq G)

[0440] 3′ITR (seq H)

[0441] p933_pAAV2.1-GRK1-3′EGFP protein

[0442] 5′ITR(seqA)

[0443] GRK1 promoter (seq N)

[0444] C-intrin Npu DnaE (seq I)

[0445] 3′EGFP(seq L)

[0446] 3xflag(seq E)

[0447] WPRE(seq F)

[0448] Bgh PolyA(seq G)

[0449] 3′ITR (seq H)

[0450] p36 pAAV2.1-CMV-5′EGFP protein_ecDHFR

[0451] 5′ITR(seqA)

[0452] CMV promoter (seq M)

[0453] 5′EGFP (seq C)

[0454] N-intrin Npu DnaE (seq D)

[0455] 3xflag(seq E)

[0456] ecDHFR (seq O) SEQ ID No. 38

[0457]

[0458] WPRE(seq F)

[0459] Bgh PolyA(seq G)

[0460] 3′ITR (seq H)

[0461] p37 pAAV2.1-CMV-5′EGFP protein_small ecDHFR

[0462] 5′ITR(seqA)

[0463] CMV promoter (seq M)

[0464] 5′EGFP (seq C)

[0465] N-intrin Npu DnaE (seq D)

[0466] 3xflag(seq E)

[0467] Small ecDHFR (seq P) SEQ ID No. 39

[0468]

[0469] WPRE(seqF)

[0470] Bgh PolyA(seq G)

[0471] 3′ITR (seq H)

[0472] p902_pAAV2.1-CMV-5′EGFP internal protein DnaB

[0473] 5′ITR(seqA)

[0474] CMV promoter (seq M)

[0475] 5′EGFP (seq C)

[0476] N-internal protein RmaDnaB(seq Q)SEQ.ID No.40

[0477]

[0478] N-intrin Npu DnaE (seq D)

[0479] 3xflag(seq E)

[0480] WPRE(seq F)

[0481] Bgh PolyA(seq G)

[0482] 3′ITR (seq H)

[0483] p903_pAAV2.1-CMV-3′EGFP internal protein DnaB

[0484] 5′ITR(seqA)

[0485] CMV promoter (seq M)

[0486] C-intrin Rma DnaB (seq R) SEQ.ID No.41

[0487]

[0488] 3′EGFP(seq L)

[0489] 3xflag(seq E)

[0490] WPRE(seq F)

[0491] Bgh PolyA(seq G)

[0492] 3′ITR (seq H)

[0493] p1256_pAAV2.1-CMV-5′EGFP internal protein mDnaE

[0494] 5′ITR(seqA)

[0495] CMV promoter (seq M)

[0496] 5′EGFP (seq C)

[0497] N-intrin mDnaE (seq S) SEQ ID No. 42

[0498]

[0499] 3xflag(seq E)

[0500] WPRE(seq F)

[0501] Bgh PolyA(seq G)

[0502] 3′ITR (seq H)

[0503] p1257 pAAV2.1-CMV-3′EGFP protein mDNAE

[0504] 5′ITR(seqA)

[0505] CMV promoter (seq M)

[0506] C-intrin mDnaE (seq T) SEQ.43

[0507]

[0508] 3′EGFP(seq L)

[0509] 3xflag(seq E)

[0510] WPRE(seq F)

[0511] Bgh PolyA(seq G)

[0512] 3′ITR (seq H)

[0513] CEP290

[0514] p1005 pAAV2.1-CMV260-5′CEP290 inner protein (Group 1)

[0515] 5′ITR(seqA)

[0516] CMV260 (seq U) SEQ ID No. 44

[0517]

[0518] 5′CEP290: SEQ ID No. 45

[0519]

[0520]

[0521] N-intrin DnaE (seq D)

[0522] 3xflag(seq E)

[0523] shPolyA (seq V) SEQ ID No. 46

[0524]

[0525] 3′ITR (seq H)

[0526] p1093 pAAV2.1-CMV260-3′CEP290 inner protein (Group 1)

[0527] 5′ITR(seqA)

[0528] CMV260(seq U)

[0529] 3′CEP290: SEQ ID No. 47

[0530]

[0531]

[0532]

[0533] C-intrin DnaE (seq I)

[0534] 3xflag(seq E)

[0535] shPolyA(seq V)

[0536] 3′ITR (seq H)

[0537] p1065 pAAV2.1-CMV260-5′CEP290 inner protein (Group 2)

[0538] 5′ITR(seqA)

[0539] CMV260(seq U)

[0540] 5′CEP290: SEQ ID No. 48

[0541]

[0542]

[0543]

[0544] N-intrin DnaE (seq D)

[0545] 3xflag(seq E)

[0546] Bgh PolyA(seq G)

[0547] 3′ITR (seq H)

[0548] p1067pAAV2.1-CMV260-3′CEP290 inner protein (Group 2)

[0549] 5′ITR(seqA)

[0550] CMV260(seq U)

[0551] 3′CEP290: SEQ ID No. 49

[0552]

[0553]

[0554]

[0555] C-intrin DnaE (seq I)

[0556] 3xflag(seq E)

[0557] Bgh PolyA(seq G)

[0558] 3′ITR (seq H)

[0559] p1087 pAAV2.1-CMV260-5′CEP290 inner protein (Group 3)

[0560] 5′ITR(seqA)

[0561] CMV260(seq U)

[0562] 5′CEP290: SEQ ID No. 50

[0563]

[0564]

[0565] N-intrin mDnaE (seq S)

[0566] 3xflag(seq E)

[0567] Bgh PolyA(seq G)

[0568] 3′ITR (seq H)

[0569] p1088 pAAV2.1-CMV260-3′CEP290 inner protein (Group 3)

[0570] 5′ITR(seqA)

[0571] CMV260(seq U)

[0572] 3′CEP290: SEQ ID No. 51

[0573]

[0574]

[0575] C-intrin mDnaE (seq T)

[0576] 3xflag(seq E)

[0577] Bgh PolyA(seq G)

[0578] 3′ITR (seq H)

[0579] p1182 pAAV2.1-CMV260-5′CEP290 inner protein (Group 4)

[0580] 5′ITR(seqA)

[0581] CMV260(seq U)

[0582] 5′CEP290: SEQ ID No. 52

[0583]

[0584] N-intrin DnaE (seq D)

[0585] 3xflag(seq E)

[0586] WPRE(seq F)

[0587] Bgh PolyA(seq G)

[0588] 3′ITR (seq H)

[0589] p1183pAAV2.1-CMV260-CEP290 in vivo protein (Group 4)

[0590] 5′ITR(seqA)

[0591] CMV260(seq U)

[0592] C-intrin DnaE (seq I)

[0593] CEP290 body: SEQ ID No.53

[0594]

[0595] N-intrin Rma DnaB (seq Q)

[0596] 3xflag(seq E)

[0597] WPRE(seq F)

[0598] Bgh PolyA(seq G)

[0599] 3′ITR (seq H)

[0600] p1181pAAV2.1-CMV260-3′CEP290 protein (Group 4 / Group 5)

[0601] 5′ITR(seqA)

[0602] CMV260(seq U)

[0603] C-intrin Rma DnaB (seq R)

[0604] 3′CEP290: SEQ ID No. 54

[0605]

[0606]

[0607] 3xflag(seq E)

[0608] WPRE(seq F)

[0609] Bgh PolyA(seq G)

[0610] 3′ITR (seq H)

[0611] p1179 pAAV2.1-CMV260-5′CEP290 inner protein (Group 5)

[0612] 5′ITR(seqA)

[0613] CMV260(seq U)

[0614] 5′CEP290: SEQ ID No. 55

[0615]

[0616] N-intrin mDnaE (seq S)

[0617] 3xflag(seq E)

[0618] WPRE(seq F)

[0619] Bgh PolyA(seq G)

[0620] 3′ITR (seq H)

[0621] p1180 pAAV2.1-CMV260-CEP290 in vivo protein (Group 5)

[0622] 5′ITR(seqA)

[0623] CMV260(seq U)

[0624] C-intrin mDnaE (seq T)

[0625] CEP290 body: SEQ ID No.56

[0626]

[0627]

[0628] N-intrin RmaDnaB (seq Q)

[0629] 3xflag(seq E)

[0630] WPRE(seq F)

[0631] Bgh PolyA(seq G)

[0632] 3′ITR (seq H)

[0633] p1152pAAV2.1-GRK1-5′CEP290 protein (Group 5)

[0634] 5′ITR(seqA)

[0635] GRK1 promoter (seq N)

[0636] 5′CEP290: SEQ ID No. 57

[0637]

[0638]

[0639] N-intrin mDnaE (seq S)

[0640] 3xflag(seq E)

[0641] WPRE(seq F)

[0642] Bgh PolyA(seq G)

[0643] 3′ITR (seq H)

[0644] p1153 pAAV2.1-GRK1-CEP290 in vivo protein (Group 5)

[0645] 5′ITR(seqA)

[0646] GRK1 promoter (seq N)

[0647] C-intrin mDnaE (seq T)

[0648] CEP290 body: SEQ ID No.58

[0649]

[0650]

[0651] N-intrin RmaDnaB (seq Q)

[0652] 3xflag(seq E)

[0653] WPRE(seq F)

[0654] Bgh PolyA(seq G)

[0655] 3′ITR (seq H)

[0656] p1156 pAAV2.1-GRK1-3′CEP290 protein (Group 5)

[0657] 5′ITR(seqA)

[0658] GRK1 promoter (seq N)

[0659] C-intrin Rma DnaB (seq R)

[0660] 3′CEP290: SEQ ID No. 59

[0661]

[0662]

[0663] 3xflag(seq E)

[0664] WPRE(seq F)

[0665] Bgh PolyA(seq G)

[0666] 3′ITR (seq H)

[0667] pzac-GRK1-5′ABCA4 protein (Group 1) SEQ ID No. 60

[0668] 5′ITR (seq A)

[0669] GRK1: Bold

[0670] 5′ABCA4: Underline

[0671] N-intrin Npu DnaE:

[0672] 3xflag: italic

[0673] SV40: Bold Underline

[0674] 3′ITR (seq H)

[0675]

[0676]

[0677] pzac-GRK1-3′ABCA4 protein (Group 1) SEQ.No.61

[0678] 5′ITR (seq A)

[0679] GRK1: Bold

[0680] 3′ABCA4: Underline

[0681] C-intrin Npu DnaE:

[0682] 3xflag: italic

[0683] SV40: Bold Underline

[0684] 3′ITR (seq H)

[0685]

[0686]

[0687] pzac-CMV260-5′ABCA4 protein (Group 1) SEQ.No.62

[0688] 5′ITR (seq A)

[0689] CMV260: Bold

[0690] 5′ABCA4: Underline

[0691] N-intrin Npu DnaE:

[0692] 3xflag: italic

[0693] SV40: Bold Underline

[0694] 3′ITR (seq H)

[0695]

[0696]

[0697]

[0698] pzac-CMV260-3′ABCA4 protein (Group 1) SEQ.No.63

[0699] 5′ITR (seq A)

[0700] CMV260: Bold

[0701] 3′ABCA4: Underline

[0702] C-intrin Npu DnaE:

[0703] 3xflag: italic

[0704] SV40: Bold Underline

[0705] 3′ITR (seq H)

[0706]

[0707]

[0708]

[0709] p38 pAAV2.1-CMV260-5′ABCA4 protein_ecDHFR (Group 1)

[0710] 5′ITR(seqA)

[0711] CMV260(seq U)

[0712] 5′ABCA4 (from group 1)

[0713] N-intrin Npu DnaE (seq D)

[0714] 3xflag(seq E)

[0715] ecDHFR(seq O)

[0716] WPRE(seq F)

[0717] SV40 PolyA (seq W)

[0718] 3′ITR (seq H)

[0719] p39 pAAV2.1-CMV260-5′ABCA4 protein_small ecDHFR (Group 1)

[0720] 5′ITR(seqA)

[0721] CMV260(seq U)

[0722] 5′ABCA4 (from group 1)

[0723] N-intrin Npu DnaE (seq D)

[0724] 3xflag(seq E)

[0725] Small ecDHFR (seq P)

[0726] WPRE(seq F)

[0727] SV40 PolyA (seq W)

[0728] 3′ITR (seq H)

[0729] p40 pAAV2.1-GRK1-5′ABCA4 protein_ecDHFR (Group 1)

[0730] 5′ITR(seqA)

[0731] GRK1(seqN)

[0732] 5′ABCA4 (from group 1)

[0733] N-intrin Npu DnaE (seq D)

[0734] 3xflag(seq E)

[0735] ecDHFR(seq O)

[0736] WPRE(seq F)

[0737] SV40 PolyA (seq W)

[0738] 3′ITR (seq H)

[0739] p41 pAAV2.1-GRK1-5'ABCA4 inner protein_small ecDHFR (Group 1) SEQ ID No. 64

[0740] 5′ITR (seq A)

[0741] GRK1: Bold

[0742] 5′ABCA4: Underline

[0743] N-intrin Npu DnaE:

[0744] 3xflag: italic

[0745] Small ecDHFR: bold underline

[0746] SV40: Bold Underline

[0747] 3′ITR (seq H)

[0748]

[0749]

[0750] pzac-CMV260-5′ABCA4 protein (Group 2) SEQ.ID No.65

[0751] 5′ITR (seq A)

[0752] CMV260: Bold

[0753] 5′ABCA4: Underline

[0754] N-intrin Npu DnaE:

[0755] 3xflag: italic

[0756] SV40: Bold Underline

[0757] 3′ITR (seq H)

[0758]

[0759]

[0760]

[0761] pzac-CMV260-3′ABCA4 protein (Group 2) SEQ.ID No.66

[0762] 5′ITR (seq A)

[0763] CMV260: Bold

[0764] 3′ABCA4: Underline

[0765] C-intrin Npu DnaE:

[0766] 3xflag: italic

[0767] SV40: Bold Underline

[0768] 3′ITR (seq H)

[0769]

[0770]

[0771]

[0772] pzac-CMV260-5′ABCA4 protein (Group 3) SEQ.No.67

[0773] 5′ITR (seq A)

[0774] CMV260: Bold

[0775] 5′ABCA4: Underline

[0776] N-intrin Npu DnaE:

[0777] 3xflag: italic

[0778] SV40: Bold Underline

[0779] 3′ITR (seq H)

[0780]

[0781]

[0782]

[0783] pzac-CMV260-3′ABCA4 protein (Group 3) SEQ.ID No.68

[0784] 5′ITR (seq A)

[0785] CMV260: Bold

[0786] 3′ABCA4: Underline

[0787] C-intrin Npu DnaE:

[0788] 3xflag: italic

[0789] SV40: Bold Underline

[0790] 3′ITR (seq H)

[0791]

[0792]

[0793]

[0794] p836 (IRBP_DsRed) SEQ ID No. 69

[0795] 5′ITR (seq A)

[0796] IRBP Bold

[0797] WPRE: italic underline

[0798] DsRed: Underline

[0799] BghpA: bold underline

[0800] 3′ITR (seq H)

[0801]

[0802]

[0803] p1232 pAAV2.1_HLP_5′F8 inner protein (Group 1)

[0804] 5′ITR(seqA)

[0805] HLP promoter (seq J) SEQ.ID No.70

[0806]

[0807] F8 signal sequence (seq K) SEQ ID No.71

[0808]

[0809] 5′F8: SEQ ID No. 72

[0810]

[0811]

[0812]

[0813] N-intrin Npu DnaE (seq D)

[0814] 3xflag(seq E)

[0815] shPolyA(seq V)

[0816] 3′ITR (seq H)

[0817] p1389 pAAV2.1_HLP_3′F8 internal protein (Group 1)

[0818] 5′ITR(seqA)

[0819] HLP promoter (seq J)

[0820] F8 signal sequence (seq K)

[0821] C-intrin Npu DnaE (seq I)

[0822] 3′F8: SEQ ID No. 73

[0823]

[0824]

[0825] 3xflag(seq E)

[0826] shPolyA(seq V)

[0827] 3′ITR (seq H)

[0828] p1207 pAAV2.1_HLP_5′F8 inner protein (Group 2)

[0829] 5′ITR(seqA)

[0830] HLP promoter (seq J)

[0831] F8 signal sequence (seq K)

[0832] 5′F8 (Group 2): SEQ ID No. 74

[0833]

[0834]

[0835] N-intrin Npu DnaE (seq D)

[0836] 3xflag(seq E)

[0837] shPolyA(seq V)

[0838] 3′ITR (seq H)

[0839] p1388 pAAV2.1_HLP_3′F8 inner protein (Group 2)

[0840] 5′ITR(seqA)

[0841] HLP promoter (seq J)

[0842] F8 signal sequence (seq K)

[0843] C-intrin Npu DnaE (seq I)

[0844] 3′F8: SEQ ID No. 75

[0845]

[0846]

[0847] 3xflag(seq E)

[0848] shPolyA(seq V)

[0849] 3′ITR (seq H)

[0850] The invention will now be illustrated by way of non-limiting examples.

[0851] Materials and methods

[0852] Generation of AAV vector plasmids

[0853] The plasmids used for AAV vector generation were derived from pAAV2.1 (36) or pZac (37) plasmids containing the ITRs of AAV serotype 2. For details on the design of AAV protein plasmids, see Figure 1A and Figure S5. The EGFP protein is cleaved at amino acid (aa) C71. The ABCA4 protein is cleaved at aaC1150 (Group 1), aaS1168 (Group 2), and aaC1090 (Group 3) within the large cytoplasmic domain CD1 (34, 35). Although aaC1150 (Group 1) and S1168 (Group 2) fall within regions not associated with known ABCA4 function, C1090 is included in the ABCA4 nucleotide binding domain spanning aa929 to aa1148. All CEP290 cleavage points fall within the coiled-coil domain (36): when CEP290 is cleaved into two polypeptides, this occurs at aaC1076 (Group 1) or S1275 (Groups 2-3), and when it is cleaved into three polypeptides, this occurs at aaC929 and C1474 (Group 4) or aaS453 and C1474 (Group 5).

[0854] The intein contained in the plasmids was either the intein of DnaE from Candida punctata (Npu) (27, 28), or an intein composed of mutant N- and C-inteins of DnaE from Npu and Synechocystis sp. PCC6803 (Ssp), respectively (30), or the intein of DnaB from Rhodothermus marinus (Rma) (29). The plasmids used in the studies were under the control of 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 the bovine growth hormone polyadenylation signal (bGHpA), while the plasmid encoding ABCA4 included the simian virus 40 (SV40) polyadenylation signal.

[0855] AAV vector generation and characterization

[0856] AAV vectors were produced by triple transfection of HEK293 cells using the TIGEM AAV Vector Core as previously described (14, 41). No difference in vector yield was observed between AAV vectors containing or excluding intein sequences.

[0857] Cell transfection and AAV infection

[0858] HEK293 cells were maintained and transfected using the calcium phosphate method (6-well plate format, 1 μg of each plasmid / well) as described previously (14). For the experiments described in Figure S9, the amount of plasmid encoding the full-length gene corresponding to the same number of molecules included in 1 μg of AAV protein plasmid was used. The total amount of DNA transfected in each well was kept equal by adding scrambled plasmids when necessary.

[0859] Lipofectamine LTX (Invitrogen) was used for transfection. Figure 2 HeLa cells (24-well plate format, 1 or 0.5 μg of each plasmid / well) from experiments in C and 2D. AAV infection was performed as described (14).

[0860] iPSC and retinal differentiation culture

[0861] Human induced pluripotent stem cells (iPSCs) were derived from fibroblasts cultured from skin biopsies using the methods described in (42). The STGD1 cell line carries the ABCA4 compound heterozygous variants c.4892T>C and c.4539+2001G>A, also described in (43), or the compound 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 segment of introns 19 and 22. This deletion is found in cis with c.4462T>C. iPSCs were maintained on Matrigel (#354277, hESC-Qualified Matrix; Corning, NY)-coated 6-well plates containing mTeSR TM Culture 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 V-bottom 96-well plates (9,000 cells / well) containing RevitaCell Supplement (#A-2644501; Gibco, ThermoFisher) and 1% Matrigel to induce aggregate formation. Aggregates were then cultured to generate 3D retinal organoids as described in (46).

[0862] Western blot analysis and ELISA

[0863] Samples (HEK293 cells, retinas, 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 Cocktail Tablets; Roche, Basel, Switzerland) and 1 mM phenylmethylsulfonyl. After lysis, ABCA4 samples were denatured in 1X Laemmli sample buffer supplemented with 2 M urea at 37°C for 15 minutes. EGFP and CEP290 samples were denatured in 1X Laemmli sample buffer at 99°C for 5 minutes. Lysates were separated by 12% (for EGFP samples) or 6% (for ABCA4 and CEP290 samples) SDS-polyacrylamide gel electrophoresis. The antibodies used for immunoblotting were as follows: anti-3xflag (1:1000, A8592; Sigma-Aldrich, St. Louis, MO, USA), for detection of EGFP, ABCA4, and CEP290 proteins; anti-ABCA4 (1:500, LS-C87292; LifeSpan BioSciences, Inc, Seattle, USA), for detection of ABCA4; anti-filam A (1:1000, #4762; Cell Signaling Technology, Danvers, MA, USA); anti-α-actin (1:1000, NB600-501; Novus Biological Technologies, Inc, Seattle, MA, USA); LLC, Littleton, Colorado, USA) for detecting filamin A and β-actin, used as loading controls in in vitro experiments; anti-dysferlin (1:500, dysferlin, clone Ham1 / 7B6, MONX10795; Tebu-bio, Le Perray-en-Yveline, France) for detecting dysferlin, used as a loading control in in vivo experiments. EGFP, ABCA4, and CEP290 bands detected by western blotting were quantified using ImageJ software (available for free download at http: / / rsbweb.nih.gov / ij / ).

[0864] for Figure 21 The experiment shown in the figure is from Abca4 injected with AAV protein vector. - / - mice and control littermates Abca4 + / -Retinal lysates from mice were lysed in 30 μl of lysis buffer as described above, and 25 or 50 μl of the lysate were subjected to Western blotting using anti-ABCA4 antibody (LS-C87292; epitope conservation: 100% for human ABCA4; 86% for mouse Abca4). The amount of ABCA4 in the retinal lysate (quantitatively measured by band intensity using ImageJ software) was then normalized to the volume of retinal lysate loaded on an acrylamide gel. Figure 9 In the experiments reported in the figures, HEK293 cells were treated daily with increasing doses of trimethoprim (T7883, Sigma-Aldrich).

[0865] ELISA was performed on cells or mouse and pig retinal lysates using the Max Discovery Green Fluorescent Protein Kit ELISA (Bioo Scientific Corporation, Austin, TX, USA).

[0866] Southern blot analysis of rAAV vector DNA.

[0867] From 1.5 to 6x10 10 DNA was extracted from 100 viral particles (measured in GC). To digest the unpackaged genome, the vector solution was incubated with 30 μl of DNase (Roche) in a total volume of 300 μl containing 50 mM Tris, pH 7.5, and 1 mM MgCl2 at 37°C for 2 hours. The DNase was then inactivated with 50 mM EDTA and the capsids were cleaved by incubation with proteinase K and 2.5% N-lauryl-sarcosil solution at 50°C for 1 hour. DNA was extracted twice with phenol-chloroform and precipitated with 2 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 marker was generated by double digestion of pF8-V3 with Smal to produce a 5102 bp band. A probe specific for the HLP promoter was used.

[0868] Activated partial thromboplastin time (aPTT)

[0869] Nine aliquots of blood were collected by retro-orbital withdrawal into one aliquot of buffered trisodium citrate 0.109 M (BD, Franklin Lakes, NJ, USA). Plasma was separated by centrifugation of the samples at 13,000 rpm for 15 minutes.

[0870] aPTT was measured on a Coatron M4 (Teco, Bunde, Germany) using the aPTT program according to the manufacturer's manual.

[0871] Immunoprecipitation and liquid chromatography / mass spectrometry analysis

[0872] Cells were seeded in 100 mm plates (1 x 10 7 Cells were plated in 4% 4% 4% 5% 6% 1% 2% 2% 3% 5% 4% 2% 5% 6% 7% 8% 9% 1% 1% 2% 3% 1% 2% 2% 3% 5% 2% 3% 1 ... Briefly, three gel slides were digested using each of the following enzymes: trypsin, chymotrypsin, Glu-C, Arg-C, Asp-N, and Lys-N. ABCA4 was also digested using pepsin. The resulting peptides were identified and quantified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS / MS). The resulting mass spectrometry data were analyzed using PEAKS STUDIO 8.5. The present inventors achieved 100% protein sequence coverage for both EGFP and ABCA4 proteins.

[0873] Animal models

[0874] Animals were housed in TIGEM animal rooms (Naples) and maintained under a 12 h light / dark cycle. C57BL / 6J mice were purchased from Envigo (Italy).

[0875] Albino Abca4 was generated by serial crosses and backcrosses with BALB / c mice (homozygous for Rpe65Leu450). - / -Mice were kept inbred. BXD24 / TyJ-Cep290 rd16 / J (referred to as rd16) mice were introduced from the Jackson Laboratory (JAX stock #000031). rd16 mice carry an 897 bp in-frame deletion encompassing exons 35-39 (46). Mice were maintained by crossing homozygous females with homozygous males. Hemophilia B6; 129S-F8 mice tm1Kaz F8tm1 mice (designated F8tm1) were introduced from the Jackson Laboratory (JAX stock #004424). F8tm1 mice carry a neomycin resistance cassette that replaces 293 bp of sequence, including 7 bp at the 3' end of exon 16 and 286 bp at the 5' end of intron 16. Mouse colonies are maintained by crossing homozygous females with hemizygous males.

[0876] Large White sows (Azienda Agricola Pasotti, Imola, Italy) used in this study were registered as purebred in the LWHerd manual of the Italian National Pig Breeders' Association and housed at Centro di Biotecnologie AORNAntonio Cardarelli (Naples, Italy) and maintained under a 12 h light / dark cycle.

[0877] Subretinal injection of AAV vectors in mice and pigs

[0878] This study was performed 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 regulations on animal procedures. All procedures involving mice were approved by the Ministry of Public Health, Animal Health, Nutrition, and Food Safety of the Italian Ministry of Health on March 6, 2015.

[0879] Subretinal injections in mice and pigs were performed as previously described (e.g., as described in 14). Mouse eyes were injected with 1 μl or 0.5 μl (for rd16 pups) of vector solution. As described in the Results section, the AAV2 / 8 dose varied between mouse experiments. Pig eyes were injected with two adjacent subretinal blebs of 100 μl of AAV2 / 8 vector solution. The AAV2 / 8 dose was 2×10^11 GC of each vector / eye, so co-injection of the two AAV vectors resulted in a total dose of 4×10^11 GC / eye.

[0880] Histology, light and fluorescence microscopy

[0881] 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 cryosections 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 acquired at 40x magnification. Due to the prevalence of red-green color blindness, the colors of the original images have been changed to avoid the appearance of red and green together. Figure 14 Modified in.

[0882] To assess the thickness of the outer nuclear layer in rd16 mice injected with an AAV CEP290 inner protein vector, eyes were fixed in 4% paraformaldehyde (PFA) overnight, then dehydrated in serial ethanol and embedded in paraffin blocks. Serial cross-sections from rd16 mice (10 μm) 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 collected at 20x magnification. For each eye, one image from the temporal injection side of the section in the central area of ​​the eye was used for analysis. Three measurements of ONL thickness were taken in each image using the "freehand line" tool of ImageJ software by an operator masked for genotype / treatment group.

[0883] Immunofluorescence analysis

[0884] HeLa cells transfected with ABCA4 or CEP290 AAV protein plasmids were fixed in 4% PFA for 10 minutes 24 hours after transfection. Cells were blocked in blocking buffer (0.05% saponin, 0.5% BSA, 50 mM NH4Cl, 0.02% NaN3 in PBS, pH 7.2) for 30 minutes and then incubated as follows:

[0885] -ABCA4 protein was detected with anti-FLAG M2 antibody (F1804, Sigma-Aldrich) for 1 hour; endoplasmic reticulum was stained with anti-VAP-B antibody [produced by Antonella De Matteis laboratory ((47)], and trans-Golgi network was stained with TGN46 (AHP-499, Serotech). After washing in PBS, cells were incubated for 30 minutes with secondary antibodies: goat anti-mouse Alexa Fluor 568; goat anti-rabbit Alexa Fluor 488, donkey anti-goat Alexa Fluor 633, against anti-FLAG, -VAP-B and -TGN46 antibodies, respectively.

[0886] - Anti-FLAG antibody (F7425, Sigma-Aldrich) was used to detect CEP290 protein overnight, and anti-acetylated tubulin antibody (T6793, Sigma-Aldrich) was used to stain microtubules. 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, directed against anti-FLAG and -Ac-tubulin antibodies, respectively.

[0887] The nuclei were stained with DAPI. Due to the prevalence of red-green color blindness, the colors of the original images have been Figure 2 CD and Figure 18 Modified in.

[0888] The antibodies used for immunofluorescence in human retinal organoids are as follows:

[0889] Anti-human cone-arrestin (CAR) (50, 51) (1:10,000, “Luminaire founders” hCAR; a gift from Dr. Cheryl M. Craft, Doheny Eye Institute, Los Angeles, CA, USA); anti-opsin, red / green (1:200, AB5405; Merck Millipore, Darmstadt, Germany); anti-recoverin (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).

[0890] Transmission and scanning electron microscopy analysis

[0891] For electron microscopy (EM) analysis, Abca4 was injected subretinally 3 months after AAV injection. - / - Mice were adapted to spend the night in the dark, and then the eyes were harvested. Eyes were fixed for 18 hours in 2% glutaraldehyde (GA)-2% PFA in 0.1M PHEM buffer pH 6.9, and then rinsed in 0.1M PHEM buffer. Eyes were then dissected under an optical microscope to select the temporal injection area of ​​the optic cup. Subsequently, this part of the optic cup was embedded in 12% gelatin and injected with 2.3M sucrose. Cryosections (60nm) were frozen in liquid nitrogen and cut using Leica Ultramicrotome EM FC7 (Leica Microsystems). For fear that data were attributed to the bias of each experimental group, the area occupied by lipofuscin particles in the retinal pigment epithelium was measured using iTEM software (Olympus SYS, Hamburg, Germany) by the operator shielded for genotype / treatment group. The "Freehand polygon" tool of iTEM software was used to image at least 20 different images (25 μm 2 The area of ​​each lipofuscin granule in each visual field was measured.

[0892] 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 on SEM specimen posts and coated with a thin layer of gold. The surface three-dimensional organization of the samples was analyzed, and images were acquired using a JEOL 6700F scanning electron microscope (JEOL Ltd., Tokyo, Japan).

[0893] For ultrastructural analysis, retinal organoids were fixed overnight with a mixture of 2% PFA and 1% GA in 0.2 M PHEM buffer, pH 7.3. Following fixation, the specimens were post-fixed as previously described. They were then dehydrated, embedded in epoxy resin, and polymerized at 60°C for 72 hours. Serial 60 nm thin sections were cut on a Leica EM UC7 microtome.

[0894] EM images were collected using a FEI Tecnai-12 electron microscope (FEI, Eindhoven, The Netherlands) equipped with a VELETTA CCD digital camera.

[0895] Electrophysiological recordings and spectral-domain optical coherence tomography

[0896] Functional and morphological analyses were performed as previously described ( 14 ).

[0897] pupil light response

[0898] The pupil light response of rd16 mice was recorded under dark conditions using the TRC-50IX retinal camera (Topcon Biomedical Systems, Oakland, New Jersey) that is connected to a charge coupled device Nikon D1H digital camera. Mice were exposed to 10 lux light stimulations for approximately 10 seconds, and IMAGEnet software (Topcon Biomedical Systems) was used to gather a photo of each eye. For each eye, pupil diameter was standardized relative to eye diameter (from temporal to nasal).

[0899] Statistical analysis

[0900] One-way ANOVA test (parametric test) or Kruskal-Wallis rank sum test (nonparametric test) was performed to determine whether there were statistically significant differences between two or more groups of independent variables on the dependent variable. P values ​​are as follows: ELISA assay for EGFP protein quantification in vitro (p Kruskal-Wallis = 0.006036), in mouse retina (p ANOVA = 0.00585), and in pig retina (p Kruskal-Wallis = 0.009005); Figure 5 A(p ANOVA=0.00585); Figure 5 B(p Kruskal-Wallis=5.547E-5); Figure 5C (p ANOVA = 5.81E-10); ERG analysis (p ANOVA or p Kruskal-Wallis > 0.05 at all intensities analyzed for both a- and b-wave amplitudes); OCT analysis in Figure S14 (p ANOVA = 0.52 for ABCA4, p ANOVA = 0.965 for CEP290). Statistically significant differences between groups, as determined by multiple pairwise comparisons between group means, were as follows: ELISA assays for EGFP protein quantification in vitro (single AAV vs. dual AAV = 0.012; AAV protein vs. dual AAV = 0.012; single AAV vs. AAV protein = 0.222), in mouse retina (single AAV vs. dual AAV = 0.0044; AAV protein vs. dual AAV = 0.3754; single AAV vs. AAV protein = 0.0561), and in porcine retina (single AAV vs. dual AAV = 0.012; AAV protein vs. dual AAV = 0.012; single AAV vs. AAV protein = 0.841); Figure 5 A: + / + vs. - / - AAV internal protein = 0.4530; + / - vs. - / - = 0.0002; Figure 5 B: Wild type relative to rd16 AAV protein = 0.00131; Figure 5 C: Wild type relative to rd16 AAV internal protein 1E-07; wild type relative to rd16 neg <1E-06. Example

[0901] Example 1: Reconstitution of AAV-EGFP Intrinsic Protein into Full-Length Protein in Vitro

[0902] The present inventors tested the efficiency of intilin-mediated protein trans-splicing in the retina; two AAV vectors were generated, each encoding a protein that is similar to that of Candida punctata [ Figure 1 The N-terminal or C-terminal half of the reporter EGFP protein was fused to the N-terminal and C-terminal halves of the DnaE split endonuclease [Npu in A]. The EGFP protein is cleaved at amino acid (aa) C71. Each AAV vector includes appropriate regulatory elements (i.e., promoter and bovine growth hormone polyadenylation signal (bGHpA) and triple flag tag (3xflag) to allow detection of these two halves as well as the full-length reconstituted EGFP protein ( Figure 1 A).

[0903] Human embryonic kidney 293 (HEK293) cells were transfected with AAV-EGFP DNA E-intrin plasmids and the production of individual N-terminal and C-terminal halves as well as full-length EGFP protein was assessed. EGFP fluorescence was detected in cells co-transfected with AAV-EGFP intrin plasmids, but not with individual N-terminal and C-terminal AAV-EGFP intrin plasmids, and was comparable to the fluorescence observed in cells transfected with a single AAV plasmid encoding full-length EGFP, as shown in Figure 2. Figure 12 Only after co-transfection of the two AAV-EGFP protein plasmids, Western blot (WB) analysis of HEK293 cell lysates confirmed the presence of the expected size (approximately 28 kDa) of trans-spliced ​​EGFP protein and the DnaE protein (approximately 17 kDa) spliced ​​from the mature protein, as shown in Figure 2. Figure 1 As shown in B. In addition, quantification of the band intensity showed that the amount of EGFP protein from the AAV protein plasmid was 76±37% of the amount of EGFP protein observed from a single AAV plasmid (n=3 independent experiments). In order to define the accuracy of protein reconstruction, EGFP was immunopurified from HEK293 cells transfected with AAV-EGFP protein plasmid and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis to define its protein sequence. 3539 peptides obtained from the proteolytic digestion of this sample, 7 of which included the cleavage point (Table 5), covered the entire protein and confirmed that the amino acid sequence of the EGFP reconstructed by the AAV protein plasmid corresponded exactly to that of the wild-type EGFP.

[0904] Table 5: Peptides encompassing the EGFP cleavage point.

[0905] C = Cysteine ​​71

[0906]

[0907]

[0908] Example 2: AAV-EGFP Intrinsic Protein is More Effective Than Dual AAV Vectors in Vitro

[0909] To confirm EGFP protein reconstitution from AAV intraprotein vectors, 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 5x10^4 genome copies (GC) / cell of each vector, which means similar dosages between the three systems, assuming that the dual vectors undergo complete DNA or protein recombination. To accurately quantify the amount of EGFP, cell lysates were harvested 72 hours after infection. EGFP expression was assessed by both WB and enzyme-linked immunosorbent assay (ELISA): the EGFP expression obtained using the AAV intraprotein vector was approximately half of that obtained using a single AAV (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 the dual AAV vector, as shown in Figure 5. Figure 1 C (dual AAV = 0.046 ± 0.01 ng EGFP / μg total lysate, n = 5 independent experiments). In addition, the intensity of full-length EGFP relative to the intensity of excised intein was quantified by WB; their relative abundance was found to be 1:0.2 (n = 6 independent experiments, Figure 13 A).

[0910] Example 3: Subretinal administration of AAV-EGFP intracellular protein vectors results in efficient full-length protein reconstitution in both mouse and pig retinas.

[0911] To investigate whether AAV intracellular protein-mediated trans-splicing reconstitutes full-length protein expression in the retina, 4-week-old C57BL / 6J mice were subretinally injected with AAV2 / 8-CMV-EGFP DNA intracellular protein vectors (dose of each vector / eye: 5.8x10^9 GC). Eyes were harvested 1 month later and analyzed by microscopy. EGFP fluorescence was detected in the retinal pigment epithelium of all eyes, and most importantly, in the photoreceptors ( Figure 1 D). To compare transgene expression of AAV-derived proteins with that of single and dual AAVs in photoreceptors, 4-week-old C57BL / 6J mice were subretinally injected with an AAV2 / 8 vector encoding EGFP under the control of the photoreceptor-specific human G protein-coupled receptor kinase 1 (GRK1) promoter (dose per vector / eye: 5 x 10^9 GC). Eyes were harvested 1 month after injection and analyzed by fluorescence microscopy, ELISA, or WB.

[0912] EGFP fluorescence was detected in the photoreceptor cell layer in eyes injected with all vector groups, as shown in Figure 5. Figure 1As seen in E. Accurate quantification of EGFP protein by ELISA confirmed that the efficiency of EGFP protein reconstitution with AAV intein was lower than that with single AAV, but about 3-fold higher than that with dual AAV (single AAV = 8.41 ± 2.48 ng EGFP / retina, n = 5 eyes; AAV intein = 3.72 ± 0.85 ng EGFP / retina, n = 7 eyes; dual AAV = 1.38 ± 0.43 ng EGFP / retina, n = 7 eyes). After quantification of WB band intensity, the relative amount of full-length EGFP to excised intein was 1:3 (n = 14 eyes analyzed, Figure 13 B).

[0913] The present inventors then evaluated the efficiency of AAV intraprotein vectors in transducing photoreceptors in the porcine retina, which, due to its size and structure, serves as an excellent preclinical model for evaluating viral vector transduction ((48)). Therefore, single, intraprotein, and dual AAV2 / 8-GRK1-EGFP vectors were injected subretinaly into Large White pigs (dose of each vector / eye: 2x10^11 GC, delivered via two adjacent subretinal blebs). Eyes were harvested 1 month after injection and analyzed by fluorescence microscopy, ELISA, or WB. Notably, AAV intraprotein-mediated reconstitution of EGFP protein in the photoreceptor cell layer was higher than dual AAV-mediated reconstitution, as assessed by EGFP fluorescence, and was indistinguishable from single AAV vectors ( Figure 1 F). Accurate quantification of EGFP in retinal lysates confirmed that AAV intein reconstituted the protein in an amount similar to that obtained with single AAV and approximately 3-fold higher than that obtained with dual AAV vectors (single AAV = 247.5 ± 45.1 ng EGFP / retina, n = 5 eyes; AAV intein = 227.0 ± 15.7 ng EGFP / retina, n = 5 eyes; dual AAV = 82.3 ± 9.6 ng EGFP / retina, n = 5 eyes). After quantification of WB band intensity, the relative amount of full-length EGFP to excised intein was 1:2 (n = 8 eyes, Figure 13 C).

[0914] Example 4: AAV-mediated protein trans-splicing reconstitutes full-length EGFP in 3D human retinal organoids.

[0915] As an additional preclinical model representing the human retina, the present 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 mature photoreceptor markers, e.g. Figure 14As shown in B; organoids were successfully transduced using AAV2 vectors with photoreceptor-specific promoters, namely AAV2 / 2CMV EGFP and AAV2 / 2IRBP DsRed vectors. Figure 14 C is shown by fluorescence analysis. Figure 14 D) and electronic ( Figure 14 EF) Microscopy shows the presence of photoreceptor outer segment buds. Nine-month-old 3D human retinal organoids incubated with AAV-GRK1-EGFP inner protein vector (dose of each vector / organoid: 1×10^12 GC) for 30 days showed EGFP fluorescence ( Figure 1 G). Western blot analysis of retinal organoid lysates ( Figure 15 ) Full-length EGFP expression, approximately 5-fold more abundant than excised intein, was confirmed after quantification of band intensity (n=4 organoids).

[0916] Example 5: Intrinsic protein-mediated trans-splicing of large proteins (Efficient AAV intrinsic protein trans-splicing requires identification of optimal ABCA4 and CEP90 cleavage sites)

[0917] To test whether protein trans-splicing could be exploited as a mechanism for remodeling large therapeutic proteins, the present inventors developed AAV-ABCA4 and -CEP290 intein vectors.

[0918] ABCA4 and CEP290 were split into two (AAV I, AAV II) or three (AAV I, AAV II, AAV III) fragments, and their coding sequences were cloned into a single AAV vector and fused to the coding sequences of the N-terminus and C-terminus of the split-intrin, e.g. Figure 16 AAV protein vectors included the ubiquitous short CMV [(shCMV), for all groups] or GRK1 promoter (group 1 for ABCA4 and group 5 for CEP290).

[0919] The cleavage point for each protein was chosen taking into account both the amino acid residue requirements at the junction for efficient protein trans-splicing (18, 51) and the maintenance of critical protein domain integrity, which should facilitate the correct folding and stability of each individual polypeptide and, therefore, the final reconstituted protein. Additional split-type endonucleases were also considered. Groups of CEP290 in which the protein was split into three polypeptides were generated (Groups 4 and 5, Figure 16B) to allow for the inclusion of a woodchuck hepatitis virus posttranscriptional regulatory element [WPRE, (52)] to increase transgene expression. To prevent unwanted trans-splicing between AAV I and AAV III that could reduce the amount of full-length protein produced, groups 4 and 5 included two different split-type inteins at the two cleavage junctions, specifically, the DnaB intein from Rhodothermus marinus and either wild-type or mutant DnaE inteins, which the inventors showed do not cross-react ( Figure 17 ).

[0920] The present inventors compared the ability of each set of AAV protein plasmids to reconstitute ABCA4 and CEP290 following transfection of HEK293 cells. Western blot analysis of cell lysates 72 hours after transfection showed that full-length ABCA4 and CEP290 proteins of the expected size (approximately 250 kDa and approximately 290 kDa, respectively) were reconstituted by each set of AAV protein plasmids, albeit with varying efficiencies ( Figure 2 AB). Groups 1 and 5 were found to be the most efficient for ABCA4 and CEP290 protein reconstitution, respectively, and were therefore used for all subsequent experiments.

[0921] To determine the accuracy of protein reconstitution, the present inventors immunopurified ABCA4 from HEK293 cells transfected with Group 1 and performed LC-MS analysis to determine its protein sequence. Proteolysis of this sample yielded 3108 peptides, 22 of which included the cleavage site (Table 6), covering the entire protein and confirming that the amino acid sequence of ABCA4 reconstructed from the AAV endonuclease precisely corresponded to that of wild-type ABCA4. The amino acid sequence of ABCA4 reconstructed from the AAV endonuclease matched that of wild-type ABCA4. An alignment of the wild-type ABCA4 sequence with the peptides identified from liquid chromatography-mass spectrometry analysis of ABCA4 reconstructed from the AAV endonuclease was performed.

[0922] Table 6: Peptides encompassing the ABCA4 cleavage point.

[0923] NB: C :Cysteine ​​1150

[0924]

[0925]

[0926] The present inventors then evaluated the intracellular localization of the protein products of the different endonuclease-containing plasmids and compared them with the localization of the full-length protein. It is known that full-length ABCA4 is localized to the endoplasmic reticulum (ER) when expressed in cultured cell lines (53, 54). The two ABCA4 polypeptides from Group 1 were found to colocalize in the ER, whereas no colocalization was found at the trans-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, confirming that exogenously expressed ABCA4 in the cell lines is primarily localized in the ER.

[0927] With regard to CEP290, the full-length protein has been reported to display a mixed distribution pattern, with a predominantly punctate and a minor fibrillar pattern (55). Dissection of the domains responsible for subcellular targeting of CEP290 revealed that the N-terminal domain (aa1-362) targets the protein to vesicular structures due to its ability to interact with membranes, whereas a region near the C-terminus of CEP290 (encompassing the myosin tail homology domain of the majority of the protein) mediates microtubule binding (aa580-2479) and, when expressed as a truncated form, has a prominent fibrillar distribution consistent with acetylated tubulin (Ac-Tub). Consistent with Drivas et al., immunofluorescence analysis of HeLa cells transfected with AAV I, II, or III protein plasmids alone or co-transfected with AAV I+II, AAV I+III, and AAV II+III showed that products from AAV I and AAV II had a predominantly punctate pattern, whereas products from AAV III (encompassing the myosin tail homology domain of the protein) exhibited a fibrillar pattern and were the only product to completely colocalize to the Ac-tub ( Figure 2 D). Thus, the product from AAV I+II has a predominantly punctate pattern, whereas the products from AAV I+III and AAV II+III have a combined microtubule fibrillar and punctate pattern. Cells co-transfected with the three AAV CEP290 protein plasmids showed a predominantly punctate signal, partially aligned along microtubules, which was comparable to the signal observed in cells transfected with plasmids encoding the full-length CEP290 protein ( Figure 2 D and Figure 18 ).

[0928] The inventors then compared the protein amounts obtained with the optimal AAV-ABCA4 and -CEP290 protein plasmid set with the protein amounts obtained from the single AAV plasmids encoding the corresponding full-length proteins. To this end, HEK293 cells were transfected with the same equimolar amounts of the single or AAV protein plasmids and cell lysates were analyzed by WB 72 hours after transfection ( Figure 19 Quantification of band intensities showed that ABCA4 and CEP290 expression from AAV protein plasmids was 61±4% (n=3 independent experiments) and 58±4% (n=3 independent experiments), respectively, of the expression observed with the corresponding single AAV plasmids.

[0929] Example 6: AAV intracellular protein vectors mediate expression of large therapeutic proteins in vitro and in the retina

[0930] The inventors compared the efficiency of AAV intein-mediated large protein reconstitution with that of dual AAV vectors in vitro and in mouse and pig retinas. HEK293 cells were infected with AAV2 / 2 dual or intein vectors encoding ABCA4 (Group 1) or CEP290 (Group 5) (moi: 5×10^4 GC / cell for each vector), and cell lysates were analyzed by WB after 72 hours. Figure 3 As shown in Figures A and 3B, both AAV-ABCA4 and -CEP290 endonuclease vectors mediated large protein reconstitution more efficiently than dual AAV vectors. As expected, in addition to full-length proteins, shorter polypeptides ( ) derived from single AAV endonuclease vectors (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).

[0931] In addition, 4-week-old wild-type mice were subretinally injected with AAV-GRK1-ABCA4 or -CEP290 proteins (Groups 1 and 5, respectively) compared with dual vectors (dose of each ABCA4 vector / eye: 3.3x10^9 GC, dose of each CEP290 vector / eye: 1.1x10^9 GC). Animals were sacrificed 4-7 weeks after injection, and protein expression in retinal lysates was evaluated by WB. In 10 / 11 (91%) eyes injected with AAV-ABCA4 proteins ( Figure 4 A and 20) and 5 / 10 (50%) eyes injected with AAV-CEP290 protein ( Figure 4 In contrast, full-length protein expression was evident in 5 / 9 (56%) and 0 / 5 eyes injected with dual AAV vectors of ABCA4 and CEP290, respectively. Similar to what was observed in vitro, polypeptides derived from single AAV intracellular protein vectors (in the case of both ABCA4 and CEP290) as well as from trans-splicing between AAV II and AAV III (in the case of CEP290) were detected. Figure 4 A and 4B).

[0932] To investigate the efficiency of protein reconstitution mediated by AAV endonuclease relative to endogenous protein, 1- to 4-month-old Abca4- / - mice were subretinally injected with AAV-GRK1-ABCA4 endonuclease vectors (Group 1) (dose of each ABCA4 vector / eye: 5.5×10^9 GC). One month later, ABCA4 expression in retinal lysates from unaffected and AAV endonuclease-injected Abca4- / - mice was analyzed by Western blotting using antibodies recognizing mouse and human ABCA4. Figure 21 ). It was found that the expression of AAV protein ABCA4 was 8.6±1.3% of endogenous ABCA4.

[0933] To confirm the efficient reconstitution of a large protein in the clinically relevant porcine retina, Large White pigs were subretinally injected with AAV2 / 8-GRK1-ABCA4 intron (Group 1) or dual vectors (dose per vector / eye: 2x10^11 GC, delivered via two adjacent subretinal blebs), and protein expression was analyzed by Western blotting 1 month after injection. Notably, AAV intron was found to be more efficient than dual AAV vectors in reconstructing full-length ABCA4 protein ( Figure 4 C).

[0934] Finally, human retinal organoids derived from iPSCs of healthy individuals or patients with STGD1 were infected with the AAV2 / 2-GRK1-ABCA4 protein vector (Group 1) (dose per vector / organoid: 1×10^12 GC) at 121 days of culture (when photoreceptor maturation begins (20)). Organoids were lysed between 20 and 40 days post-infection and analyzed by Western blotting. ABCA4 (A) was detected in all infected organoids at the expected size. Figure 4 D and Figure 22 ; n=3 and n=4 from normal control and STGD1 organoids, respectively).

[0935] Example 7: Subretinal administration of AAV intraprotein vectors improves retinal phenotypes in STGD1 and LCA10 mouse models

[0936] To determine whether photoreceptor transduction achieved with AAV intracellular protein vectors is therapeutically relevant, we tested it in the retina of STGD1 (Abca4- / -) and LCA10 (rd16) mouse models.

[0937] One-month-old Abca4- / - mice were injected subretina with an AAV2 / 8-GRK1-ABCA4 intracellular vector (Group 1) (dose per vector / eye: 4.3-4.8 x 10^9 GC). Three months later, the eyes were harvested and ultrathin sections of the retina were analyzed by transmission electron microscopy to measure the amount of lipofuscin, which accumulates in the retinal pigment epithelium (RPE) of Abca4- / - mice (56, 57). Notably, RPE lipofuscin accumulation was significantly reduced in Abca4- / - eyes injected with the AAV intracellular vector, but not in eyes injected with the negative control (p value = 0.0163; Figure 5 A and Figure 23 ).

[0938] In parallel, 4-6 day old rd16 mice were subretinally injected with AAV2 / 8-GRK1-CEP290 protein vectors (Group 5) (dose per vector / eye: 5.5x10^8 GC). Retinal section microscopy analysis performed 1 month after injection showed that the thickness of the outer nuclear layer (ONL), including the photoreceptor nuclei, was significantly reduced in rd16 mice compared with wild-type mice (p value = 0.00048; p value = 0.00048) as a result of progressive retinal degeneration (55). Figure 5 B). Notably, ONL thickness in rd16 retinas injected with AAV intracellular protein vector was significantly higher (approximately 60%, p-value = 0.00281) than in rd16 retinas injected with negative control ( Figure 5 B). Thus, retinal function testing based on pupillary light response (PLR) showed that pupil constriction was significantly greater in rd16 mice injected with AAV intracellular protein vectors than in rd16 eyes injected with negative controls (approximately 20%, p-value = 0.00073) ( Figure 5 C).

[0939] In addition, the inventors studied the safety of AAV protein vectors in the retina. To this end, wild-type C57BL / 6J mice were subretinally injected with AAV2 / 8-GRK1-ABCA4 or -CEP290 protein vectors (Group 1 and Group 5, respectively) (dose of each ABCA4 vector: 4.3x10^9GC; dose of each CEP290 vector / eye: 1.1x10^9GC), and retinal electrical activity was measured by Ganzfeld electroretinography (ERG) 6 months and 4.5 months after injection, respectively. In both studies, the a-wave amplitude and b-wave amplitude were similar between the eyes of mice injected with AAV protein vectors (n=14-15 and n=11 for ABCA4 and CEP290, respectively) and the eyes injected with negative control AAV vectors (n=8 and n=5 for ABCA4 and CEP290, respectively) or PBS (n=6-7 and n=6 for ABCA4 and CEP290, respectively). Similarly, ONL thickness measured by optical coherence tomography was similar between eyes injected with AAV protein, negative control, and PBS ( Figure 24 ).

[0940] Example 8: Safe AAV Intrinsic Protein-Mediated Large Gene Delivery

[0941] Although no overt signs of toxicity were observed in wild-type mice injected with AAV intein, the present inventors have evaluated the inclusion of a degron in the trans-splicing system, which, once incorporated into the excised intein, leads to rapid ubiquitination of the fusion protein and subsequent proteasomal destruction ( Figure 6 Most of the described degrons are functional at the N-terminal or C-terminal positions (i.e., CL1, SMN, CIITA, ODC), and these degrons cannot be fused to N-terminal or C-terminal internal proteins because this would result in degradation of a single host protein, thus eliminating polypeptides required for protein trans-splicing (PTS) reactions. Therefore, the present inventors selected a mutant form of dihydrofolate reductase (ecDHFR) from Escherichia coli that includes three amino acid mutations, R12Y, Y100I, and G67S, that confer functional activity only at the N-terminal or internal positions (69).

[0942] To test the efficiency of ecDHFR in reducing the amount of excised intein, the inventors generated an AAV vector encoding the N-terminal half of EGFP fused to the N-terminal half of Npu DnaE and ecDHFR (pAAV2.1-CMV-5′EGFP intein ecDHFR). Thus, the degradation determinant would be located at its C-terminus where it should be inactive. HEK293 cells were transfected using a combination of an AAV-EGFP-ecDHFR intein plasmid and vector II encoding the C-terminal half of EGFP fused to the C-terminal half of Npu DnaE (pAAV2.1-CMV-3′EGFP intein) and the production of full-length EGFP protein and excised intein was assessed. Trans-spliced ​​EGFP protein with similar protein levels compared to AAV intein was detected by WB analysis. Furthermore, the amount of excised intein in HEK293 cell lysates was significantly reduced after co-transfection of the AAV-EGFP-ecDHFR intein plasmid ( Figure 7 The inventors then decided to apply the same strategy to the large ABCA4 protein (pAAV2.1-CMV260-5′ABCA4 intein ecDHFR). In the case of EGFP, the inventors found that similar amounts of full-length ABCA4 ( Figure 8 A). Importantly, complete elimination of the excised intein was observed ( Figure 8 B).

[0943] To demonstrate that the present inventors were observing ecDHFR-mediated DnaE degradation, cells were treated with trimethoprim (TMP). TMP is an antibiotic that binds to ecDHFR and prevents protein degradation, thereby allowing the fusion protein to escape degradation (69). HEK293 cells co-transfected with AAV-ABCA4-ecDHFR protein plasmids were treated with increasing doses of TMP and found that the DnaE protein was no longer degraded. TMP stabilized ecDHFR in a dose-dependent manner, suggesting that the reduction of DnaE protein is mediated by ecDHFR ( Figure 9 ).

[0944] One limitation of including a degron in the vector (outside the intein) is that the cloning capacity of AAV is further reduced, resulting in AAV vectors that are too large for certain applications. In fact, ecDHFR is 159aa in length. Therefore, the inventors designed a shorter 105aa ecDHFR variant that retains amino acids that are said to be critical for its activity at the N-terminus or internal positions. The inventors tested this small ecDHFR in both EGFP and ABCA4 intein plasmids (pAAV2.1-CMV-5′EGFP intein_small ecDHFR; pAAV2.1-CMV260-5′ABCA4 intein_small cDHFR). After co-transfection of AAV-EGFP- or ABCA4-small ecDHFR intein plasmids, they found similar full-length protein expression compared to AAV intein plasmids ( Figure 10 and 11 A) and a significant decrease in DnaE protein ( Figure 10 and 11 B).

[0945] These results demonstrate that inclusion of ecDHFR or mini-ecDHFR in the PTS system mediates selective endonuclease degradation without significantly affecting the efficacy of protein trans-splicing and therapeutic protein production.

[0946] Example 9: Intrinsic protein trans-splicing in the liver

[0947] To test the efficiency of intein-mediated protein trans-splicing in the liver, two AAV vectors were generated, each encoding the N-terminal or C-terminal half of the reporter EGFP protein fused to the N-terminal and C-terminal halves of the DnaE split-type intein from Candida punctata.

[0948] Five-week-old C57 / BL6 mice were retroorbitally injected with AAV2 / 8 vectors carrying the liver-specific human thyroxine-binding globulin (TBG) promoter (dose of each vector / kg: 5 × 10 11 GC). 4 weeks after injection, livers were harvested and lysed for analysis by Western blotting with an anti-3xflag antibody to detect EGFP-3xflag and intron-3xflag. Quantification of EGFP band intensity demonstrated that AAV intron transduced the liver more efficiently than dual AAV, with approximately 6-7 times higher protein levels.

[0949] Example 10: AAV intracellular protein vectors can be used to deliver the large F8 gene affected in hemophilia A

[0950] The F8 gene, which is mutated in hemophilia A, is too large (approximately 7 kb) to be delivered by a single AAV in its wild-type conformation. Due to this, only the B domain deleted (BDD) conformation of the gene is suitable for the context of AAV gene therapy. Recently, a 5 kb expression cassette containing BDD-F8 and a short liver-specific promoter and polyA signal has been packaged into AAV5 and shown to produce therapeutic levels of FVIII in mice and cynomolgus monkeys (70) as well as in HemA patients (71). However, the genome of this vector is slightly too large and is packaged into AAV capsids as a library of heterogeneous truncated genomes, which, after reconstitution in target cells, induce efficient transduction. Compared to normal size, oversized AAV vectors are less efficient and the quality of products with heterogeneous truncated genomes may hinder their further commercial development.

[0951] To overcome the limited AAV cargo capacity, a protein trans-splicing strategy was designed involving two independent AAV vectors with regularly sized genomes, each encoding one of the two halves of the large FVIII protein flanked by a split Npu DnaE intron.

[0952] The wild-type F8 gene is split into two different split points in the B domain, namely group 1 and group 2. The liver-specific hybrid liver promoter (HLP) and the F8 protein vector under the short synthetic polyA were generated ( Figure 25 A). As shown by Southern blot, the vector genome is properly packaged into AAV capsids, unlike its ultra-large AAV BDD-F8 control ( Figure 25 B).

[0953] To determine the therapeutic relevance of this strategy, AAV2 / 8F8 protein vectors were administered retroorbitally (dose of each vector / animal: 4-5 x 10 11 GC) was injected systemically into 7-8 week old hemophilia A knockout mice. Analysis of plasma aPTT (activated partial thromboplastin time) 8 weeks after injection showed a slight correction of the bleeding phenotype, although not at the same level as the oversized single AAVBDD-F8 control ( Figure 25 C).

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Claims

1. A vector system for expressing a coding sequence in a cell, wherein the coding sequence encodes ABCA4, the coding sequence consisting of a first part (CDS1) and a second part (CDS2) or consisting of a first part (CDS1), a second part (CDS2) and a third part (CDS3), the vector system comprising: a) a first carrier, the first carrier comprising: - said first part of said coding sequence (CDS1), - a first intein nucleotide sequence encoding an N-intein, said sequence being located at the 3' end of CDS1; and b) a second carrier, the second carrier comprising: - said second part of the coding sequence (CDS2), - a second intein nucleotide sequence encoding C-intein, said sequence being located at the 5' end of CDS2; wherein when the first vector and the second vector are inserted into a cell, the protein product of the coding sequence is produced by protein splicing; or The carrier system comprises: a′) a first vector, the first vector comprising: - said first part of said coding sequence (CDS1), - a first intein nucleotide sequence encoding a first N-intein, said sequence being located at the 3' end of CDS1; and b') a second carrier, the second carrier comprising: - said second part of the coding sequence (CDS2), - a second intein nucleotide sequence encoding the first C-intein, said sequence being located at the 5' end of CDS2; - a third intein nucleotide sequence encoding a second N-intein, said sequence being located at the 3' end of CDS2; and c') a third vector, the third vector comprising: - said third part of said coding sequence (CDS3) - a fourth intein nucleotide sequence encoding a second C-intein, said sequence being located at the 5' end of CDS3, wherein the first intein nucleotide sequence is different from the third intein nucleotide sequence, when present, and the second intein nucleotide sequence is different from the fourth intein nucleotide sequence, when present, wherein when the first vector, the second vector, and the third vector, when present, are inserted into a cell, the protein product of the coding sequence is produced by protein splicing.

2. The vector system of claim 1, wherein the first intein nucleotide sequence, the second intein nucleotide sequence, the third intein nucleotide sequence when present, and the fourth intein nucleotide sequence encode a split-type intein.

3. The vector system according to claim 2, wherein the maximum length of the split-type intein is 150 amino acids.

4. The vector system according to claim 2, wherein the split-type intein is DnaE or DnaB intein.

5. The vector system according to claim 1, wherein - the first intein nucleotide sequence encodes an intein selected from the group consisting of SEQ ID No 1, 3, 5, 7, 9, 11, 13 or a variant thereof, or a fragment thereof or a homolog thereof; - the second intein nucleotide sequence encodes an intein selected from the group consisting of SEQ ID No 2, 4, 6, 8, 10, 12, 14 or a variant thereof, or a fragment thereof or a homolog thereof; - said third intein nucleotide sequence when present encodes an intein selected from the group consisting of SEQ ID No 1, 3, 5, 7, 9, 11, 13 or a variant thereof, or a fragment thereof or a homologue thereof; - said fourth intein nucleotide sequence when present encodes an intein selected from the group consisting of SEQ ID No 2, 4, 6, 8, 10, 12, 14 or a variant thereof, or a fragment thereof or a homologue thereof.

6. The vector system of claim 1 , wherein the first vector, the second vector, and when present, the third vector further comprises a promoter sequence operably linked to the 5' terminal portion of the first portion of the coding sequence (CDS1), the second portion of the coding sequence (CDS2), or the third portion of the coding sequence (CDS3).

7. The vector system of claim 1, wherein the first vector, the second vector, and when present, the third vector further comprise a 5'-terminal repeat (5'-TR) nucleotide sequence and a 3'-terminal repeat (3'-TR) nucleotide sequence. 8 . The vector system according to claim 7 , wherein 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.

9. The vector system of claim 1 , wherein the first vector, the second vector, and when present, the third vector further comprise a polyadenylation signal nucleotide sequence, and / or wherein at least one of the first vector or the second vector or the third vector further comprises a nucleotide sequence encoding a degradation signal.

10. The vector system according to claim 9, wherein the degradation signal is selected from the group consisting of CL1, PB29, SMN, CIITA, ODc, ecDHFR or fragments thereof.

11. The vector system according to claim 1 , wherein the coding sequence is split into the first part and the second part or the first part, the second part and the third part at the following position, which consists of a nucleophilic amino acid that does not fall within the structural or functional domain of the encoded protein product, wherein the nucleophilic amino acid is selected from serine, threonine or cysteine.

12. The vector system of claim 1, wherein at least one of the first vector, the second vector, and when present, the third vector further comprises at least one enhancer or regulatory nucleotide sequence operably linked to the coding sequence.

13. The carrier system according to claim 1, comprising: a) a first vector, said first vector comprising in a 5'-3' direction: -5'-inverted terminal repeat (5'-ITR) sequence; - promoter sequence; - a 5' terminal portion of the coding sequence (CDS1), said 5' terminal portion being operably linked to and under the control of said promoter; - a first intein nucleotide sequence encoding an N-intein; and -3'-inverted terminal repeat (3'-ITR) sequence; and b) a second vector comprising, in a 5'-3' direction: -5'-inverted terminal repeat (5'-ITR) sequence; - promoter sequence; - a second intein nucleotide sequence encoding C-intein; - the 3' terminal portion of the coding sequence (CDS2); and -3'-inverted terminal repeat (3'-ITR) sequence; Or include: a') a first vector comprising in the 5'-3' direction: -5'-inverted terminal repeat (5'-ITR) sequence; - promoter sequence; - a 5' terminal portion of the coding sequence (CDS1'), said 5' terminal portion being operably linked to and under the control of said promoter; - a first intein nucleotide sequence encoding a first N-intein; and -3'-inverted terminal repeat (3'-ITR) sequence; and b') a second vector comprising in the 5'-3' direction: -5'-inverted terminal repeat (5'-ITR) sequence; - promoter sequence; - a second intein nucleotide sequence encoding the first C-intein; - the second part of the coding sequence (CDS2'); and - a third intein nucleotide sequence encoding a second N-intein; -3'-inverted terminal repeat (3'-ITR) sequence; and c') a third vector, said third vector comprising in the 5'-3' direction: -5'-inverted terminal repeat (5'-ITR) sequence; - promoter sequence; - a fourth intein nucleotide sequence encoding a second C-intein; - the third part of the coding sequence (CDS3'); and -3'-inverted terminal repeat (3'-ITR) sequence.

14. The vector system of claim 1, wherein the coding sequence is cleaved at nucleotides corresponding to aaCys1150, Ser1168, Ser1090 of the ABCA4 protein, and the split-in protein is inserted at the cleavage point.

15. The vector system of claim 1, wherein the first vector, the second vector, and when present, the third vector are independently viral vectors.

16. The vector system of claim 15, wherein the viral vector is an adenoviral vector or an adeno-associated virus (AAV) vector.

17. The vector system of claim 16, wherein the first, the second, and the third adeno-associated virus (AAV) vectors are selected from the same or different AAV serotypes.

18. The vector system of claim 17, wherein the serotype is selected from serotype 2, serotype 8, serotype 5, serotype 7 or serotype 9, serotype 7m8, serotype sh10; or serotype 2quad YF.

19. A host cell transformed with the vector system according to claim 1.

20. The vector system according to claim 1 or the host cell according to claim 19, for use in medical applications.

21. The vector system according to claim 1 or the host cell according to claim 19, for use in gene therapy.

22. The vector system or host cell for use according to claim 21, wherein the gene therapy is for the treatment and / or prevention of a pathology or disease characterized by retinal degeneration.

23. The vector system or host cell for use according to claim 22, wherein the retinal degeneration is hereditary.

24. The vector system or host cell for use according to claim 22, wherein the pathology or disease is a disease caused by a mutation in the ABCA4 gene.

25. The vector system or host cell for use according to claim 22, wherein the pathology or disease is Stargardt's disease.

26. Use of the vector system according to claim 1 or the host cell according to claim 19 for the preparation of a medicament for the treatment and / or prevention of a pathology or disease characterized by retinal degeneration, The pathology or disease is a disease caused by a mutation in the ABCA4 gene.

27. Use of a vector system according to claim 1 or a host cell according to claim 19 for the preparation of a medicament for the treatment and / or prevention of Stargardt's disease (STGD).

28. A pharmaceutical composition comprising the vector system according to claim 1 or the host cell according to claim 19 and a pharmaceutically acceptable carrier.

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