Method for producing large proteins by co-delivery of multiple vectors

Abstract

Provided herein is a method and composition for delivering large protein to a subject in need thereof for the treatment of disease or disorder.In some embodiments, the method and composition described herein is useful for delivering large protein to a subject by using a protein expression system comprising a first and a second AAV vector to treat muscle disease or muscle disorder, or neuromuscular disease or neuromuscular disorder. TIFF2024526938000059.tif75134

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 225,212, filed July 23, 2021, and U.S. Provisional Patent Application No. 63 / 256,819, filed October 18, 2021, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Technical Field The field of the invention relates to methods for delivering or inducing the production of large therapeutic proteins using multiple vectors. [Background technology]

[0003] background Gene therapy using AAV vectors shows promise for the treatment of various loss-of-function genetic disorders (Li 2020). However, this therapeutic modality is challenged by the small packaging capacity of the viral vectors (approximately 5 kb). Summary of the Invention

[0004] overview The methods and compositions described herein are based in part on the discovery that split inteins enable the delivery of large polypeptides using AAV vectors, where the large polypeptides include, but are not limited to, dystrophin.

[0005] In one aspect, a method for delivering an exogenous polypeptide to a cell is described herein, the method comprising: a first adeno-associated virus (AAV) vector particle comprising a first nucleic acid encoding a first fusion polypeptide, the first fusion polypeptide comprising a first portion of a foreign polypeptide fused to a first portion of a split intein; and A second AAV vector particle comprising a second nucleic acid encoding a second fusion polypeptide, the second fusion polypeptide comprising a second portion of an exogenous polypeptide fused to a second portion of a split intein. contacting a cell with the first and second fusion polypeptides are produced in the cell from the first and second nucleic acids, and the first and second portions of the split intein facilitate ligation of the first portion of the foreign polypeptide with the second portion of the foreign polypeptide, thereby delivering the foreign polypeptide to the cell; The exogenous polypeptide to be delivered is larger than can be encoded by a single AAV vector particle.

[0006] In one embodiment of this aspect and all other aspects described herein, the first and second nucleic acids comprise a muscle-specific expression cassette (MSEC).

[0007] In another embodiment of this aspect and all other aspects described herein, the split intein is a naturally occurring split intein.

[0008] In another embodiment of this and all other aspects described herein, the split intein is a genetically modified split intein, hi another embodiment, the genetic modification of the split intein is selected from codon optimization for expression and / or stability in mammalian cells, shortening or lengthening the split intein, or altering the encoded amino acids in the split intein to more closely match the sequence of the foreign protein to be delivered.

[0009] In another embodiment of this aspect and all other aspects described herein, the first and second portions of the foreign polypeptide are substantially the same size.

[0010] In another embodiment of this aspect and all other aspects described herein, the first and second portions of the foreign polypeptide differ in size by no more than 50 amino acids.

[0011] In another embodiment of this aspect and all other aspects described herein, the foreign polypeptide comprises less than four amino acids of the footprint from the split intein.

[0012] In another embodiment of this aspect and all other aspects described herein, the foreign polypeptide comprises no more than 3 amino acids of the footprint from the split intein.

[0013] In another embodiment of this and all other aspects described herein, the split site separating the first and second portions of the exogenous polypeptide is selected at a site having the same sequence as the footprint of the split intein, thereby producing the exogenous polypeptide without extra amino acids from the split intein.

[0014] In another embodiment of this aspect and all other aspects described herein, the exogenous polypeptide is a therapeutic polypeptide.

[0015] In another embodiment of this and all other aspects described herein, the therapeutic polypeptide is selected from dystrophin, mini-dystrophin, utrophin, and dysferlin, nebulin, titin, myosin, spectrin repeat containing nuclear envelope protein 1 (Syne-1), dystroglycan, ATP synthase, coagulation factor IIX, lamin A / C, thyroglobulin, epidermal growth factor receptor (EGFR), α-spectrin and / or β-spectrin, muscle target of rapamycin (mTOR), and ryanodine receptor 1. In another embodiment, the mini-dystrophin is greater than 160 kDa and smaller than full-length dystrophin.

[0016] In another embodiment of this aspect and all other aspects described herein, the therapeutic polypeptide is dystrophin and the N-terminal portion of the dystrophin extein is linked to the N-terminal portion of the split intein in or adjacent to the hinge domain of dystrophin.

[0017] In another embodiment of this aspect and all other aspects described herein, the hinge domain comprises hinge 1, hinge 2, hinge 3, or hinge 4 of dystrophin.

[0018] In another embodiment of this and all other aspects described herein, the therapeutic polypeptide is dystrophin and the N-terminal portion of the dystrophin extein is linked to a loop domain connecting helix b to helix c or helix c to helix a' in one of the 24 spectrin-like repeat domains of dystrophin.

[0019] In another embodiment of this and all other aspects described herein, the therapeutic polypeptide is dystrophin and the C-terminal portion of the dystrophin extein is linked to the C-terminal portion of a split intein in or next to the hinge domain of dystrophin, or to a loop domain connecting helix b to helix c, or helix c to helix a', in one of the 24 spectrin-like repeat domains of dystrophin. In another embodiment of this and all other aspects described herein, the hinge domain comprises hinge 1, hinge 2, hinge 3, or hinge 4 of dystrophin.

[0020] In another embodiment of this aspect and all other aspects described herein, the exogenous polypeptide is functional in the cell.

[0021] In another aspect, described herein is a method for delivering an exogenous polypeptide to a cell, the method comprising: a first adeno-associated virus (AAV) vector particle comprising a first nucleic acid encoding a first fusion polypeptide, the first fusion polypeptide comprising a first portion of a foreign polypeptide fused to a first portion of a first split intein, the first portion of the split intein being fused to a carboxy terminus of the first portion of the foreign polypeptide; a second AAV vector particle comprising a second nucleic acid encoding a second fusion polypeptide, (i) a second portion of a foreign polypeptide that is fused to a second portion of a first split intein at the amino terminus of the second portion of the foreign polypeptide, and (ii) a first portion of a second split intein that is fused to a carboxy terminus of the second portion of the foreign polypeptide. a second AAV vector particle, wherein the second fusion polypeptide comprises: A third AAV vector particle comprising a third core encoding a third fusion polypeptide, the third fusion polypeptide comprising a third portion of a foreign polypeptide fused at an amino terminus of the third portion of the foreign polypeptide to a second portion of a second split intein. contacting a cell with the first, second, and third fusion polypeptides are produced in the cell from the first, second, and third nucleic acids, and each portion of the first and second split intein facilitates (a) ligation of the carboxy terminus of the first portion of the foreign polypeptide to the amino terminus of the second portion of the foreign polypeptide, and (b) ligation of the carboxy terminus of the second portion of the foreign polypeptide to the amino terminus of the third portion of the foreign polypeptide, thereby delivering the foreign polypeptide to the cell; The exogenous polypeptide delivered is larger than can be encoded by a single AAV vector particle. In one embodiment of this and all other aspects described herein, the first and second split inteins do not cross-splice.

[0022] In another aspect, a protein expression system is provided herein comprising a set of AAV vector particles comprising a first and a second AAV particle, the first AAV vector particle comprises a first nucleic acid encoding a first fusion polypeptide, the first fusion polypeptide comprising a first portion of a foreign polypeptide fused to a first portion of a split intein; and The second AAV vector particle comprises a second nucleic acid encoding a second fusion polypeptide, the second fusion polypeptide comprising a second portion of the foreign polypeptide fused to a second portion of the split intein.

[0023] In one embodiment of this and all other aspects described herein, coinfection of a cell with the first and second AAV vector particles promotes linkage of the first portion of the foreign polypeptide with the second portion of the foreign polypeptide, accompanied by removal of the first and second portions of the split intein.

[0024] In another embodiment of this and all other aspects described herein, linking a first portion of a foreign polypeptide to a second portion of a foreign polypeptide, with removal of the first and second portions of the split intein, results in a larger foreign polypeptide than can be encoded in one AAV particle.

[0025] In another aspect, a protein expression system is provided herein comprising a set of AAV vector particles comprising a first, second, and third AAV particles, the first AAV vector particle comprises a first nucleic acid encoding a first fusion polypeptide, the first fusion polypeptide comprising a first portion of a foreign polypeptide fused to a first portion of a first split intein, the first portion of the split intein fused to a carboxy terminus of the first portion of the foreign polypeptide; the second AAV vector particle comprises a second nucleic acid encoding a second fusion polypeptide; (i) a second portion of a foreign polypeptide that is fused to a second portion of a first split intein at the amino terminus of the second portion of the foreign polypeptide, and (ii) a first portion of a second split intein that is fused to a carboxy terminus of the second portion of the foreign polypeptide. the second fusion polypeptide comprises; The third AAV vector particle comprises a third core encoding a third fusion polypeptide, the third fusion polypeptide comprising a third portion of a foreign polypeptide fused at the amino terminus of the third portion of the foreign polypeptide to a second portion of a second split intein.

[0026] In one embodiment of this and all other aspects described herein, coinfection of a cell with the first, second, and third AAV vector particles promotes ligation of the first portion of the foreign polypeptide to the second portion of the foreign polypeptide, with removal of the first and second portions of the first split intein, and ligation of the second portion of the foreign polypeptide to the third portion of the foreign polypeptide, with removal of the first and second portions of the second split intein.

[0027] In another embodiment of this and all other aspects described herein, the linking of a first portion of a foreign polypeptide to a second portion of a foreign polypeptide, with removal of the first and second portions of the first split intein, and the linking of the second portion of a foreign polypeptide to a third portion of a foreign polypeptide, with removal of the first and second portions of the second split intein, results in a foreign polypeptide that is larger than can be encoded in one AAV particle.

[0028] In another embodiment of this aspect and all other aspects described herein, expression of the first and second fusion polypeptides, or expression of the first, second, and third fusion polypeptides, is driven by a muscle-specific expression cassette.

[0029] In another aspect, described herein is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering a protein expression system described herein, thereby treating the subject.

[0030] In one embodiment of this aspect and all other aspects described herein, the subject in need thereof has a muscle disease or disorder, or a neuromuscular disease or disorder.

[0031] In another embodiment of this aspect and all other aspects described herein, the exogenous polypeptide is dystrophin or mini-dystrophin, and the subject in need thereof has Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).

[0032] In another embodiment of this aspect and all other aspects described herein, the dystrophin or mini-dystrophin increases muscle strength in a dystrophic muscle by at least 10%.

[0033] In another embodiment of this aspect and all other aspects described herein, expression of the first and second fusion polypeptides, or expression of the first, second, and third fusion polypeptides, is driven by a muscle-specific expression cassette.

[0034] In another embodiment of this and all other aspects described herein, the protein expression system is administered by injection into the vascular system or by direct injection into tissue.

[0035] In another aspect, described herein is a method for inducing production of an exogenous polypeptide in a cell, the method comprising: a first adeno-associated virus (AAV) vector particle comprising a first nucleic acid encoding a first fusion polypeptide, the first fusion polypeptide comprising a first portion of a foreign polypeptide fused to a first portion of a split intein; and A second AAV vector particle comprising a second nucleic acid encoding a second fusion polypeptide, the second fusion polypeptide comprising a second portion of an exogenous polypeptide fused to a second portion of a split intein. contacting a cell with the first and second fusion polypeptides are produced in the cell from the first and second nucleic acids, and the first and second portions of the split intein facilitate ligation of the first portion of the foreign polypeptide with the second portion of the foreign polypeptide, thereby inducing production of the foreign polypeptide in the cell; The foreign polypeptide produced is larger than can be encoded by a single AAV vector particle.

[0036] In one embodiment of this aspect and all other aspects described herein, the first and second nucleic acids comprise a muscle-specific expression cassette (MSEC).

[0037] In another embodiment of this aspect and all other aspects described herein, the split intein is a naturally occurring split intein.

[0038] In another embodiment of this and all other aspects described herein, the split intein is a genetically modified split intein, hi another embodiment, the genetic modification of the split intein is selected from codon optimization for expression and / or stability in mammalian cells, shortening or lengthening the split intein, or altering the encoded amino acids in the split intein to more closely match the sequence of the foreign protein to be produced.

[0039] In another embodiment of this aspect and all other aspects described herein, the first and second portions of the foreign polypeptide are substantially the same size.

[0040] In another embodiment of this aspect and all other aspects described herein, the first and second portions of the foreign polypeptide differ in size by no more than 50 amino acids.

[0041] In another embodiment of this aspect and all other aspects described herein, the foreign polypeptide comprises less than four amino acids of the footprint from the split intein.

[0042] In another embodiment of this aspect and all other aspects described herein, the exogenous polypeptide comprises no more than 3 amino acids of the footprint of the split intein.

[0043] In another embodiment of this and all other aspects described herein, the split site separating the first and second portions of the exogenous polypeptide is selected at a site having the same sequence as the footprint of the split intein, thereby producing the exogenous polypeptide without extra amino acids from the split intein.

[0044] In another embodiment of this aspect and all other aspects described herein, the exogenous polypeptide is a therapeutic polypeptide.

[0045] In another embodiment of this and all other aspects described herein, the therapeutic polypeptide is selected from dystrophin, mini-dystrophin, utrophin, and dysferlin, nebulin, titin, myosin, spectrin repeat-containing nuclear membrane protein 1 (Syne-1), dystroglycan, ATP synthase, coagulation factor IIX, lamin A / C, thyroglobulin, epidermal growth factor receptor (EGFR), α-spectrin and / or β-spectrin, muscle target of rapamycin (mTOR), and ryanodine receptor 1.

[0046] In another embodiment of this aspect and all other aspects described herein, the mini-dystrophin is larger than 160 kDa and smaller than full-length dystrophin.

[0047] In another embodiment of this aspect and all other aspects described herein, the therapeutic polypeptide is dystrophin and the N-terminal portion of the dystrophin extein is linked to the N-terminal portion of the split intein in or adjacent to the hinge domain of dystrophin.

[0048] In another embodiment of this aspect and all other aspects described herein, the hinge domain comprises hinge 1, hinge 2, hinge 3, or hinge 4 of dystrophin.

[0049] In another embodiment of this and all other aspects described herein, the therapeutic polypeptide is dystrophin and the N-terminal portion of the dystrophin extein is linked to a loop domain connecting helix b to helix c or helix c to helix a' in one of the 24 spectrin-like repeat domains of dystrophin.

[0050] In another embodiment of this and all other aspects described herein, the therapeutic polypeptide is dystrophin and the C-terminal portion of a dystrophin extein is linked to the C-terminal portion of a split intein in or next to the hinge domain of dystrophin, or to a loop domain connecting helix b to helix c, or helix c to helix a', in one of the 24 spectrin-like repeat domains of dystrophin.

[0051] In another embodiment of this aspect and all other aspects described herein, the hinge domain comprises hinge 1, hinge 2, hinge 3, or hinge 4 of dystrophin.

[0052] In another embodiment of this aspect and all other aspects described herein, the exogenous polypeptide is functional in the cell.

[0053] In another aspect, described herein is a method for inducing production of an exogenous polypeptide in a cell, the method comprising: a first adeno-associated virus (AAV) vector particle comprising a first nucleic acid encoding a first fusion polypeptide, the first fusion polypeptide comprising a first portion of a foreign polypeptide fused to a first portion of a first split intein, the first portion of the split intein being fused to a carboxy terminus of the first portion of the foreign polypeptide; a second AAV vector particle comprising a second nucleic acid encoding a second fusion polypeptide, (i) a second portion of a foreign polypeptide that is fused to a second portion of a first split intein at the amino terminus of the second portion of the foreign polypeptide, and (ii) a first portion of a second split intein that is fused to a carboxy terminus of the second portion of the foreign polypeptide. a second AAV vector particle, wherein the second fusion polypeptide comprises: A third AAV vector particle comprising a third core encoding a third fusion polypeptide, the third fusion polypeptide comprising a third portion of a foreign polypeptide fused at an amino terminus of the third portion of the foreign polypeptide to a second portion of a second split intein. contacting a cell with the first, second, and third fusion polypeptides are produced in the cell from the first, second, and third nucleic acids, and each portion of the first and second split intein promotes (a) ligation of the carboxy terminus of the first portion of the foreign polypeptide to the amino terminus of the second portion of the foreign polypeptide, and (b) ligation of the carboxy terminus of the second portion of the foreign polypeptide to the amino terminus of the third portion of the foreign polypeptide, thereby producing the foreign polypeptide in the cell; The exogenous polypeptide produced is larger than can be encoded by a single AAV vector particle. In one embodiment of this and all other aspects described herein, the first and second split inteins do not cross-splice.

[0054] In another aspect, provided herein is a composition described herein for use in treating a disease or disorder in a subject in need thereof (e.g., a subject having a muscular or neuromuscular disorder). [Brief description of the drawings]

[0055] [Figure 1]1A-1B. (FIG. 1A) Schematic of the coding sequence of DMD (top) encoding the full-length "muscle-specific" isoform of dystrophin (bottom), which consists of an amino-terminal globular domain that binds to the actin cytoskeleton, followed by a flexible and stretchable rod-like domain composed of 24 spectrin-like repeats interspersed with four proline-rich "hinge" regions. The dystroglycan-binding domain (DgBD) is located after the rod-like domain, followed by a carboxy-terminal (CT) domain that contains binding sites for the syntrophin and dystrobrevin protein families. The DgBD and CT domains nucleate the assembly of the dystrophin-glycoprotein complex (DGC). (FIG. 1B) Various μDys constructs being evaluated in clinical trials by Pfizer, Sarepta Therapeutics, and Solid Biosciences. [Diagram 2] Dual AAV vector homologous recombination strategy to reconstitute mini-Dys(ΔH2-SR19). The two AAV vectors encode either the N-terminal fragment (top) or the C-terminal fragment (bottom) of mini-Dys. Both vectors carry recombination sequences (exons 51-53), which allow the formation of the larger and functional mini-Dys(ΔH2-SR19). [Diagram 3] Schematic diagram of protein trans-splicing mediated by consecutive inteins (more common) or split inteins. [Figure 4]Figures 4A-4B. Examples of GFP reconstitution with the split intein Npu in HEK293 cells. (Figure 4A) Brightfield and fluorescence micrographs of live HEK293 cells transfected with a wild-type (WT) GFP plasmid or with the N- and / or C-terminal plasmids of the GFP / Npu intein. (Figure 4B) GFP fluorescence intensity was measured using a spectrophotometer. Values ​​are expressed as mean ± sem (n = 3). [Figure 5A] Figures 5A-5C. In vitro validation of mini-Dys reconstitution. (Figure 5A) Schematic of intein-mediated reconstitution of mini-Dys. Left: N-terminal vector encoding exons 1-50 of the human DMD sequence, but lacking exons 21-41. Right: C-terminal vector encoding exons 51-79 of the human DMD sequence. The mini-Dys sequence is fused to the N- or C-terminal half of the selected intein. (Figure 5B) Western blot analysis of HEK293 cell lysates showing the 290 kDa mini-Dys. For the control mini-dys, cells were transfected with a plasmid expressing the entire mini-Dys ΔSR5-15. For the split mini-Dys / intein, cells were co-transfected with both the N- and C-terminal vectors. Each lane represents the selected split site between SR19 and hinge 3. (FIG. 5C) Densitometry of mini-Dys quantification normalized to the loading control, GAPDH (n = 4–5 independent experiments). Data are shown as mean ± sem. MW: molecular weight. kDa: kilodaltons. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6A]6A-6B. Schematic of AAV-based dystrophin replacement using the SIMPL-GT (Split Intein-Mediated Protein Ligation for Gene Therapy) approach. (FIG. 6A) Dual vector strategy consisting of co-administration of two AAV vectors expressing two halves of mini-Dys(ΔSR5-15) fused to a split intein. The intein polypeptide self-excises after in-frame transcription and translation together with the N- or C-terminal fragments of mini-Dys and ligates the adjacent peptide, resulting in expression of highly functional mini-Dys(ΔSR5-15). (FIG. 6B) Expression of full-length dystrophin by administration of triple AAV vectors. The first AAV vector encodes the N-terminal to SR8 dystrophin protein fused to the N-terminal fragment of split intein1. The second AAV vector encodes an intermediate dystrophin fragment (SR9-19) flanked by both the C-terminal half of intein 1 and the N-terminal half of intein 2, while the third AAV vector encodes a C-terminal fragment of dystrophin fused to the C-terminal half of intein 2. Two rounds of trans-splicing between intein 1 and intein 2 ligate the three dystrophin fragments into the full-length protein. [Figure 6B] See legend to Figure 6A. [Figure 7]Screening of split inteins using the split GFP system. The N- or C-terminal half of GFP was cloned in frame to the N- or C-terminal half of our codon-optimized split intein. Human embryonic kidney 293 (HEK293) cells were co-transfected with both the N- and C-terminal GFP / intein plasmids. After 24 h, the fluorescence intensity of GFP was measured using a spectrophotometer. Values ​​are expressed as the mean ± sem of the percentage relative to WT GFP (n = 5–6). The efficiency of protein ligation of each split intein (GFP fluorescence of a given intein / GFP fluorescence of the internal control) is marked in the bar. [Figure 8] Specificity and cross-reactivity of split inteins using the split-GFP system. To test the cross-reactivity of the N-terminus of a given split intein with the C-terminus of another split intein, the N- and C-termini of the split GFP·inteins were tested in HEK293 cells. The first group of split inteins showed amino acid similarity, so they showed low specificity and cross-reacted with other inteins of the same group. On the other hand, the second group of split inteins, namely gp41.1, IMPDH, and Nrdj1, were more specific for the other half of the same intein and did not cross-react with any other split inteins. Values ​​are expressed as the mean ± sem of the percentage relative to WT GFP (n = 3–4). These observations are particularly important with respect to the triple vector strategy, which requires two highly specific split inteins to ligate three dystrophin fragments into a full-length protein. [Figure 9A]Figures 9A-9C. Importance and optimization of split intein footprint for dystrophin reconstitution. (Figure 9A) Intein-mediated protein trans-splicing is highly dependent on a sequence of amino acids found in natural bacterial extein proteins. When the N- and C-terminal fragments of split inteins are fused and spliced ​​out, these natural extein amino acids (AEY and CFN for both Aha and Sel; and SGY and SSS for gp41.1; GGG and SIC for IMPDH; NPC and SEI for Nrdj1) are left as footprints in the reconstituted protein. Therefore, we tested several combinations to reduce this footprint to a minimum. These data show that when tested in the split GFP system, with the split intein Aha, the same splicing efficiency can be achieved with AEY as with AEY / CFN, while with gp41.1, GY / S is sufficient for efficient GFP ligation. The footprint was shorter with IMPDH and Nrdj1, G / S only, and C / S only, respectively. Values ​​are expressed as the mean ± sem of the percentage relative to WT GFP (n = 4). Illustrated examples of protein trans-splicing with Aha and / or gp41.1 leaving minimal footprints for reconstitution of full-length dystrophin (Figure 9B) or mini-dystrophin (Figure 9C). [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 10] Identification of several cleavage sites in the human dystrophin protein where several naturally occurring amino acids can be used as part of the footprint of the intein. [Figure 11]11A-11B. (FIG. 11A) Western blot analysis of HEK293 cell lysates showing the 290 kDa mini-Dys. For control mini-dys, cells were transfected with a plasmid expressing the entire mini-Dys ΔSR5-15. For split mini-Dys / intein, cells were co-transfected with both N- and C-terminal vectors. Each lane represents a selected split site between SR19 and hinge 3. (FIG. 11B) Densitometry of mini-Dys quantification normalized to the loading control GAPDH (n = 4-5 independent experiments). Data are shown as mean ± sem. MW: molecular weight. kDa: kilodaltons. These data show that mini-dystrophin ΔSR-5-15 can be efficiently reconstituted when split gp41.1 is inserted into four different split sites. [Figure 12A]Figures 12A-12C. In vivo expression of mini-dystrophin ΔSR-5-15 after intramuscular injection of AAV. The split mini-dystrophin / intein clone was inserted into a pAAV plasmid, which contained a muscle-specific creatine kinase 8 (CK8) regulatory cassette and a short synthetic polyA flanked by two inverted terminal repeats (ITRs) of AAV serotype 2. The final pAAV plasmid was co-transfected with pDG6 packaging plasmid to generate recombinant AAV2 / 6 vectors, which were purified using heparin affinity column chromatography and then concentrated using sucrose gradient centrifugation. AAV encoding the N- and / or C-terminal ends of the split mini-dystrophin / intein at a dose of 5 x 1010 viral genomes (vg) was administered into the tibialis anterior (TA) muscle of 3-week-old C57BL / 6-mdx4cv mice. Four weeks after injection, the injected muscles were harvested and total proteins were extracted, which were resolved on SDS gels for Western blotting (Figure 12A). Strong expression of mini-dystrophin ΔSR5-15 was detected in the four TA muscles tested, highlighting the effectiveness of the SIMPLI-GT approach. Muscles were cryosectioned and immunostained for dystrophin (Figure 12B) or stained with hematoxylin and eosin to measure myofiber size and nuclear location (Figure 12C). Reconstituted mini-dystrophin ΔSR5-15 was correctly localized in the sarcolemma of mdx4cv injected with dual N- and C-terminal AAV vectors. These muscles showed an overall improvement in muscle histology, with no inflammation present. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 13]In vitro proof of concept for expression of full-length dystrophin by a triple vector strategy. Western blot analysis of HEK293 cell lysates transfected with three plasmids expressing either the N-, C-, or intermediate fragments of human dystrophin. Split intein gp41.1 was used to ligate the intermediate fragment to the C-terminal fragment, while six different split inteins were tested for ligation of the N-terminal and intermediate fragments. [Figure 14] Figures 14A-14B. In vitro proof of concept of full-length dysferlin expression. (Figure 14A) Western blot analysis of HEK293 cell lysates transfected with plasmids expressing either full-length human dysferlin, or split dysferlin / gp41.1 intein, or split dysferlin / IMPDH intein. Three different cleavage sites were tested. (Figure 14B) Densitometry quantification of full-length dysferlin expression normalized to GAPDH loading control (n = 5 independent experiments). Data are shown as mean ± sem. These data show that full-length dysferlin is efficiently reconstituted when split gp41.1 is inserted into two different cleavage sites. [Figure 15A]Figures 15A-15W. DNA and protein sequences of split inteins. Figure 15A is Aha (SEQ ID No: 1 and 2). Figure 15B is Aov (SEQ ID No: 3 and 4). Figure 15C is Asp (SEQ ID No: 5 and 6). Figure 15D is Ava (SEQ ID No: 7 and 8). Figure 15E is Cra (SEQ ID No: 9 and 10). Figure 15F is Csp-CCY (SEQ ID No: 11 and 12). Figure 15G is Csp-PCC7424 (SEQ ID No: 13 and 14). Figure 15H is Csp-PCC8801 (SEQ ID No: 15 and 16). Figure 15I is Cwa (SEQ ID No: 17 and 18). Figure 15J is gp41.1 (SEQ ID No: 19 and 20). Figure 15K is gp41.8 (SEQ ID No: 21 and 22). Figure 15L is IMPDH (SEQ ID No: 23 and 24). Figure 15M is Maer (SEQ ID No: 25 and 26). Figure 15N is Mcht (SEQ ID No: 27 and 28). Figure 15O is Npu (SEQ ID No: 29 and 30). Figure 15P is Nrdj (SEQ ID No: 31 and 32). Figure 15Q is Oli (SEQ ID No: 33 and 34). Figure 15R is Sel (SEQ ID No: 35 and 36). Figure 15S is Ssp-PCC6803 (SEQ ID No: 37 and 38). Figure 15T is Ssp-PCC7002 (SEQ ID No: 39 and 40). Figure 15U is Tel (SEQ ID No: 41 and 42). Figure 15V is Ter (SEQ ID No: 43 and 44). Figure 15W is Tvu (SEQ ID No: 45 and 46). [Figure 15B] See legend to Figure 15A. [Figure 15C] See legend to Figure 15A. [Figure 15D] See legend to Figure 15A. [Figure 15E] See legend to Figure 15A. [Figure 15F] See legend to Figure 15A. [Figure 15G] See legend to Figure 15A. [Figure 15H] See legend to Figure 15A. [Figure 15I] See legend to Figure 15A. [Figure 15J] See legend to Figure 15A. [Figure 15K] See legend to Figure 15A. [Figure 15L] See legend to Figure 15A. [Figure 15M] See legend to Figure 15A. [Figure 15N] See legend to Figure 15A. [Figure 15O] See legend to Figure 15A. [Figure 15P] See legend to Figure 15A. [Figure 15Q] See legend to Figure 15A. [Figure 15R] See legend to Figure 15A. [Figure 15S] See legend to Figure 15A. [Figure 15T] See legend to Figure 15A. [Figure 15U] See legend to Figure 15A. [Figure 15V] See legend to Figure 15A. [Figure 15W] See legend to Figure 15A. [Figure 16] Cleavage site of full-length dysferlin. [Figure 17] Cleavage site of full-length dystrophin (IMPDH intein). [Figure 18] Cleavage site of full-length dystrophin (Nrdj intein). [Figure 19] Cleavage site of full-length dystrophin. [Figure 20] Cleavage site of full-length dystrophin (gp41.1 intein). [Figure 21-1] Cleavage site of mini-dystrophin ΔSR5-15. [Figure 21-2] See description of Figure 21-1. [Figure 22A]Figures 22A-22D. In vivo expression of full-length dystrophin after intramuscular administration of three intein vectors. Each combination of split dystrophin / intein clones was packaged into an AAV6 vector using the CK8e promoter and administered locally into the TA muscle of 3-week-old mdx4cv mice at 5 x 1010 vg per construct. Four weeks after injection, total protein was analyzed by Western blot using an antibody that recognizes the C-terminal end of dystrophin (Figure 22A). Similar to the in vitro observations, a 427 kDa band was detected in both combinations, with higher expression detected with the combination of split Nrdj1 and split gp41.1 (2-4 times higher than WT expression). More importantly, a dramatic reduction in centrally-nucleated myofibers was observed at this early time point, approximately 65% ​​in untreated mdx4cv TA muscles, compared to 30-40% in muscles treated with this triple-vector strategy, and there was a marked improvement in overall muscle histology at this short-term time point (Figures 22B, 22C). Reconstituted full-length dystrophin was correctly localized in the sarcolemma as assessed with one of three different antibodies that recognize the N-terminal, C-terminal, or intermediate fragments of the full-length dystrophin protein. Figure 22A, Western blot showing full-length dystrophin expression after triple-vector administration in mdx4cv TA muscles (top). Figure 22B, Visualization of centrally-nucleated myofibers in cross-sections of mdx4cv TA muscles treated with various combinations of vectors (or saline) and stained with hematoxylin and eosin. Also shown are untreated wild type (WT) or mdx4cv TA muscles from age-matched mice. N-terminal only: muscles injected with one vector, in this case only the N-terminal vector; intermediate only: muscles injected with one vector, in this case only the intermediate vector; C-terminal only: muscles injected with one vector, in this case only the C-terminal vector. Other panels show muscles injected with a combination of two vectors or a combination of all three vectors (triple).FIG. 22C, Quantification of centrally nucleated myofibers in cross sections of mdx4cv TA muscles treated with the indicated combinations of triple vectors (or saline) and stained with hematoxylin and eosin. Values ​​from untreated wild-type (WT) mouse TA muscles are also shown. Data are derived from counting approximately 400 myofibers in the different muscles. FIG. 22D, Triple immunolabeling of TA muscle cryosections with antibodies against the N-terminal, C-terminal, or intermediate fragments of dystrophin. Wild-type muscles were uninjected; mdx4cv muscles were injected with saline or two combinations of triple vectors as in FIG. 22A. [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 22D] See legend to Figure 22A. [Figure 23A] 23A-23C. In vivo expression of mini-Dys and full-length dystrophin after intravenous injection of dual or triple vectors. Eight-week-old mdx4cv mice were treated systemically with a total dose of 2 x 1014 vg / kg over a 3-month treatment period. Contractile properties of both hindlimb and diaphragm muscles were assessed using muscle force transducers (Fig. 23A, Fig. 23B). Mice treated with dual or triple vectors showed significant improvement in intrinsic force development of tibialis anterior and diaphragm muscles compared to saline-treated mdx4cv mice and wild-type mice. These muscles showed robust expression of mini-Dys and full-length dystrophin using Western blot (Fig. 23C). Fig. 23A, In vivo intrinsic force of tibialis anterior. Fig. 23B, In vitro intrinsic force of isolated diaphragm muscle fragments. FIG. 23C, Western blot showing expression of mini-Dys and full-length dystrophin in tibialis anterior muscle following systemic administration of dual or triple vectors. [Figure 23B] See legend to Figure 23A. [Figure 23C] See legend to Figure 23A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] Detailed Description Provided herein is a method and composition that is useful for delivering foreign polypeptides that are too large to fit into one adenovirus vector, one adeno-associated virus vector, one lentivirus vector, or one retrovirus vector.The method and composition described herein employs the use of split intein, which mediates the fusion of the first and second parts of the large foreign polypeptide to be delivered by using at least two types of virus vectors (e.g., AAV vectors), thereby enabling the delivery of large foreign polypeptides to cells (e.g., muscle cells).The method and composition also relates to the muscle-specific expression of such foreign polypeptides (e.g., dystrophin, utrophin, and dysferlin, etc.) in cells.

[0057] definition For convenience, some terms used throughout this application (including the specification, examples, and appended claims) are collected below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0058] As used herein, the term "splice" or "splices" refers to the excision of an internal portion of a polypeptide and the joining of adjacent portions of the internal portion to form two or more smaller polypeptide molecules (e.g., a truncated polypeptide and a spliced ​​polypeptide). In some instances, splicing also includes fusing two or more smaller polypeptides together to form a new polypeptide. Splicing can also refer to the joining of two polypeptides encoded by two separate nucleic acid sequences or two separate vectors through the action of a split intein.

[0059] As used herein, the term "cleave" or "cleaves" refers to the cutting of one polypeptide to form two or more smaller polypeptide molecules. In some instances, cleavage is mediated by the addition of an exogenous endopeptidase, which is often referred to as "proteolytic cleavage". In other instances, cleavage can be mediated by an activity that is endogenous to one or both of the peptide sequences to be cleaved, which is often referred to as "autocleavage". Cleavage also refers to the autocleavage of two polypeptides induced by the addition of a third peptide that is not proteolytic, such as in the action of the split intein system described herein.

[0060] The term "fused" means covalently linked, for example, a first peptide and a second peptide are fused when the two peptides are covalently linked to one another (e.g., via a peptide bond).

[0061] As used herein, the term "intein" refers to a naturally occurring, self-splicing protein subdomain that can simultaneously excise itself from a larger protein structure and join two previously adjacent peptide regions ("exteins") together to form one mature host protein. In some inteins, the precursor protein is derived from two genes, and is referred to as a "split intein."

[0062] As used herein, the term "split intein" refers to an intein that is composed of two or more separate elements that are not fused to each other. Split inteins can be naturally occurring or engineered by splitting a contiguous intein. Typically, the term "split intein" refers to any intein that has one or more peptide bond breaks between the N-terminal segment of the intein and the C-terminal segment of the intein, resulting in separate molecules that can be non-covalently recombined or reconstituted as an intein that is functional for splicing or cleavage reactions. Any catalytically active intein or fragment thereof can be used to derive a split intein for use in the systems and methods disclosed herein. For example, in one aspect, the split intein can be derived from a eukaryotic intein. In another aspect, the split intein can be derived from a bacterial intein. In another aspect, the split intein may be derived from an archaeal intein. Preferably, such derived split inteins retain only the amino acid sequences necessary to catalyze the splicing reaction.

[0063] As used herein, "N-terminal segment of an intein" refers to any intein sequence that includes an N-terminal amino acid sequence that is functional for splicing and / or cleavage reactions when combined with a corresponding C-terminal segment of an intein. Thus, the N-terminal segment of an intein also includes a sequence that is removed by splicing when splicing occurs. The N-terminal segment of an intein may include a sequence that is a modified version of the N-terminal portion of a naturally occurring (natural) intein sequence. For example, the N-terminal segment of an intein may include additional amino acid residues and / or mutated residues, provided that the inclusion of such residues does not render the intein non-functional for splicing or cleavage. Preferably, the inclusion of the additional residues and / or mutated residues improves or enhances the splicing activity and / or controllability of the intein. It is also possible to genetically fuse non-intein residues to intein segments to confer additional functionality, such as the ability to undergo affinity purification or covalent immobilization.

[0064] As used herein, "C-terminal segment of intein" refers to any intein sequence that includes a C-terminal amino acid sequence that is functional for splicing and / or cleavage reactions when combined with the corresponding N-terminal segment of intein. In one aspect, the C-terminal segment of intein includes a sequence that is removed by splicing when splicing occurs. In another aspect, the C-terminal segment of intein is cleaved from the peptide sequence fused to its C-terminus. The sequence cleaved from the C-terminus of the C-terminal intein is a protein for treating muscle disorders, such as dystrophin, utrophin, dysferlin, mini-dystrophin, etc. The C-terminal segment of intein may include a sequence that is a modified version of the C-terminal portion of a naturally occurring intein sequence (natural intein sequence). For example, the C-terminal segment of an intein may contain additional amino acid residues and / or mutated residues, provided that the inclusion of such residues does not render the C-terminal segment of the intein non-functional with respect to splicing or cleavage. Preferably, the inclusion of the additional and / or mutated residues improves or enhances the splicing and / or cleavage activity of the intein.

[0065] As used herein, the term "larger than can be encoded by one AAV vector particle" refers to a polypeptide whose encoding nucleic acid exceeds the packaging limit of AAV vector particle.Although the exact packaging limit may vary slightly depending on the serotype or variant of the AAV vector used, the maximum genome packaging capacity of AAV vectors that efficiently infect and transduce target cells is about 5 kb (wild type AAV genome is about 4.7 kb; under certain conditions, it is possible to package genomes up to 5.5 kb or larger, but they do not efficiently infect and transduce target cells).By excluding ITRs, it is possible to incorporate about 3.5 kb of DNA for promoter, transgene coding region, polyadenylation sequence, and other regulatory elements to carry transgene constructs by one AAV vector particle.Therefore, a transgene that requires more than about 3.5 kb to express a desired protein is larger than can be encoded by one AAV vector particle as the term is used herein. In some embodiments, proteins larger than can be encoded by a single vector particle require at least 4 kb, at least 4.5 kb, at least 5 kb, at least 5.5 kb, at least 6 kb, at least 6.5 kb, at least 7 kb, at least 7.5 kb, at least 8 kb, at least 8.5 kb, at least 9 kb, at least 9.5 kb, at least 10 kb, at least 10.5 kb, at least 11 kb, at least 11.5 kb, at least 12 kb, at least 12.5 kb, at least 13 kb, at least 13.5 kb, at least 14 kb, or more, to encode a transgene polypeptide.As discussed elsewhere herein, if a target protein requires a longer nucleic acid sequence than can fit into two separate viral vectors to generate a full-length target polypeptide (or a shorter-than-full-length polypeptide that has functional improvements over the more truncated mini- or microgene constructs), the polypeptide may be split between separate vectors, including three, or potentially more, split intein constructs. In this example, coinfection of the vector set may produce a full-length polypeptide, or an improved shorter-than-full-length polypeptide.

[0066] As used herein, the terms "a first portion of a foreign polypeptide fused to a first portion of a split intein" and "a second portion of a foreign polypeptide fused to a second portion of a split intein" refer to a fragment of a target polypeptide that is larger than can be encoded by one AAV vector particle when used in connection with a method for delivering a foreign polypeptide to a cell, in connection with a method for producing a foreign polypeptide in a cell, or in connection with a method of treatment or prevention based on or a composition for such delivery or production, as described herein. The fragments of the first and second portions of the target polypeptide are fused to the amino-terminal and carboxy-terminal portions of the split intein, respectively, in a manner that allows excision of the intein and covalent attachment of the first and second portions of the polypeptide (the engineered exteins) to reconstitute the target protein when both fusion proteins are expressed in a cell. The size of the first and second portions of the target polypeptide may be different, for example, the amino-terminal fragment is shorter, about the same size, or longer than the carboxy-terminal fragment (and the corresponding carboxy-terminal fragment is changed to be longer, about the same size, or shorter, respectively) but when the target is cleaved into two fragments, it is preferred that the target is split approximately near the middle of the target protein. When the target protein is cleaved into three fragments as described herein, they may be different in size, but it is also preferred that the three fragments are about the same length. It may be considered to split the target protein between structural domains, or at the junction of structural domains, rather than within the structural domains, such as between alpha helices, beta sheets, or any two such structural domains. As a non-limiting example, in the context of a dystrophin or utrophin polypeptide, it is intended that the protein is split between spectrin-like repeat domains, or between the spectrin-like repeat domain and the hinge domain.The various domains of the exemplary large proteins dystrophin, utrophin, and dysferlin are discussed further herein below. The boundaries of the various domains for the dystrophin and dysferlin polypeptides are also described herein below, and one skilled in the art can determine the boundaries between domains in other proteins.

[0067] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and individual components thereof that are essential to the invention, but allow for the inclusion of unspecified elements that may or may not be essential.

[0068] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that are characteristic of that embodiment of the invention and do not materially affect the basic and novel or functional characteristics.

[0069] The term "consisting of" refers to compositions, methods, and individual components thereof described herein, excluding any element not recited in the description of the embodiment.

[0070] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods and / or one or more steps of the type described herein and / or that would be apparent to one of ordinary skill in the art upon reading this disclosure, etc. It is understood that the above detailed description and the following examples are merely illustrative and should not be construed as limitations on the scope of the invention. Various changes and modifications to the disclosed embodiments that are apparent to those skilled in the art can be made without departing from the spirit and scope of the invention. Moreover, all patents, patent applications, and publications identified are expressly incorporated herein by reference for purposes of illustration and disclosure, such as the methodologies described in such publications that may be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. Any statement of the dates of these documents or any description of the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the accuracy of the dates or contents of these documents.

[0071] In preferred aspects, the disclosure described herein does not relate to processes for cloning humans, processes for altering the genetic identity of the human germ line, the use of human embryos for industrial or commercial purposes, or processes for altering the genetic identity of animals that may cause suffering to the animals without providing any substantial medical benefit to either humans or animals, as well as the animals resulting from such processes.

[0072] Muscular dystrophies Muscular dystrophies are a group of inherited disorders characterized by progressive muscle weakness and loss of muscle tissue.

[0073] Muscular dystrophy includes many genetic disorders, including Becker muscular dystrophy and Duchenne muscular dystrophy (or DMD), both of which are caused by mutations in dystrophin gene.Both disorders have similar symptoms, but Becker muscular dystrophy is one of the more slowly progressive forms of the disease.Duchenne muscular dystrophy is one of the rapidly progressive forms of muscular dystrophy.

[0074] Both of these disorders are characterized by progressive muscle weakness in the legs and pelvis, which is associated with loss of muscle mass. Muscle weakness also occurs in the arms, neck, and other areas, but is not as severe as in the lower body. Calf muscles are the first to hypertrophy (the body's attempt to compensate for muscle loss), and the hypertrophied muscle tissue is eventually replaced by fat and connective tissue (pseudohypertrophy). Muscle contraction occurs in the legs and heels, leading to muscle inactivity, which is due to muscle fiber shortening and connective tissue fibrosis. Skeletal development becomes abnormal, causing skeletal deformation in the chest and other areas. Cardiomyopathy occurs in almost all cases. Mouse models for DMD exist and are proving to be useful for better understanding both the normal function of dystrophin and the pathology of the disease. In particular, experiments to enhance production of utrophin, a close relative of dystrophin, to compensate for the loss of dystrophin are promising and may lead to the development of effective treatments for this devastating disease.

[0075] Dysferlinopathy is a muscular dystrophy caused by mutations in the dysferlin gene. Symptoms of dysferlinopathy vary significantly between individuals. The most common clinical manifestations associated with dysferlinopathy include limb-girdle muscular dystrophy (LGMD2B), Miyoshi myopathy, distal myopathy with anterior tibial onset (DMAT), proximodistal weakness, pseudometabolic myopathy, and hyperCKemia. Most commonly, patients present with distal weakness in their teenage years and lose distal motor function over the course of the next decade. Patients typically require a wheelchair for mobility, but have a variable degree of overall physical control. Because dysferlinopathy is often misdiagnosed, its incidence has not been determined. To date, there are no effective treatments to slow the loss of muscle function or to reverse / ameliorate the dystrophic phenotype.

[0076] Foreign Polypeptides A distinct advantage of the methods and compositions described herein is their ability to encode and deliver large proteins to cells, such as, inter alia, muscle cells. Vectors, such as adenoviral associated vectors (AAV), are limited in their ability to package nucleic acids and therefore cannot encode and deliver large proteins with a single AAV vector. The methods and compositions described herein utilize split inteins, where the N-terminal region of the split intein and a portion of a desired foreign polypeptide are encoded in a first AAV vector, and the C-terminal region of the split intein and a second portion of a desired foreign polypeptide are encoded in a second AAV vector. Co-expression of the products from each AAV in a cell allows the first and second portions of the split intein to facilitate the linkage of the first portion of the foreign polypeptide with the second portion of the foreign polypeptide. As will be appreciated by one of skill in the art, the methods and compositions described herein can be utilized for any large gene product, and the gene product need not be limited by function.

[0077] The methods and compositions described herein are illustrated in the examples using muscle proteins including dystrophin, utrophin, and dysferlin.

[0078] Dystrophin: Dystrophin is a 427 kDa cytoskeletal protein and a member of the spectrin / α-actinin superfamily (see, e.g., Blake et al, Brain Pathology, 6:37 (1996); Winder, J. Muscle Res. Cell. Motil., 18:617 (1997); and Tinsley el al., PNAS, 91:8307 (1994)). The N-terminus of dystrophin binds to actin, with a higher affinity for non-muscle actin than for sarcomeric actin. Dystrophin is involved in a submembranous network of non-muscle actin beneath the plasma membrane. Dystrophin is associated with the dystrophin-associated protein complex (DPC), an oligomeric, transmembrane complex of proteins and glycoproteins. The C-terminus of dystrophin binds to the cytoplasmic tail of β-dystroglycan and cooperates with actin to anchor dystrophin to the sarcolemma. The C-terminus of dystrophin also binds to the cytoplasmic members of the DPC, thereby providing a link between the actin-based cytoskeleton of muscle fibers and the extracellular matrix, and it is this link that is disrupted in muscular dystrophies.

[0079] The central rod domain of dystrophin consists of a stretch of 24 weakly repeated units of approximately 110 amino acids, which are similar to those found in spectrin (i.e., spectrin-like repeats). This domain constitutes the major part of dystrophin and gives it its flexible rod-like structure. The rod domain is interrupted by four proline-rich hinge regions. The rod domain is thought to provide a structural link between members of the DPC.

[0080] Table 1. Domains of full-length dystrophin cDNA TIFF2024526938000002.tif215127

[0081] Dystrophin homologues have been identified in various organisms, including mouse (Genbank accession number M68859); dog (Genbank accession number AF070485); and chicken (Genbank accession number X13369). Similar comparisons can be made with homologues from other species, including but not limited to those mentioned above, by using any of a variety of available computer programs (e.g., BLAST by NCBI). Candidate homologues can be screened for biological activity using any suitable assay, including but not limited to those described herein.

[0082] Utrophin: Utrophin is considered to be the autosomally encoded homologue of dystrophin and to play a similar physiological role (for a recent review, see, e.g., Blake et al., Brain Pathology, 6:37

[1996] ). Human utrophin shows substantial homology to dystrophin, with the main differences occurring in the rod domain, where utrophin lacks repeats 15 and 19, as well as two hinge regions (see, e.g., Love et al., Nature 339:55

[1989] ; Winder et al., FEBS Lett., 369:27

[1995] ). Thus, utrophin contains 22 spectrin-like repeats and two hinge regions.

[0083] Dysferlin: Dysferlin contains the following domains: C2A, C2B, C2C, FerA, DysF, C2D, C2E, C2F, C2G, and TM. The exact boundaries of each domain may vary among orthologs and variants. The approximate amino acid ranges for each domain in human dysferlin are shown in Table 2. The boundaries of the listed domains may vary by up to about 20 residues, e.g., by about 5, 10, 15, or 20 residues.

[0084] Table 2. Dysferlin domains TIFF2024526938000003.tif58128

[0085] Protein variants: As mentioned above, variants (e.g. mutants) of exogenous polypeptides are also intended to be used in the methods and compositions described herein, where the exogenous polypeptide is, for example, dystrophin, utrophin, mini-dystrophin, or dysferlin. For example, it is believed that a single substitution of leucine with isoleucine or valine, a single substitution of aspartic acid with glutamic acid, a single substitution of threonine with serine, or a similar substitution of an amino acid with another structurally related amino acid (i.e., conservative mutation) does not necessarily have a significant effect on the biological activity of the resulting molecule. Thus, in some embodiments, exogenous polypeptides may contain one or more conservative substitutions of amino acids. Conservative substitutions are those that occur within a family of amino acids whose side chains are related. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified collectively as aromatic amino acids. In a similar manner, the amino acid repertoire can be classified as follows: (1) acidic (aspartic acid, glutamic acid); (2) basic (lysine, arginine, histidine); (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), where serine and threonine may optionally be classified separately as aliphatic hydroxyls; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur (cysteine ​​and methionine) (see, e.g., Stryer (ed.) "Biochemistry," 2nd ed., W. H. Freeman and Co.

[1981] ).Whether changes in the amino acid sequence of a peptide have resulted in a functional homologue can be readily determined by assessing the ability of the variant peptide to function in a manner similar to the wild-type protein. Peptides in which multiple substitutions have occurred can also be readily tested in the same manner.

[0086] In some embodiments, variants of the exogenous polypeptide are engineered to contain enhanced biological activity. Such polypeptides, when expressed from recombinant DNA constructs, can be used in the therapeutic aspects described herein.

[0087] In some embodiments, the variant of the exogenous polypeptide may comprise an extended intracellular half-life compared to the corresponding wild-type protein. For example, such variant proteins may have higher or lower stability against proteolytic degradation or other intracellular processes that lead to the destruction or inactivation of the variant. Such variants and the genes encoding them can be used to change the pharmaceutical activity of the construct expressing the exogenous polypeptide variant by adjusting the half-life of the protein. For example, a shorter half-life can produce a more transient biological effect. As mentioned above, such proteins are used in pharmaceutical applications or to treat muscle diseases or disorders.

[0088] A variety of techniques are known in the art for screening the gene products of combinatorial libraries made by point mutation, and for screening cDNA libraries for gene products with certain properties.Such techniques can generally be adapted to rapidly screen gene libraries made by combinatorial mutagenesis of a given foreign polypeptide.The most widely used techniques for screening large gene libraries typically include: cloning gene libraries into replicable expression vectors, transforming appropriate cells with the resulting vector library, and expressing combinatorial genes under conditions that relatively facilitate easy isolation of vectors encoding genes whose products are detected by detecting the desired activity.

[0089] In some embodiments, the exogenous polypeptide comprises a mini-dystrophin or a micro-dystrophin. As used herein, a "mini-dystrophin" comprises an amino-terminal actin-binding domain, a β-dystroglycan-binding domain, and multiple (e.g., at least two) spectrin-like repeat domains.

[0090] Adenoviral Associated Vector (AAV) AAV is a small virus that shows very low immunogenicity and is not associated with any known human disease, which makes it attractive as a vector for delivering foreign genetic material (e.g., for gene therapy).However, due to the size of AAV capsid, there is a limit to the length of DNA that can be packaged therein.The AAV genome is approximately 4.7 kilobases (kb) in size.

[0091] The methods and compositions described herein allow for the delivery of large (e.g., greater than 4.7 kb) proteins by administering two (or more) AAV vectors, each of which has a portion of the foreign polypeptide to be expressed and a portion of a split intein. In one embodiment, the methods and compositions described herein use at least two different adeno-associated virus (AAV) vectors. The first AAV vector comprises the N-terminal portion of the split intein fused to a first portion of the foreign polypeptide (e.g., dystrophin, dysferlin, utrophin, or other desired therapeutic protein, e.g., a therapeutic protein for a muscle disease or disorder or other disease or disorder), and the second AAV vector comprises the C-terminal portion of the split intein fused to a second portion of the foreign polypeptide. Upon expression of the first and second fusion polypeptides in the cell, the first and second portions of the split intein facilitate linkage of the first portion of the foreign polypeptide with the second portion of the foreign polypeptide, thereby delivering the foreign polypeptide to the cell. This system or arrangement allows for the delivery of foreign polypeptides larger than can be encoded by a single AAV vector particle.

[0092] The first and second AAV vector embodiments are provided herein, and include the following non-limiting embodiments. The AAV vector used herein can be in the form of AAV mature particle or virion, i.e., in the form of nucleic acid surrounded by AAV protein capsid. The AAV vector can comprise the AAV genome, or a part or derivative thereof. The AAV genome is a polynucleotide that encodes the functions required to produce AAV particles. These functions include the functions that operate in the AAV replication and packaging cycle in host cells, including the function that encapsidates the AAV genome into AAV particles. Naturally occurring AAV is replication-incompetent and needs to provide helper functions in trans to complete the replication and packaging cycle. Therefore, the AAV genome of the vector used herein is typically replication-incompetent.

[0093] AAV genome can be either positive or negative sense single-stranded, or double-stranded. Using double-stranded allows to avoid the DNA replication step in target cells, and thus accelerates the expression of transgene. In one embodiment, AAV genome is single-stranded. AAV genome can be derived from any serotype, isolate, or clade of AAV that is naturally derived. Thus, AAV genome can be the full-length genome of naturally occurring AAV, or the full-length genome of recombinant, engineered AAV. As known to those skilled in the art, naturally occurring AAV can be classified according to various biological systems.

[0094] Generally, AAV is expressed in terms of its serotype. Serotype corresponds to a variant subspecies of AAV, which has a unique reactivity that can be used to distinguish it from other variant subspecies due to its profile of capsid surface antigen expression. Typically, a virus with a certain AAV serotype does not cross-react efficiently with neutralizing antibodies specific for other AAV serotypes. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, and also include recombinant serotypes such as Rec2 and Rec3. Any of these AAV serotypes can be used in the methods and compositions described herein. Reviews of AAV serotypes can be found in Choi et al. (2005) Curr. Gene Ther. 5: 299-310, and Wu et al. (2006) Molecular Therapy 14: 316-27. The sequence of the AAV genome, or the sequence of elements of the AAV genome, including ITR sequences, the rep gene, or the cap gene, may be derived from the following accession numbers for the complete AAV genome sequence: NC_002077, AF063497 for adeno-associated virus 1; NC_001401 for adeno-associated virus 2; NC_001729 for adeno-associated virus 3; NC_001863 for adeno-associated virus 3B; NC_001829 for adeno-associated virus 4; Y18065, AF085716 for adeno-associated virus 5; NC_001862 for adeno-associated virus 6; AY186198, AY629583, NC_004828 for avian AAV ATCC VR-865; avian AAV NC_006263, AY629583 for strain DA-1; NC_005889, AY388617 for bovine AAV.

[0095] AAVs can also be expressed in terms of clades or clones, which refer to the phylogenetic relationships of naturally occurring AAVs and typically also refer to lineages of AAVs that can be traced back to a common ancestor and include all its descendants.

[0096] In addition, AAV can be described in terms of specific isolates, i.e., specific genetic isolates of AAV found in nature. The term genetic isolate refers to a population of AAV that has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining a population that is recognizably distinct at the genetic level.

[0097] One of skill in the art can select an appropriate serotype, clade, clone, or isolate of AAV for use in the methods and compositions described herein based on the general knowledge the artisan has about the characteristics of a particular AAV.

[0098] The serotype of AAV determines the tissue specificity (or tropism) of AAV viral infection. Thus, preferred AAV serotypes for use as AAV administered to patients according to the methods and compositions described herein are those that have a natural tropism or the ability to infect target cells with high efficiency, for example, in muscle.

[0099] Typically, the AAV genome of a naturally occurring AAV serotype, isolate, or clade contains at least one inverted terminal repeat (ITR), which acts in cis to provide a functional origin of replication and allows integration of the vector into and excision of the vector from the genome of a cell.

[0100] AAV genomes also typically contain packaging genes, such as the rep and / or cap genes, which code for packaging functions for AAV particles. The rep genes code for one or more of the proteins Rep78, Rep68, Rep52, and Rep40, or variants thereof. The cap genes code for one or more of the capsid proteins, such as VP1, VP2, and VP3, or variants thereof. These proteins constitute the capsid of the AAV particle. Capsid variants are discussed below. Each of the packaging genes may be operably linked to a promoter. Specific examples of such promoters include the p5 promoter, the p19 promoter, and the p40 promoter (Laughlin et al. (1979) Proc. Natl. Acad. Sci. USA 76: 5567-5571). For example, the p5 and p19 promoters are typically used to express the rep gene, while the p40 promoter is typically used to express the cap gene.

[0101] Typically, the AAV genome for use in the methods and compositions described herein is derivatized for the purpose of administration to a patient. Such derivatization is common in the art (see, for example, Coura and Nardi (2007) Virology Journal 4: 99). Derivatives of AAV genome include any truncated or modified AAV genome that allows transgene expression in vivo. Typically, AAV genome can be significantly truncated so as to contain minimal viral sequences but retain the above-mentioned functions. This is preferred for safety reasons, since it reduces the risk of vector recombination with wild-type virus and also avoids the cellular immune response caused by the presence of viral gene proteins in target cells.

[0102] Typically, the derivative of the AAV genome comprises at least one terminal inverted repeat (ITR), preferably a plurality of ITRs, such as two or more ITRs. One or more of the ITRs may be derived from AAV genomes with different serotypes, or may be chimeric or mutant ITRs. A preferred mutant ITR is one that has a trs (terminal resolution site) deletion. This deletion allows the genome to be replicated continuously to produce a single-stranded genome that contains both coding and complementary sequences, i.e., a self-complementary AAV genome. This allows DNA replication in target cells to be avoided, and therefore allows the expression of the transgene to be accelerated.

[0103] Inclusion of one or more ITRs is preferred to aid in vector concatemer formation in the host cell nucleus, for example, following conversion of single-stranded vector DNA to double-stranded DNA by the action of host cell DNA polymerases. Formation of such episomal concatemers protects the vector construct for the life of the host cell, thereby allowing extended expression of the transgene in vivo.

[0104] In some embodiments, the ITR elements are the only sequences that remain in the derivative from the native AAV genome. Thus, preferably, the derivative does not contain the rep and / or cap genes of the native genome, and does not contain any other sequences of the native genome. This is preferred for the reasons mentioned above, and also because it reduces the possibility of the vector integrating into the genome of the host cell. Thus, the following parts may be removed in the derivative: one inverted terminal repeat (ITR) sequence, the replication (rep) gene, and the capsid (cap) gene. However, in some embodiments, the derivative may additionally contain one or more of the rep and / or cap genes of the AAV genome or other viral sequences. Since naturally occurring AAV integrates at a high frequency into a specific site on human chromosome 19 and shows a negligible frequency of random integration, the fact that the vector retains the ability to integrate may be tolerated in a therapeutic environment. Where the derivatives include capsid proteins, i.e., VP1, VP2, and / or VP3, the derivatives may be chimeric, shuffled, or capsid-modified derivatives of one or more naturally occurring AAVs. In particular, the methods and compositions described herein encompass those in which capsid protein sequences from different serotypes, clades, clones, or isolates of AAV are provided in the same vector (i.e., pseudotyped vectors).

[0105] Chimeric, shuffled or capsid modified derivatives are typically selected to provide one or more desired functionalities as a viral vector. Thus, these derivatives may exhibit: increased gene delivery efficiency, reduced immunogenicity (humoral or cellular), altered tropism range, and / or improved targeting of specific cell types compared to AAV vectors that contain naturally occurring AAV genomes, such as those of AAV2. Increased gene delivery efficiency may be affected by: improved binding of receptors or co-receptors on the cell surface, improved internalization, improved trafficking within cells and trafficking to the nucleus, improved uncoating of viral particles, and / or improved conversion of single-stranded genome to double-stranded form. Increased efficiency may also be associated with altered tropism range or targeting of specific cell populations, so that the vector dose is not diluted by administration to tissues that do not need it.

[0106] Chimeric capsid proteins include those produced by recombination between two or more of the capsid coding sequences of naturally occurring AAV serotypes.This can be carried out, for example, by a marker rescue approach, in which a non-infectious capsid sequence of one serotype is co-transfected with the capsid sequence of another serotype, and directional selection is used to select the capsid sequence with desired properties.The capsid sequences of various serotypes can be changed in cells by homologous recombination to produce novel chimeric capsid proteins.

[0107] Chimeric capsid proteins also include those created by engineering capsid protein sequences to transfer particular capsid protein domains, surface loops, or particular amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes.

[0108] Shuffled or chimeric capsid proteins can also be produced by DNA shuffling or error-prone PCR.AAV capsid hybrid genes can be produced by randomly fragmenting the sequences of related AAV genes, such as the sequences of genes encoding capsid proteins of multiple different serotypes, and then re-linking the fragments in a self-priming polymerase reaction, which method can also cause crossover in regions where sequences have homology.The library of AAV hybrid genes produced in this manner by shuffling the capsid genes of several serotypes can be screened to identify viral clones with desired functionality.Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to produce a diverse library of variants, which can then be selected for desired properties.

[0109] The sequence of the capsid gene can also be genetically modified so that specific deletions, substitutions, or insertions are introduced relative to the native wild-type sequence. In particular, the capsid gene may be modified by inserting the sequence of another protein or peptide within the open reading frame of the capsid coding sequence or at the N-terminus and / or C-terminus of the capsid coding sequence.

[0110] As used herein, vectors may include those that provide the sequence of the AAV genome in a different order and configuration from that of the natural AAV genome.Vectors may also include the replacement of one or more of the AAV sequences or genes with sequences from another virus, or with chimeric genes that are composed of sequences from multiple viruses.Such chimeric genes may be composed of sequences from two or more related viral proteins of different virus species.

[0111] AAV vectors for use as described herein may include transcapsidated forms, in which the AAV genome or AAV derivatives with ITRs of one serotype are packaged in the capsid of another serotype.Such AAV vectors may also include mosaic forms, in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid.AAV vectors may also include chemically modified forms that carry ligands adsorbed on the capsid surface.For example, such ligands may include antibodies for targeting certain cell surface receptors.

[0112] The first and second AAV vectors of the AAV vector system described herein contain all the elements necessary for reconstituting a fully functional foreign polypeptide in target cells after transduction with both vectors.Those skilled in the art will recognize additional genetic elements that are commonly used to ensure the expression of transgenes in cells transduced with viral vectors.These can be referred to as expression control sequences.Thus, the AAV vector of the AAV viral vector system described herein typically contains an expression control sequence (e.g., one that includes a promoter sequence) that is functionally linked to the nucleotide sequence that codes for the desired foreign polypeptide (e.g., dystrophin, utrophin, dysferlin, etc.).

[0113] Any suitable promoter can be used. The promoter sequence may be constitutively active (i.e., functional in any host cell background), or it may be active only in a particular host cell environment, thereby allowing expression of the transgene to be targeted to a particular cell type (e.g., a tissue-specific promoter). The promoter may exhibit inducible expression in response to the presence of another factor, e.g., a factor present in the host cell. In any case, when the vector is administered for therapeutic purposes, it is preferred that the promoter is functional in the target cell background.

[0114] In some embodiments, the promoter is preferably highly effective in muscle cells to allow the transgene to be selectively expressed or expressed only in a muscle cell population. As a result, expression from the promoter can be specific to muscle cells. In one embodiment, the muscle-specific promoter is included in a muscle-specific expression cassette, as that term is used herein.

[0115] At least one of the vectors described herein may include an untranslated region (UTR) (i.e., 5' UTR) located between the promoter and the upstream of the nucleic acid sequence encoding the polypeptide. Any suitable UTR sequence may be used. The UTR may include one or more of the following elements: an intron 1 fragment of Gallus gallus β-actin (CBA), an intron 2 fragment of Oryctolagus cuniculus β-globin (RBG), and an exon 3 fragment of Oryctolagus cuniculus β-globin. The UTR may include a Kozak consensus sequence. Any suitable Kozak consensus sequence may be used.

[0116] At least one of the vectors described herein may further comprise a post-transcriptional response element (also known as a post-transcriptional regulatory element), i.e., PRE. Any suitable PRE may be used. The presence of a suitable PRE may enhance the expression of a desired transgene. In one embodiment, the PRE is the Woodchuck Hepatitis Virus PRE (WPRE). One or more vectors may also comprise a polyadenylation sequence located 3' to the nucleic acid sequence encoding a protein. Any suitable polyadenylation sequence may be used. In one embodiment, the polyadenylation sequence is the bovine growth hormone (bGH) polyadenylation sequence.

[0117] The expression of a given foreign protein requires that both the first and second AAV vectors are used to transduce target cells; however, the order is not important.Therefore, target cells can be transduced with the first and second AAV vectors in any order (first AAV vector followed by the second AAV vector, or second AAV vector followed by the first AAV vector), or can be transduced simultaneously.The method for transducing target cells with AAV vectors is known in the art, and those skilled in the art will be familiar with it.The target cell is preferably a muscle cell, and is preferably a skeletal muscle cell or a cardiac muscle cell.

[0118] While the methods and compositions described herein involve the use of at least two types of adeno-associated vectors, the methods and compositions may also utilize additional vectors, including, for example, second generation adenoviral, lentiviral, or retroviral vectors.

[0119] Second generation adenovirus vectors have deleted the early region (E2A, E2B, and E4) of the Ad genome. Highly modified second generation adenovirus vectors are less likely to generate replication-competent virus during large-scale vector preparation. Thus, the host immune response to late viral proteins is reduced (see Amalfitano et al., "Production and Characterization of Improved Adenovirus Vectors With the E1, E2b, and E3 Genes Deleted," J. Virol. 72:926-933 (1998)). The removal of E2A, E2B, and E4 genes from the adenovirus genome also results in increased cloning capacity. This can be combined with the split intein approach described herein to further increase the size of the exogenously encoded introduced polypeptide.

[0120] Lentivirus-based vectors are produced by expression of packaging vector constructs in cell lines that infect non-dividing cells and express viral proteins as part of the normal life cycle of the virus. The small size of lentivirus particles limits the length of foreign DNA that the virus can carry to approximately 10 kb.

[0121] Moloney murine leukemia virus (MMLV) and other retrovirus-based vectors have proven useful for gene therapy applications. These vectors stably transduce actively dividing cells and are integrated into the host cell chromosomes as part of the normal life cycle of the virus. As an example, retroviruses may be utilized as described herein in the context of infecting and transducing muscle precursor cells, such as myoblasts, satellite cells, or other muscle stem cells.

[0122] Split intein Inteins are naturally occurring, self-splicing protein subdomains that are capable of excising themselves from a larger protein structure and simultaneously linking together two previously adjacent peptide regions ("exteins") to form one mature host protein.

[0123] The ability of inteins to rearrange adjacent peptide bonds while retaining activity when fused to proteins other than their natural exteins has led to a number of intein-based biotechnologies, including various types of applications in protein ligation and activation, and protein labeling and tracking. One important application of inteins is in the production of purified recombinant proteins. Inteins in particular have the ability to confer self-cleavage activity to many conventional affinity and purification tags, and thus provide a major advance in the production of recombinant protein products for research, medical, and other commercial applications.

[0124] The use of split inteins in the methods and compositions provided herein allows for the separation of sequences encoding large proteins between two (or more) different vectors, such as AAV vectors, which are ligated together to form full-length proteins when expressed in cells. Given the limited size that AAV vectors can carry with respect to protein-encoding sequences, the use of split inteins allows for the delivery of large proteins into cells that could not be encoded by a single AAV vector alone.

[0125] Any catalytically active intein or fragment thereof can be used to derive a split intein for use in the methods of the present invention. For example, in one aspect, the split intein may be derived from a eukaryotic intein. In another aspect, the split intein may be derived from a bacterial intein. In another aspect, the split intein may be derived from an archaeal intein. Preferably, such derived split inteins retain only the amino acid sequence required to catalyze the trans-splicing reaction.

[0126] The N-terminal side of the split intein, as that term is used herein, may comprise a sequence that is a modified version of the N-terminal portion of a naturally occurring intein sequence. For example, the N-terminal sequence of the split intein may comprise additional amino acid residues and / or mutated residues, provided that the inclusion of such residues does not render the intein non-functional with respect to splicing the two portions of a foreign polypeptide. Preferably, the inclusion of the additional residues and / or mutated residues improves or enhances the splicing activity of the intein.

[0127] The C-terminal side of the split intein used in the methods and compositions described herein can be any intein sequence that includes a C-terminal amino acid sequence that is functional with respect to a trans-splicing reaction. In one aspect, the C-terminal side of the split intein includes 4-7 consecutive amino acid residues, at least 4 of which are derived from the last β-strand of the intein from which it is derived. Thus, the C-terminal region of the split intein also includes a sequence that is removed by splicing when trans-splicing occurs. The C-terminal region of the split intein may include a sequence that is a modified version of the C-terminal portion of a naturally occurring intein sequence. For example, the C-terminal region of the split intein may include additional amino acid residues and / or mutated residues, provided that the inclusion of such residues does not render the intein non-functional with respect to splicing. Preferably, the inclusion of the additional residues and / or mutated residues improves or enhances the splicing activity of the C-terminal region of the split intein.

[0128] In some embodiments, the peptide linked to the C-terminal or N-terminal region of the split intein may contain additional chemical moieties, including, among others, fluorescent groups, biotin, polyethylene glycol (PEG), amino acid analogs, unnatural amino acids, phosphate groups, glycosyl groups, radioisotope labels, and pharmaceutical molecules. In other embodiments, the peptide linked to the C-terminal region of the split intein may contain one or more chemically reactive groups, including, among others, ketones, aldehydes, Cys residues, and Lys residues. The N-intein and C-intein of the split intein non-covalently bind to form an active intein, capable of catalyzing a splicing reaction in the presence of an "intein-splicing polypeptide (ISP)". An "intein splicing polypeptide (ISP)" is a portion of the amino acid sequence of a split intein that remains when the C-terminal or N-terminal region, or both, of the split intein are removed from the split intein. In one embodiment, the N-terminal region of the split intein comprises an ISP. In another embodiment, the C-terminal region of the split intein comprises an ISP. In yet another embodiment, the ISP is a separate peptide that is not covalently linked to either the C-terminal or N-terminal region of the split intein.

[0129] In protein trans-splicing, one precursor protein consists of an N-extein portion followed by an N-intein, and the other precursor protein consists of a C-intein portion followed by a C-extein portion, and the two intein sequences are excised and linked by a peptide bond in a trans-splicing reaction (catalyzed jointly by the N-intein and the C-intein). Protein trans-splicing, which is an enzymatic reaction, can act on very low concentrations (e.g., micromolar concentrations) of proteins and can be performed under physiological conditions.

[0130] In some embodiments, the sequence of the split intein used herein is codon-optimized for expression in a certain cell, for example, a eukaryotic cell (e.g., a eukaryotic muscle cell). The eukaryotic cell can be of a certain organism or derived from a certain organism, for example, a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest, by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons, or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the native sequence with the codon that is more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Different species of organisms show a certain bias for some codons of certain amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), and therefore codon bias is thought to be dependent, among other things, on the properties of the codon to be translated and on the availability of certain transfer RNA (tRNA) molecules. The preference of selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis. Thus, genes can be tailored based on codon optimization to optimally express them in a given organism. Codon usage tables are readily available, for example in the "Codon Usage Database," and such codon usage tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, Pa.).

[0131] In some embodiments, the methods and compositions described herein utilize one or more split inteins as provided in the table below. Exemplary split inteins for use herein are depicted in Figures 15A-15U herein.

[0132] Table 3. Exemplary split inteins TIFF2024526938000004.tif191166TIFF2024526938000005.tif229166TIFF2024526938000006.tif245166TIFF2024526938000007.tif245166TIFF2024526938000008.tif254166TIFF2024526938000009.tif241166TIFF2024526938000010.tif241166TIFF2024526938000011.tif238166TIFF2024526938000012.tif243166TIFF2024526938000013.tif254166TIFF2024526938000014.tif242166TIFF2024526938000015.tif243166TIFF2024526938000016.tif243166TIFF2024526938000017.tif243166TIFF2024526938000018.tif241166TIFF2024526938000019.tif248166TIFF2024526938000020.tif244166TIFF2024526938000021.tif248166TIFF2024526938000022.tif237166TIFF2024526938000023.tif230166TIFF2024526938000024.tif241166TIFF2024526938000025.tif244166TIFF2024526938000026.tif196166TIFF2024526938000027.tif234166TIFF2024526938000028.tif244166TIFF2024526938000029.tif237166TIFF2024526938000030.tif242166TIFF2024526938000031.tif232166TIFF2024526938000032.tif241166TIFF2024526938000033.tif254166TIFF2024526938000034.tif229166

[0133] In some embodiments, the coding sequence for the foreign polypeptide may be divided into three (or more) parts.

[0134] Split inteins can mediate efficient post-translational splicing of two or more heterologous extein polypeptides. However, the resulting spliced ​​product usually contains an intein sequence of 3-5 amino acids introduced at the amino- and carboxy-terminal junction of the spliced ​​extein polypeptide. In some instances, these 3-5 "intein footprint" (or simply "footprint") amino acids do not significantly affect the function of the final spliced ​​polypeptide, but in other instances, the presence of such inserted amino acids may have a negative effect on the structure and function of the final product. Therefore, it may be beneficial to minimize or even completely avoid the intein footprint in the trans-spliced ​​product.

[0135] In one aspect, intein footprint inserts may be minimized or even avoided entirely in the methods and compositions described herein. To accomplish this, for example, the sequence of the target protein may be analyzed against the footprints of known split inteins to identify sequences within the target that match or closely resemble the footprint of the split intein.

[0136] Table 4. Exemplary sequences for minimizing the footprint of split inteins TIFF2024526938000035.tif83163Amino acid abbreviations: A: Alanine C: Cysteine E: Glutamic acid F: Phenylalanine G: Glycine I: Isoleucine N: Asparagine P: Proline S: Serine Y: Tyrosine

[0137] Such split inteins with naturally occurring footprints in a given target protein can then be used to design and express multiple heterologous extein-intein fusions separately, thereby minimizing or even avoiding the insertion of non-naturally occurring amino acids in the spliced ​​polypeptide product. For example, after screening the sequence of the target protein for sequences matching the footprint sequence of the split intein, sequences can be prepared that encode amino- and carboxy-terminal fusions in which the target polypeptide fragment is fused to the amino- and carboxy-terminal fragments of the split intein, respectively, and in which the footprint amino acids are not present in the extein fusion polypeptide sequence. In this situation, upon cleavage and joining of the extein sequence, the intein footprint insert reconstitutes the native target polypeptide sequence, resulting in a spliced ​​target polypeptide that has no amino acid sequence differences from the native target polypeptide. That is, the footprint insert characteristic of the split intein is still technically present, but its sequence matches a sequence present in the target protein, such that the resulting spliced ​​polypeptide product is free of the unnatural footprint.

[0138] In some instances, a given target polypeptide may lack a perfect match with the footprint of a split intein, or the perfect match may be located so close to the amino or carboxy terminus of the target protein that splitting the target protein coding sequence at such a location would not separate the target protein coding sequence into separate fragments that fit into a delivery vector. In such instances, it may still be useful to identify sequences within the target protein that are similar, but not identical, to the footprint sequence of the split intein. Such similarity may be, for example, a match of four out of five footprint amino acids, a match of three out of five footprint amino acids, or even two out of five footprint amino acids. Similarity in this context may also include, for example, the inclusion of amino acids with similar properties to those in the footprint, such as amino acids that are conservative substitutions for naturally occurring amino acids, or a combination of matches and conservative substitutions. Such footprint similarity based approaches can be used to minimize the footprint of the intein and / or its effect on the function of the spliced ​​target protein, using analogy to the situation where perfect matches with the footprint of the intein can be identified at informative locations of the target protein. Thus, splicing products having intein footprints with four or less, three or less, two or less, one or less, or no differences compared to the naturally occurring or desired target protein sequence can be produced as described herein.

[0139] In some embodiments, an "engineered" split intein differs from a naturally occurring polypeptide or nucleic acid by deletion, addition, substitution, or side chain modification of one or more amino acids or nucleic acids, yet retains one or more of the specific functions or biological activities of the naturally occurring split intein sequence. Amino acid substitutions include alterations in which an amino acid is replaced with another naturally occurring amino acid residue or with a non-conventional amino acid residue. Some substitutions can be classified as "conservative", where an amino acid residue present in a polypeptide is replaced with another naturally occurring amino acid that has similar characteristics in terms of either polarity, side chain functionality, or size. Substitutions encompassed by the variants described herein can also be "non-conservative", where an amino acid residue present in a peptide is replaced with an amino acid with different properties (e.g., a charged or hydrophobic amino acid is replaced with an uncharged or hydrophilic amino acid) or a naturally occurring amino acid is replaced with a non-conventional amino acid.

[0140] In one embodiment, the split intein comprises at least two of SEQ ID Nos: 1-46. In another embodiment, the split intein comprises SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, the split intein comprises SEQ ID NO: 3 and SEQ ID NO: 4. In another embodiment, the split intein comprises SEQ ID NO: 5 and SEQ ID NO: 6. In another embodiment, the split intein comprises SEQ ID NO: 7 and SEQ ID NO: 8. In another embodiment, the split intein comprises SEQ ID NO: 9 and SEQ ID NO: 10. In another embodiment, the split intein comprises SEQ ID NO: 11 and SEQ ID NO: 12. In another embodiment, the split intein comprises SEQ ID NO: 13 and SEQ ID NO: 14. In another embodiment, the split intein comprises SEQ ID NO: 15 and SEQ ID NO: 16. In another embodiment, the split intein comprises SEQ ID NO:17 and SEQ ID NO:18. In another embodiment, the split intein comprises SEQ ID NO:19 and SEQ ID NO:20. In another embodiment, the split intein comprises SEQ ID NO:21 and SEQ ID NO:22. In another embodiment, the split intein comprises SEQ ID NO:23 and SEQ ID NO:24. In another embodiment, the split intein comprises SEQ ID NO:25 and SEQ ID NO:26. In another embodiment, the split intein comprises SEQ ID NO:27 and SEQ ID NO:28. In another embodiment, the split intein comprises SEQ ID NO:29 and SEQ ID NO:30. In another embodiment, the split intein comprises SEQ ID NO:31 and SEQ ID NO:32. In another embodiment, the split intein comprises SEQ ID NO:33 and SEQ ID NO:34. In another embodiment, the split intein comprises SEQ ID NO:35 and SEQ ID NO:36.In another embodiment, the split intein comprises SEQ ID NO:37 and SEQ ID NO:38. In another embodiment, the split intein comprises SEQ ID NO:39 and SEQ ID NO:40. In another embodiment, the split intein comprises SEQ ID NO:41 and SEQ ID NO:42. In another embodiment, the split intein comprises SEQ ID NO:43 and SEQ ID NO:44. In another embodiment, the split intein comprises SEQ ID NO:45 and SEQ ID NO:46.

[0141] Exemplary sequences of specific dystrophin, dysferlin, utrophin, and mini-dystrophin fragments with their corresponding inteins are provided in Example 2 herein.

[0142] MSEC In certain embodiments, the split intein constructs described herein may benefit from cell type specific expression. Such a design can ensure expression of the target protein where it is most needed, including high, medium, low or regulated expression, but also avoid or limit potential negative effects of ectopic expression in non-target cells or tissues. Thus, the inclusion of a tissue specific expression cassette can maximize the therapeutic benefit of transgene introduction. Such a design can also facilitate or allow for systemic administration of the vector, for example, where infection may occur in non-target cells or tissues, but expression of the transgene polypeptide occurs substantially only in the desired cell or tissue type. For example, when used in combination with a vector with tropism or enhanced tropism for transduction into a given tissue or cell type, the use of a tissue specific expression cassette to drive expression of each of the target protein-split intein constructs described herein can be highly beneficial. When used in the context of delivery of more than one vector, multiple tissue-specific expression cassettes can be used, e.g., such that mRNA production or accumulation, or protein translation, production, or accumulation, etc., occurs in a balanced ratio.

[0143] A "tissue-specific expression cassette", as the term is used herein, provides expression of a target protein in a manner restricted to a particular tissue or cell type. In this context, "specific" or "in a specific manner" means that expression from the construct in the target tissue or target cell type is at least 5 times higher than in other tissues or other cell types, e.g., at least 5 times higher, 10 times higher, 15 times higher, 20 times higher, or even higher. Expression can be measured, for example, at the level of mRNA production or accumulation, or at the level of protein translation, production, or accumulation. In one embodiment, the tissue-specific expression cassette is a "muscle-specific expression cassette" or "MSEC" as described herein. The MSEC drives the expression of the linked construct in a manner specific to muscle cells or muscle tissue, as that term is defined herein above.

[0144] MSEC generally comprises muscle-specific promoter and enhancer elements. For examples and discussion of muscle-specific expression cassettes designed for use in rAAV vectors to drive heterologous protein expression in skeletal and cardiac muscle, see, for example, Salva et al., Molecular Therapy 15: 320-329 (2007), which is incorporated herein by reference. Muscle-specific expression cassettes include, among others, promoter and enhancer elements from muscle-specific genes, including, for example, muscle creatine kinase (MCK), skeletal α-actin, and α-myosin heavy chain genes. The mouse MCK gene comprises a 206 bp enhancer located approximately 1.2 kb upstream of the transcription start site, and a 358 bp proximal promoter. However, for use in gene therapy vectors, such as AAV, as discussed herein, due to viral packaging limitations, it is necessary to minimize the regulatory elements designed to drive muscle-specific expression (approximately 800 bp or less) in order to maximize the length of the coding sequence of the payload protein of a given vector. Thus, muscle-specific expression cassettes useful in the methods and compositions described herein are composed of truncated / modified muscle-specific regulatory elements that provide binding sites for myogenic regulatory factors, as well as Inr (initiator element) and / or TATA box sequences, and the cassettes may contain additional sequences, for example, from the 5' untranslated region of muscle-specific genes. The MHCK7 cassette described by Salva et al. is just one example of an MSEC useful in the methods and compositions described herein. The cassette drives expression in MM14 myocytes to a higher degree than the constitutively active CMV promoter, but is essentially inactive in non-muscle cells (e.g., HEK 293 fibroblasts, mouse L-cell fibroblasts, and JAWSII dendritic cells). See also the expression cassettes described in US Pat. No. 10,479,821, which is incorporated herein by reference.As just one example, SEQ ID NO: 19, described herein and referred to as CK8, is highly active in cardiac and skeletal muscles. It is contemplated that variants of such MSEC sequences can also be highly active and provide muscle-specific expression of therapeutic transgenes. For example, sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to such MSECs can also be useful in the methods and compositions described herein. Those skilled in the art can determine the activity of a given MSEC in muscle cells or muscle tissue, for example, using the assay described in the publication of Salva et al.

[0145] Pharmaceutical Compositions Provided herein is a vector composition that is useful for treating or preventing a variety of different diseases and / or disorders in subjects.An important subset of diseases and disorders is muscle disease and muscle disorders.In one embodiment, the composition is a pharmaceutical composition.The composition can comprise a therapeutically effective amount or a prophylactically effective amount of at least two kinds of vectors that code for exogenous polynucleotides or therapeutic substances.The at least two kinds of vectors utilize split inteins to help deliver nucleic acids that code for large proteins to a given cell.

[0146] The composition may optionally include a carrier, such as a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the specific composition to be administered, and in part by the specific method used to administer the composition. Thus, there are a variety of suitable formulations for pharmaceutical compositions. For example, formulations suitable for parenteral administration can be formulated for intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes. Carriers can include sterile, isotonic aqueous injection solutions, which can include antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of intended recipient, and carriers can also include aqueous sterile suspensions and non-aqueous sterile suspensions, which can include suspending agents, solubilizers, thickeners, stabilizers, preservatives, liposomes, microspheres, and emulsions.

[0147] In one embodiment, the composition is formulated for delivery intramuscularly.

[0148] The therapeutic composition includes a physiologically tolerable carrier together with the vector described herein, in which the vector is dissolved or dispersed as an active ingredient. As used herein, the terms "pharmaceutical acceptable" and "physiologically tolerable" and their grammatical variants are used interchangeably when referring to compositions, carriers, diluents, and reagents, and refer to a material that can be administered to a mammal without causing or does not cause undesirable physiological effects in a mammal, such as nausea, dizziness, stomach upset, etc. A pharmaceutical acceptable carrier does not promote the development of an immune response against an agent mixed with the carrier, unless such development is desired. The preparation of a pharmaceutical composition that includes an active ingredient dissolved or dispersed therein is understood in the art, and the preparation need not be limited based on the formulation. Typically, such compositions are prepared to be injectable as either a liquid solution or suspension; however, they may also be prepared in solid forms suitable for solution or suspension in liquid prior to use. The preparation may also be emulsified or provided as a liposomal composition. The active ingredient may be mixed with an excipient, where the excipient is pharma- ceutically acceptable and compatible with the active ingredient, and in an amount appropriate for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient. The therapeutic composition used in the methods described herein may contain pharma- ceutically acceptable salts of its components. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide), which are formed with inorganic acids, such as, for example, hydrochloric acid or phosphoric acid, or with organic acids, such as, for example, acetic acid, tartaric acid, mandelic acid, and the like.Salts formed with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and from organic bases such as, for example, isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Physiologically tolerable carriers are well known in the art. Exemplary carriers that are liquids are sterile aqueous solutions that contain only the active ingredient and water, or that contain a buffer at a physiological pH value, such as, for example, sodium phosphate, or that contain saline, or that contain both, such as, for example, phosphate-buffered saline. In addition, aqueous carriers may contain multiple buffer salts, and may contain salts such as, for example, sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions may also contain liquid phases in addition to water and without water. Examples of such additional liquid phases are glycerin, vegetable oils such as, for example, cottonseed oil, and emulsions of water and oil. The amount of vector to be administered herein that will be effective to treat a particular disorder or condition will vary depending on the nature of the disorder or condition and the manifestations of the therapeutic agent, and can be determined by standard clinical techniques.

[0149] In pharmaceutical compositions, any suitable carrier known to those skilled in the art can be used, but the type of carrier varies depending on the mode of administration.Compositions for use as described herein can be formulated for any suitable mode of administration, including, for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration.For parenteral administration, such as intramuscular or subcutaneous injection, the carrier preferably comprises water, saline, alcohol, oil, wax, or buffer.Alternatively, the compositions as described herein can be formulated as lyophilizates.The compound can also be encapsulated in liposomes.

[0150] Dosage and Administration Treatment with the methods and compositions described herein includes both prophylaxis / prevention of disease onset and treatment of ongoing disease. Prevention or treatment can be achieved by a single direct injection at one or multiple time points. Administration can also be near-simultaneous to multiple sites. Patients or subjects include mammals, such as humans, bovine, equine, canine, feline, porcine, and ovine animals, and other veterinary subjects. Preferably, patients or subjects are human.

[0151] In one aspect, the method described herein provides a method for treating a disease or disorder (e.g., a muscle disease or muscle disorder) in a subject. In one embodiment, the subject can be a mammal. In another embodiment, the mammal can be a human, although the approach is effective for all mammals. The method includes administering to the subject an effective amount of a pharmaceutical composition comprising a vector described herein in a pharma- ceutically acceptable carrier.

[0152] The dosage range of the agent varies depending on the efficacy and expression level of the therapeutic protein, and includes an amount that is large enough to produce the desired effect, for example, to reduce at least one symptom of the disease to be treated. The dosage should not be so large as to cause unacceptable adverse side effects. In general, the dosage varies according to the type of foreign protein expressed from the vector (e.g., recombinant polypeptide, peptide, peptidomimetic, small molecule, etc.), according to the characteristics of the therapeutic protein (e.g., dystrophin, utrophin, dysferlin, etc.), and according to the age, condition, and sex of the patient. The dosage can be determined by a person skilled in the art, and can be adjusted by an individual physician in the case of any complication.

[0153] In some embodiments, the vectors are administered at a multiplicity of infection (MOI) of at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 500, or higher.

[0154] In one embodiment, the vector comprises at least 1 x 10 5 Pieces, 1 x 10 6 Pieces, 1 x 10 7 Pieces, 1 x 10 8 Pieces, 1 x 10 9 Pieces, 1 x 10 10 Pieces, 1 x 10 11 Pieces, 1 x 10 12 The vaccine is administered at a titer of 10 or more viral particles.

[0155] Repeated administration can be performed as necessary to maintain the effectiveness of treatment. As used herein, the term "therapeutically effective amount" refers to the amount of vector or the amount of expressed therapeutic agent that is sufficient to cause a statistically significant and measurable change in at least one symptom of disease (see "Measurement of efficacy" below). Alternatively, the therapeutically effective amount is the amount of vector or the amount of expressed therapeutic protein that is sufficient to cause a statistically significant and measurable change in the expression level of biomarkers related to the disease of a subject. Such effective amount can be evaluated in clinical trials and animal tests for a given agent.

[0156] The vector composition may be administered directly to a particular site (e.g., intramuscular injection, intravenously, into a particular organ) or orally. It is also contemplated herein that the agent may be delivered intravenously (by bolus injection or continuous infusion), by inhalation, intranasally, intraperitoneally, intramuscularly, subcutaneously, intracavity, and the agent may be delivered by peristaltic means, if desired, or by other means known to those of skill in the art. The agent may be administered systemically, if desired.

[0157] Therapeutic compositions containing at least one active substance can generally be administered as unit dosage forms. The term "unit dosage form", when used in relation to therapeutic compositions, refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined amount of active substance calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0158] The exact amount of active ingredient required to be administered is subject to the judgment of the physician and is specific to each individual.However, the range of suitable dosage for systemic application is disclosed herein and varies according to the route of administration.The suitable regimen for administration is also variable, but typically includes an initial administration followed by repeated administration at one or more intervals by subsequent infusion or other administration methods.Alternatively, continuous intravenous infusion sufficient to maintain blood concentration within the range specified for in vivo therapy is contemplated.

[0159] Measuring effectiveness The effectiveness of a given treatment for a disease can be determined by a skilled physician. However, if any one or all of the signs or symptoms of the condition being treated are changed in a beneficial manner after treatment with the vectors described herein, or if other clinically recognized symptoms or disease markers are improved or even reversed, for example by at least 10%, the treatment is considered to be an "effective treatment" as the term is used herein. Efficacy can also be measured by the absence of deterioration in an individual (i.e., the progression of the disease is stopped or at least delayed) as assessed by disease stability, hospitalization, or need for medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment for a disease in an individual or animal (some non-limiting examples include humans or mammals) and includes: (1) inhibiting the disease, e.g., arresting or slowing the progression of the disease; or (2) alleviating the disease, e.g., causing the amelioration of symptoms; and (3) preventing the onset of the disease or reducing the likelihood of developing the disease, or preventing secondary problems associated with the disease.

[0160] In some embodiments, the effectiveness of the treatment of a muscle disease or disorder can be determined by assessing one or more parameters of muscle function, including, but not limited to, inherent muscle force generation, mobility, spasticity, tension, stability, etc. In some embodiments, clinical tests to determine improvement in muscle function, such as electromyography, magnetic resonance imaging (MRI), or muscle biopsy, can be used to assess the effectiveness of the treatment methods described herein.

[0161] It is understood that the above description and the following examples are merely illustrative, and that they should not be construed as limitations on the scope of the present invention. Various changes and modifications to the disclosed embodiments, which are apparent to those skilled in the art, can be made without departing from the spirit and scope of the present invention. Furthermore, all patents, patent applications, and publications identified are expressly incorporated herein by reference for purposes of illustration and disclosure, such as the methodologies described in such publications that may be used in connection with the present invention. These publications are merely provided for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior invention or for any other reason. Any mention of the dates of these documents, or any description of the contents of these documents, is based entirely on the information available to the applicants, and does not constitute any admission as to the accuracy of the dates or contents of these documents. EXAMPLES

[0162] The following non-limiting examples are provided to demonstrate and support the techniques described herein.

[0163] Example 1 overview Duchenne muscular dystrophy (DMD) is one of the most common genetic disorders in humans, affecting approximately 1 in 5,000 male newborns (Emery 2003). The disease is caused by genetic mutations in the DMD gene that prevent the expression of functional dystrophin (Monaco 1985, Kunkel 1986), where dystrophin is one of the largest proteins produced by human cells. Adeno-associated virus (AAV) vector-based gene delivery has been actively used to treat DMD (Crudele 2019). However, the main limitation for the delivery of the DMD gene is that the coding sequence of the DMD gene is large (11 kb) (Koenig 1989, Chamberlain 1989), while the maximum AAV cargo capacity is less than 5 kb (Srivastava 1983). Our previous work includes the development of miniaturized "micro-dystrophins" (μDys) that can be packaged into an AAV vector (Harper 2002, Gregorevic 2006). These truncated but functional dystrophins have been shown to improve skeletal muscle strength and histology when administered to dystrophin-deficient animal models, despite the lack of key domains required for dystrophin's mechanical and signaling functions and the impossibility of delivering them all in a single AAV vector.

[0164] Gene therapy using AAV vectors shows promise for the treatment of various genetic disorders with loss of function (Li 2020). However, this therapeutic modality is challenged by the small packaging capacity of this viral vector (-5 kb). Due to the large size of the DMD coding sequence (-11.2 kb), currently under development therapeutic approaches for DMD using AAV vectors aim to either express a miniaturized μDys or to express genetic elements that restore the open reading frame (CRISPR / Cas9-mediated gene editing or exon skipping induced by the small nuclear RNA U7) (Harper 2002, Goyenvalle 2004, Long 2016, Nelson 2016, Bengtsson 2017). In both of these strategies, a smaller dystrophin than normal is produced. In addition, when one or more exons are skipped using CRISPR / Cas9 or U7 tools, very low expression and unstable secondary structures are observed (likely due to improperly "placed" repeats; Harper 2002). Previous studies have shown the feasibility of reconstituting larger mini- or full-length dystrophins via homologous recombination and doing so after administering two or three vectors (Figure 2) (Odom 2011, Koo 2014, Lostal 2014). However, these events occurred rarely and in some cases led to unwanted and potentially toxic products that could jeopardize its clinical use.

[0165] Herein, a novel therapeutic approach is provided that allows for the delivery of larger dystrophin, possibly up to full-length dystrophin, in a precise manner with high expression. The method is not limited by unwanted recombinant products and can be adapted for clinical use for any patient with Duchenne or Becker muscular dystrophy (BMD). This improved strategy allows for the expression of large and stable proteins with high specificity and high efficiency (SIMPLI-GT). (Split Intein-Mediated Protein Li Split Intein-Mediated Protein Ligation for Gene Therapy. This approach exploits the ability endogenous to split inteins to mediate protein trans-splicing and thus reconstitute larger therapeutic constructs, which extends the use of AAV-based gene replacement approaches to any gene that exceeds the maximum cargo capacity of the AAV vector.

[0166] test Gene replacement therapy using AAV vectors holds great promise for the treatment of genetic disorders caused by loss-of-function mutations. Currently, hundreds of primate serotypes have been isolated, and additional serotypes are in development (Li 2020). We have previously shown that AAV can be used to deliver genes systemically with high efficiency to both skeletal and cardiac muscles (Gregorevic 2004). Numerous studies have also demonstrated highly stable expression in various animal models, lasting up to 8 years (Rivera 2005, Niemeyer 2009). In the clinic, several patients suffering from neuromuscular disorders, such as myotubular myopathy or spinal muscular atrophy, have recently been treated with a single dose of AAV vector replacing the respective defective genes, MTM or SMN (NCT03199469, NCT02122952), and the clinical data show very encouraging physiological outcomes with a lack of cellular immunogenicity against both the transgene and the vector.

[0167] However, one of the main drawbacks of AAV vectors is their limited packaging capacity (approximately 5 kb), which excludes many genetic disorders when used as gene transporters. Due to the large coding sequences of the defective genes in muscular dystrophies such as Duchenne or limb-girdle type 2B, respectively, one AAV vector cannot be used to deliver the DMD or DYSF genes to the affected muscles. For DMD, a series of miniaturized μDys have been previously developed that can be delivered by one AAV vector (Figure 1) (Harper 2002, Gregorevic 2006, Banks 2010, Ramos 2019). These truncated but functional dystrophins have been shown to improve skeletal muscle strength and histology when administered to dystrophin-deficient animal models, and three constructs are currently being tested in human clinical trials by Sarepta and Solid Biosciences (NCT03375164, NCT03368742).

[0168] It is noteworthy that the majority of μDys developed so far contain 4–6 spectrin repeats (SRs) with 2 or 3 hinges, plus an N-terminal actin-binding domain (ABD) and a dystroglycan-binding domain (DgBD) (Ramos, 2019). From the 24 SR domains and 4 hinge domains that make up the original full-length dystrophin, thousands of combinations are possible to create μDys. This large number of possible combinations, along with the limited carrying capacity of AAV, makes it difficult to predict whether μDys will have the best physiological outcomes. Indeed, previous studies have revealed that many μDys are not functional, and even the best constructs cloned to date have been shown to be incomplete in rescuing skeletal and cardiac muscles (Wasla 2018, Ramos 2019). This suggests that therapeutic candidates should contain additional domains to stabilize the dystrophin structure and provide additional protein functions.

[0169] On the other hand, two patients with a 46% deleted DMD gene (Δexon 17-48) were reported to express a truncated but highly functional dystrophin lacking most of the region between hinge 2 and the middle of SR19, and to display a very mild phenotype with normal life span (England 1990), which prompted the development of a larger "mini-dystrophin" (mini-Dys) construct (Phelps 1995, Harper, 2002, Odom 2011). Co-administration of two AAV vectors expressing two halves of mini-Dys (ΔH2-SR19) arranged with short homologous regions in each allows recombination of the AAV genome, which results in the rearrangement of mini-Dys (Figure 2). This highly functional protein is expressed as mdx 4cvWhen tested in mouse models, it showed high therapeutic potential, leading to improvements in muscle histology and physiological performance. However, unwanted products, likely resulting from non-specific annealing of single-stranded genomes and formation of concatemers, were detected by Southern blot analysis and by polymerase chain reaction. These non-specific products may be potentially toxic and may cause immune responses that would prevent clinical use.

[0170] In this study, the inventors used SIMPLI-GT ( S plit I ntein- M edited P Rotein Li gation for G ene T This paper describes a novel method, named 'split intein-mediated protein ligation for gene therapy' (Split Intein-Mediated Protein Ligation for Gene Therapy), which aims to reconstitute larger therapeutic constructs using an intein-mediated protein trans-splicing mechanism. This approach can overcome the major obstacles associated with AAV-based gene replacement and extends its application to a number of genetic disorders caused by the loss of function of large genes.

[0171] Inteins are genetic elements found in unicellular organisms. They are embedded in important genes involved in DNA transcription, replication, and maintenance (e.g., DNA or RNA polymerase subunits, helicases, gyrases, and ribonucleotide reductases) or in other housekeeping genes, including important proteases and metabolic enzymes (Shah 2014). Intein polypeptides (varying in size from 138 to 844 amino acids) are self-excised from precursor proteins (also called exteins) after their in-frame transcription and translation together with the host gene, and link adjacent peptides. This post-translational modification, known as protein splicing, does not require energy supply, cofactors, or the intervention of exogenous proteases. Over 600 inteins have been identified so far, and approximately 30 have the particularity of being encoded by two separate genes. Such split inteins, unlike the more common sequential inteins, are transcribed and translated separately as N- and C-intein fragments, which then combine to form a reconstituted complex (N-extein / N-intein / C-intein / C-extein) that subsequently undergoes spontaneous splicing to generate a reconstituted and fully functional extein (host protein) (Figure 3).

[0172] This protein trans-splicing mechanism has been used in biotechnological applications, including protein purification and labeling steps (Li 2015). We propose to utilize split inteins to reconstitute larger proteins that cannot be delivered by one AAV vector due to packaging limitations. To this end, we generated a library of 23 split inteins to screen them for their ability to reconstitute two polypeptide fragments into one functional protein. This preliminary screening was performed using green fluorescent protein (GFP) as a screening platform, which allows several inteins to be tested under the same conditions in an unbiased and reliable manner. GFP is a widely used protein that has revolutionized various fields of biology due to its small size (238 amino acids), ease of use, specificity, and lack of cytotoxicity. GFP has previously been adapted as a scaffold for screening aptamers and small antibacterial peptides (Abedi 1988, Soundrarajan 2016).

[0173] First, we identified split sites in the GFP protein sequence where the N- and C-terminal ends of the intein could be inserted. In preliminary studies, two plasmids were cloned, encoding either the N- or C-terminal half of GFP fused to the N- or C-terminal half of the Npu intein (one of the most studied inteins and found in the cyanobacterium Nostoc punctiforme). Human embryonic kidney 293 (HEK293) cells were then cotransfected with both or either the GFP / intein N- and C-terminal plasmids. After 24 h, GFP fluorescence was only detected in cells transfected with either WT GFP (full-length GFP expressed from one plasmid) or the split GFP / intein dual plasmids, but not in cells transfected with the N- or C-terminal plasmids alone (Figure 4A). These data indicate that GFP was efficiently reconstituted by protein trans-splicing mediated by Npu intein. Fluorescence intensity of GFP was measured in living cells using a spectrophotometer. The GFP signal from the reconstituted protein was found to be lower than that from WT GFP (Figure 4B). This may be due to the short transfection time and the time taken for various steps before the formation of functional GFP (i.e., transcription, translation, fusion of N-terminal and C-terminal fragments, splicing of the intein, and ligation of the two N-terminal and C-terminal ends of GFP). In addition, we observed that other inteins currently being screened have higher activity. This initial test demonstrated the feasibility of using split GFP to test the ability of inteins to reconstitute full-length GFP capable of emitting measurable fluorescence. We continued screening of intein libraries with this refined system, and results from various additional split inteins are shown in Figure 7.

[0174] Next, the intein's ability to link the two mini-Dys halves was tested. Due to the small size of the split intein, the dual AAV vector approach allows the expression of the largest mini-Dys constructs tested so far. In silico modeling showed that the two AAV vectors were capable of transporting the coding sequences of two dystrophin fragments, respectively: the N-terminal clone encodes the protein from the N-terminus to the end of SR19 but lacks SR5-SR15, while the C-terminal clone encodes the sequence from hinge 3 to the C-terminal domain. Thus, the reconstituted mini-Dys mediated by trans-splicing of the split intein contains 4 hinges, 13 SRs, the ABD, the CR domain, and the CT domain. Unlike the misplaced repeats often produced in dystrophin using exon skipping or gene editing (Harper, 2002), this novel mini-Dys(ΔSR5-15) retains only the complete spectrin-like repeats, which stabilize its secondary structure and molecular folding. More importantly, this mini-Dys(ΔSR5-15) is larger than the highly functional ΔH2-SR19 dystrophin found in very mild Becker patients (discussed in the "Background" section). This novel mini-Dys retains several functional domains, including binding sites for actin, dystroglycan, dystrobrevin, syntrophin, and neuronal nitric oxide synthase (nNOS), which are important for its mechanical and signaling roles.

[0175] In the study, we cloned two plasmids expressing either the N- or C-terminal fragments of the mini-Dys / intein (Figure 5A). We tested four different split sites located in the linker between SR19 and hinge 3. (See, for example, Figure 10). HEK293 cells were transfected with a control plasmid encoding the entire mini-Dys (ΔSR5-15) or with N- and C-terminal vectors encoding split mini-Dys (ΔSR5-15) fused to a split intein. After 48 hours, total protein was harvested for Western blot analysis. Surprisingly, the levels of mini-Dys protein with the split vector were found to be higher (5-11-fold) compared to the control plasmid (Figures 5B and 5C). This could be explained by the shorter time required to simultaneously process the two halves encoded by the two vectors compared to the long construct expressed by one vector, or by transfection efficiency. Interestingly, we noted that all four selected split sites led to efficient formation of mini-Dys, and the highest expression was obtained in site #2. In addition, no other bands were detected on the Western blot membrane even after excessive exposure, highlighting the specificity of the method.

[0176] These in vitro data indicate that it is possible to use this novel split mini-Dys system to validate preselected inteins in vitro. Once the most efficient intein is identified, the split mini-Dys / intein set will be cloned into an AAV vector for in vivo validation.

[0177] Preliminary screening of an intein library using a split-GFP system Many split intein pairs have been described, and we compared the splicing of many of them using the split intein-GFP system (compare data in Figures 4 and 5). The following were cloned, transfected into HEK293 cells, and relative fluorescence was monitored: a plasmid expressing CMV-eGFP (control), a dual plasmid carrying the N- or C-terminal half of eGFP together with the N- or C-terminal half of a codon-optimized split intein, and one plasmid carrying CMV-eGFP with a footprint that could be left by ligation of the split inteins of the dual vector (Figure 7). The data show that this system worked well for comparing the splicing of split inteins. Although no pair produced fluorescence as intense as that of WT GFP, several split intein vectors produced similar levels of fluorescence to that from a single vector carrying the corresponding footprint left after ligation of the exteins (e.g., split intein Aha; Figure 7 ).

[0178] To assess whether it was possible to use the two sets of split inteins in the three vectors to generate full-length Dys, combinations of split intein halves were tested to see if any could cross-splice with another split intein, as cross-splicing would likely prevent the joining of exteins from the three vectors by skipping the middle extein. Many split inteins were observed to cross-splice, but a newer class, "group 2 inteins," do not generally cross-splice (Figure 8).

[0179] Next, it was tested whether the footprint size could be reduced from its usual 6 amino acids (AA), and several split inteins were found to splice efficiently even when engineered to leave a footprint of only 3 AA (Figure 9A). These results demonstrate that it is possible to efficiently screen "wild-type" and synthetic split inteins using a rapid GFP assay, and that several split intein pairs exhibit efficient and specific splicing while leaving only a minimal footprint after extein ligation.

[0180] In vitro validation of preselected inteins using the split mini-Dys system This intein system was adapted to mini- and full-length dystrophin (Dys). Two or three vectors with one or two sets of split inteins were prepared and tested in HEK293 cells. Controls were the corresponding mini-Dys (ΔSR5-15) or full-Dys expressing plasmids alone. All split intein vectors produced the correct protein at higher levels than the single vectors (presumably reflecting the reduced transfection efficiency of larger plasmids; Figures 9B, 9C; Figures 5A-5C; Figure 13). Some smaller products accumulated in cells (dual vectors) and some "mini-Dys" accumulated in triple vector studies, the latter suggesting that the N-terminal side of the extein was directly spliced ​​to the C-terminal side of the extein.

[0181] In vivo validation of the best set of mini-Dys / intein by intramuscular injection mdx 4cvAn example set of dual vectors tested in muscle reveals efficient expression of ΔSR5-15 mini-dystrophin ( FIG. 12 ). In this example, the split mini-dystrophin / intein clone was inserted into an AAV plasmid and used to generate an AAV vector that contained a muscle-specific creatine kinase 8 (CK8) regulatory cassette and a short synthetic polyA flanked by two inverted terminal repeats (ITRs) of AAV serotype 2. 5 x 10 10 AAV encoding the N- and / or C-terminal ends of the split mini-dystrophin / intein was administered in a dose of viral genome (vg) to 3-week-old C57BL / 6-mdx mice. 4cv The reconstituted mini-dystrophin ΔSR5-15 was injected into the tibialis anterior (TA) muscle of mdx mice injected with dual AAV vectors, N- and C-terminal, respectively. Four weeks after injection, the injected muscles were harvested and analyzed. Strong expression of mini-dystrophin ΔSR5-15 was detected in the four TA muscles tested, highlighting the efficacy of the SIMPLI-GT approach (Figure 12A). The muscles were also cryosectioned and immunostained for dystrophin or stained with hematoxylin and eosin. The reconstituted mini-dystrophin ΔSR5-15 was expressed in mdx mice injected with dual AAV vectors, N- and C-terminal. 4cv The sarcolemma of the muscles was correctly localized in the sarcolemma of ...

[0182] For any given split intein, there is flexibility as to the exact location where the mini-Dys extein is split, which can affect splicing efficiency; andIt may also minimize the footprint left after extein ligation (Figures B-E). As part of that effort, we focused on ΔSR5-15 mini-Dys (Figures 10 and 9C), in which spectrin-like repeat 4 (SR4) is linked to SR16. This mini-Dys was modified from Dys produced in a mildly diseased patient with a deletion of exons 17-48, who was ambulatory into her late 70s (England, 1989). Meanwhile, the design described here removes the partial SR encoded on exon 49 (Harper, 2002) and adds an nNOS localization domain to SR16-SR17 (Adams et al, 2018; Lai et al., 2009). This ΔSR5-15 mini-Dys is predicted to assemble a complete DGC. Unlike exon skipping or Cas9 cleavage methods, it is not necessary to split the Dys sequence exactly at the exon (where a partial domain is often left behind). Instead, either vector may insert a portion of the SR16-SR19 region followed by a 5' split intein, while the 3' vector may carry a 3' split intein linked to the remaining SR. The only difference is the location of the footprint. There are multiple split sites and these can be tested in 293 cells while monitoring splicing efficiency to minimize the footprint (Figures 10, 9B, 9C, and 13). Engineered split inteins were also developed to create smaller inteins whose footprints more closely match the candidate split sites (e.g., near the middle of the mini-Dys clone; FIG. 10). The gist of this is that there is a wide range of choices for the split target that maximizes splicing efficiency (and protein stability / function) while allowing the intein footprint to match the Dys sequence. Ideally, a Dys region containing 6 amino acids that perfectly match the intein footprint would be used. While we did not find such a 6 amino acid region at a useful position, we did find regions where the footprint differed from Dys by only 1 or 2 amino acids.Since each extein vector must fit within the cloning capacity of AAV (<5 kb), including the ITRs (360 bp), polyA site (approximately 60 bp), and MSEC, considering the size of the ideal mini-Dys protein, at least 600 bp in the middle of Dys is targeted by the split intein. A further way to minimize the footprint is to target Dys sequences that display different conservative amino acids from the given footprint.

[0183] To adapt this system to genes other than dystrophin, we split the dysferlin cDNA into two fragments using three different split sites, as was done in the dystrophin dual vector study, and cloned three sets of plasmids, each carrying one of the sets of split inteins. In this study, HEK293 cells were co-transfected with the three sets of split intein-dysferlin plasmids separately, followed by cell harvesting and Western blot analysis for dysferlin protein (Figure 14). As shown in Figure 14A, full-length dysferlin protein was produced in HEK293 cells when both sets of split intein-dysferlin clones were used. Both sets produced similar levels of dysferlin as the control plasmid carrying the full-length dysferlin cDNA. Figure 14B shows the quantification of the protein levels, indicating that similar efficiencies were achieved.

[0184] This novel SIMPLI-GT approach offers several advantages and can be applied to any genetic disorder with defective genes that are larger than the packaging capacity of AAV vector.This approach is made using AAV vector, which is widely used in the field of gene therapy due to its efficiency, serotype diversity and tissue tropism.Unlike CRISPR-Cas9 gene editing and U7 exon skipping, this method promotes the high expression of larger dystrophin with appropriately placed domains, which stabilize the structure of dystrophin.This strategy can be applied to any patient with DMD or BMD, regardless of their genetic mutation, and ultimately produces therapeutic candidates with less variability and fewer regulatory obstacles.

[0185] References: TIFF2024526938000036.tif24165TIFF2024526938000037.tif232165TIFF2024526938000038.tif225165TIFF2024526938000039.tif78165

[0186] Example 2: Exemplary sequences of split inteins combined with mini-dystrophin, dystrophin, dysferlin, or utrophin TIFF2024526938000040.tif134165TIFF2024526938000041.tif241165TIFF2024526938000042.tif241165TIFF2024526938000043.tif231165TIFF2024526938000044.tif241165TIFF2024526938000045.tif241165TIFF2024526938000046.tif241165TIFF2024526938000047.tif241165TIFF2024526938000048.tif241165TIFF2024526938000049.tif241165TIFF2024526938000050.tif240165TIFF2024526938000051.tif237165TIFF2024526938000052.tif241165TIFF2024526938000053.tif241165TIFF2024526938000054.tif241165TIFF2024526938000055.tif241165TIFF2024526938000056.tif241165TIFF2024526938000057.tif241165TIFF2024526938000058.tif116165

Claims

**Claim 1** A method for delivering an exogenous polypeptide to a cell or inducing the production of an exogenous polypeptide in a cell, the method comprising: a first adeno-associated virus (AAV) vector particle comprising a first nucleic acid encoding a first fusion polypeptide, the first fusion polypeptide comprising a first portion of an exogenous polypeptide fused to a first portion of a split intein; and a second AAV vector particle comprising a second nucleic acid encoding a second fusion polypeptide, the second fusion polypeptide comprising a second portion of an exogenous polypeptide fused to a second portion of a split intein contacting the cell with; the first and second fusion polypeptides are produced from the first and second nucleic acids in the cell, and the first and second portions of the split intein facilitate the ligation of the first portion of the exogenous polypeptide to the second portion of the exogenous polypeptide, whereby the exogenous polypeptide is delivered to the cell or the production of the exogenous polypeptide in the cell is induced, the exogenous polypeptide to be delivered or produced is larger than can be encoded by one AAV vector particle, method. **Claim 2** the first and second nucleic acids comprise a muscle-specific expression cassette (MSEC), and / or the split intein is a naturally occurring split intein, and / or the split intein is a genetically modified split intein, optionally wherein the genetic modification of the split intein is selected from codon optimization for expression and / or stability in mammalian cells, shortening or lengthening of the split intein, or alteration in the split intein of the encoded amino acids to more closely match the sequence of the exogenous protein to be delivered, The method according to claim 1. **Claim 3** the first and second portions of the exogenous polypeptide are of substantially the same size, or the first and second portions of the exogenous polypeptide differ by 50 amino acids or less in size, The method according to claim 1 or 2. **Claim 4** The exogenous polypeptide contains a footprint derived from a split intein of less than 4 amino acids, optionally the exogenous polypeptide contains a footprint derived from a split intein of 3 amino acids or less, and further optionally, the cleavage site that separates the first and second parts of the exogenous polypeptide is selected at a site having the same sequence as the footprint of the split intein, whereby the exogenous polypeptide is produced without surplus amino acids derived from the split intein, the method according to any one of claims 1 to 3.

5. The exogenous polypeptide is a therapeutic polypeptide, optionally the therapeutic polypeptide is selected from dystrophin, minidystrophin, utrophin, dysferlin, nebulin, titin, myosin, spectrin repeat containing nuclear envelope protein 1 (Syne-1), dystroglycan, ATP synthase, coagulation factor VIII, lamin A / C, thyroglobulin, epidermal growth factor receptor (EGFR), α-spectrin and / or β-spectrin, muscle target of rapamycin (mTOR), and ryanodine receptor 1, the method according to any one of claims 1 to 4.

6. Minidystrophin is larger than 160 kDa and smaller than full-length dystrophin, or the therapeutic polypeptide is dystrophin, and the N-terminal part of the dystrophin extein is linked to the N-terminal part of the split intein in or adjacent to the hinge domain of dystrophin, optionally the hinge domain contains hinge 1, hinge 2, hinge 3, or hinge 4 of dystrophin, or the therapeutic polypeptide is dystrophin, and the N-terminal part of the dystrophin extein is linked to the loop domain that connects helix b to helix c, or the loop domain that connects helix c to helix a' in one of the 24 spectrin-like repeat domains of dystrophin, or The therapeutic polypeptide is dystrophin, and the C-terminal portion of dystrophin extain is linked to the C-terminal portion of a split intein within or adjacent to the hinge domain of dystrophin, or to the loop domain that links helix b to helix c, or the loop domain that links helix c to helix a' within one of the 24 spectrin-like repeat domains of dystrophin, and optionally, the hinge domain includes hinge 1, hinge 2, hinge 3, or hinge 4 of dystrophin. The method according to claim 5.

7. The method according to any one of claims 1 to 6, wherein the foreign polypeptide is functional in a cell.

8. A method for delivering a foreign polypeptide to a cell or inducing the production of a foreign polypeptide in a cell, the method comprising: A first adeno-associated virus (AAV) vector particle comprising a first nucleic acid encoding a first fusion polypeptide, wherein the first fusion polypeptide comprises a first portion of a foreign polypeptide fused to a first portion of a first split intein, and the first portion of the split intein is fused to the carboxy terminus of the first portion of the foreign polypeptide; A second AAV vector particle comprising a second nucleic acid encoding a second fusion polypeptide, wherein the second fusion polypeptide comprises: (i) a second portion of a foreign polypeptide fused to a second portion of a first split intein at the amino terminus of the second portion of the foreign polypeptide, and (ii) a first portion of a second split intein fused to the carboxy terminus of the second portion of the foreign polypeptide of the second portion of the foreign polypeptide; and A third AAV vector particle comprising a third nucleic acid encoding a third fusion polypeptide, wherein the third fusion polypeptide comprises a third portion of a foreign polypeptide fused to a second portion of a second split intein at the amino terminus of the third portion of the foreign polypeptide comprising contacting the cell with. The first, second, and third fusion polypeptides are produced from the first, second, and third nucleic acids in a cell, and each part of the first and second split inteins facilitates (a) the ligation of the carboxy terminus of the first part of the foreign polypeptide to the amino terminus of the second part of the foreign polypeptide, and (b) the ligation of the carboxy terminus of the second part of the foreign polypeptide to the amino terminus of the third part of the foreign polypeptide, whereby the foreign polypeptide is delivered to the cell or the foreign polypeptide is produced in the cell, wherein the foreign polypeptide to be delivered or produced is larger than can be encoded by one AAV vector particle, Method. **Claim 9** The method according to claim 8, wherein the first and second split inteins do not cross-splice. **Claim 10** A protein expression system comprising a set of AAV vector particles comprising the first and second AAV particles, wherein the first AAV vector particle comprises a first nucleic acid encoding a first fusion polypeptide, and the first fusion polypeptide comprises a first part of a foreign polypeptide fused to a first part of a split intein; and the second AAV vector particle comprises a second nucleic acid encoding a second fusion polypeptide, and the second fusion polypeptide comprises a second part of a foreign polypeptide fused to a second part of a split intein, Protein expression system. **Claim 11** Co-infection of the first and second AAV vector particles into a cell facilitates the ligation of the first part of the foreign polypeptide with the second part of the foreign polypeptide, accompanied by the removal of the first and second parts of the split intein, and / or the ligation of the first part of the foreign polypeptide with the second part of the foreign polypeptide, accompanied by the removal of the first and second parts of the split intein, results in a foreign polypeptide larger than can be encoded by one AAV particle, and / or the expression of the first and second polypeptides is driven by a muscle-specific expression cassette, The protein expression system according to claim 10. **Claim 12** A protein expression system comprising a set of AAV vector particles comprising the first, second, and third AAV particles, The first AAV vector particle comprises a first nucleic acid encoding a first fusion polypeptide, the first fusion polypeptide comprising a first portion of a foreign polypeptide fused to a first portion of a first split intein, the first portion of the split intein being fused to the carboxy terminus of the first portion of the foreign polypeptide; The second AAV vector particle comprises a second nucleic acid encoding a second fusion polypeptide, the second fusion polypeptide being (i) fused to a second portion of the first split intein at the amino terminus of the second portion of the foreign polypeptide and (ii) fused to a first portion of the second split intein at the carboxy terminus of the second portion of the foreign polypeptide, a second portion of the foreign polypeptide comprising; and The third AAV vector particle comprises a third nucleus encoding a third fusion polypeptide, the third fusion polypeptide comprising a third portion of the foreign polypeptide fused to a second portion of the second split intein at the amino terminus of the third portion of the foreign polypeptide, a protein expression system.

13. Co-infection of the first, second, and third AAV vector particles into a cell promotes the ligation of the first portion of the foreign polypeptide with the second portion of the foreign polypeptide, with removal of the first and second portions of the first split intein, and the ligation of the second portion of the foreign polypeptide with the third portion of the foreign polypeptide, with removal of the first and second portions of the second split intein, and / or The ligation of the first portion of the foreign polypeptide with the second portion of the foreign polypeptide, with removal of the first and second portions of the first split intein, and the ligation of the second portion of the foreign polypeptide with the third portion of the foreign polypeptide, with removal of the first and second portions of the second split intein, results in a larger foreign polypeptide than can be encoded in one AAV particle, and / or Expression of the first, second, and third fusion polypeptides is driven by a muscle-specific expression cassette, The protein expression system according to claim 12.

14. A protein expression system for use according to any one of claims 8 to 13 for treating a disease or disorder in a subject in need thereof.

15. The subject in need has a muscle disease or disorder, or a neuromuscular disease or disorder, and / or the exogenous polypeptide is dystrophin or minidystrophin, and the subject in need has Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD), and optionally, the dystrophin or minidystrophin increases the muscle strength of dystrophic muscle by at least 10%, and / or the expression of the first and second fusion polypeptides, or the expression of the first, second, and third fusion polypeptides, is driven by a muscle-specific expression cassette, and / or the protein expression system is administered by injection into the vasculature or by direct injection into tissue, The protein expression system according to claim 14.

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