Use of split intein for the treatment of ABCA3-associated disease

Abstract

The present disclosure relates to the use of split inteins for expressing phospholipid-transporting ATPase ABCA3 protein encoded by ABCA3 gene in a subject in need thereof for gene therapy in particular for the treatment of ABCA3-associated disease, preferably ABCA3-surfactant dysfunction such as respiratory distress syndrome, interstitial lung disease or pulmonary fibrosis.

Description

USE OF SPLIT INTEIN FOR THE TREATMENT OF ABCA3-ASSOCIATEDDISEASETECHNICAL FIELDThe present disclosure relates to the use of split inteins for expressing phospholipid-transporting ATPase ABCA3 protein encoded by ABCA3 gene in a subject in need thereof for gene therapy in particular for the treatment of A8G43-associated disease, preferably ABC A3 -surfactant dysfunction such as respiratory distress syndrome, interstitial lung disease or pulmonary fibrosis.BACKGROUNDPulmonary surfactant is a complex mixture of phospholipids and proteins secreted into the alveolar space, that reduces surface tension, prevents end expiratory alveolar collapse and is required for gas exchange. Surfactant is synthesized in lamellar bodies, specialized intracellular organelles derived from lysosomes in alveolar epithelial type II cells (AEC2, aka ATII or AT2 cells). Phospholipids are transported into the lamellar bodies by ABCA3 and assemble with surfactant proteins B and C to form surfactant. The lamellar bodies are released into the alveolar lumen via exocytosis. Loss of ABCA3 results in surfactant lacking phosphatidylcholine and increased surface tension (Garmany et al., 2006; Pediatr. Res. 59, 801-805). Pathogenic variants in ATP-binding cassette transporter A3 gene (ABCA3 gene) lead to a surfactant dysfunction such as neonatal respiratory distress syndrome (RDS) and childhood interstitial lung disease (chILD) and pulmonary fibrosis (Garmany et al., 2008, Cooney AL et al. Front Genome Ed. 2021; 3: 785829).Current available medical therapies such as exogeneous surfactant replacement, antiinflammation therapies or lung transplantation are limited and generally ineffective. There therefore remains a need for new and advanced therapies for ABCA3 surfactant disorder.Gene therapy is a promising option to treat monogenic lung diseases (Cooney AL et al. Front Genome Ed. 2021; 3: 785829).Adeno-associated viruses (AAV) have been widely used for viral delivery in gene therapy. However, the size of a gene that can be encapsulated into AAV has been reported to be limited to ~4.7 kb (Grieger and Samulski 2005, J Virol 79: 9933-9944). Gene therapy strategies fortreating ABCA3 associated disease are complicated by the size of the protein and the difficulty of packaging large sequences (5.1 kb) into currently used gene therapy vectors. Due to its large size, ABCA3 gene cannot be encapsulated into a single AAV, and there remains a need to develop new strategies to deliver ABCA3 gene inside target cells and efficiently express the full-length protein for therapeutic applications.A dual vector approach using split inteins has been employed to deliver gene editing tools. Inteins are genetic elements that carry out trans-splicing, where two protein fragments bind to form a catalytically competent enzyme, then catalyze their own excision and the ligation of their flanking sequences. Split inteins have been mainly used to fuse different functional protein domains in protein purification system and labeling steps. However, this approach may not be feasible to reconstitute in vivo complex proteins with multiple transmembrane domains such as ABCA3.SUMMARYIn the present application, the inventors showed that split intein-mediated protein trans-splicing can be used to efficiently reconstitute phospholipid-transporting ATPase ABCA3 protein encoded by ABCA3 gene in a cell.Phospholipid-transporting ATPase ABCA3 reconstitution with split inteins represents an efficient strategy to treat ABC A3 -associated disease such as H C43-associated disease, preferably ABC A3 -surfactant dysfunction such as respiratory distress syndrome, interstitial lung disease or pulmonary fibrosis.The present disclosure relates to a combination of polynucleotides for use in the treatment of ABC A3 -associated disease, preferably ABC A3 -surfactant dysfunction, more preferably selected from the group consisting of: respiratory distress syndrome, interstitial lung disease or pulmonary fibrosis, in a subject in need thereof wherein the combination comprises: i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’ : a N-terminal fragment of phospholipid-transporting ATPase ABCA3 (ABCA3) protein and N-split intein, fused directly or indirectly via a linker, ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker,wherein expression of first and second polynucleotides in said subject generates ABC A3 by protein splicing.In a preferred embodiment, the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively, the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively, the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively, the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABCA3 protein from residue 875 respectively, the N-terminal fragment of ABC A3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, the N-terminal fragment of ABCA3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively, the N-terminal fragment of ABC A3 protein up to residue 1022 and the C-terminal fragment of ABC A3 protein from residue 1023 respectively, the N-terminal fragment of ABC A3 protein up to residue 1065 and the C-terminal fragment of ABC A3 protein from residue 1066, or the N-terminal fragment of ABC A3 protein up to residue 1255 and the C-terminal fragment of ABCA3 protein from residue 1256, wherein said residue is numbered according to SEQ ID NO: 15, preferably the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively, the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or the N-terminal fragment of ABC A3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, wherein said residue is numbered according to SEQ ID NO: 15.In a preferred embodiment, said ABCA3 protein is human ABCA3 protein, preferably comprising or consisting of SEQ ID NO: 15 or any functional variant thereof having at least 90% identity to SEQ ID NO: 15.In a particular embodiment, the first and second fusion proteins according to the present disclosure comprise amino acid sequences selected from any one of the following pairs: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33 or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 16-33, preferably SEQ ID NO; 20 and 21, SEQID NO: 22 and 23 or SEQ ID NO: 24 and 25 or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 20-25.In a more particular embodiment, said N-split intein is a N-Cfa-intein of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity to SEQ ID NO: 1; and said C-split intein is a C-Cfa intein of SEQ ID NO: 2 or any functional variant thereof having at least 90% identity to SEQ ID NO: 2, preferably wherein amino acid residues 20 to 22 of SEQ ID NO: 2 are GEP, more preferably C-Cfamut intein of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity to SEQ ID NO: 13.In a more preferred embodiment, the first and second fusion proteins according to the present disclosure comprise amino acid sequences selected from any one of the following pairs: SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, SEQ ID NO: 42 and 43, SEQ ID NO: 44 and 45, SEQ ID NO: 46 and 47, SEQ ID NO: 48 and 49, and SEQ ID NO: 50 and 51 or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 34-51, preferably SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, or SEQ ID NO: 42 and 43 or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 38 to 43.In a particular embodiment, the first fusion protein or second fusion protein may further comprise a degron, preferably selected from the group consisting of: SEQ ID NO: 52 to 82 or any functional variant thereof having at least 90% identity to any one of sequences SEQ ID NO: 52 to 82, more preferably the first fusion protein further comprises a degron located at the 3 ’end of the N-split-intein, and / or the second fusion protein further comprises a degron located at 5 ’end of the C-split-intein, fused directly or indirectly via a linker.According to the present disclosure, each polynucleotide of the combination further comprises some regulatory elements, for example, promoters, transcription termination sequences, translation termination sequences, introns, enhancers, signal peptides, and polyadenylation elements, preferably a promoter selected from the group consisting of: Cytomegalovirus (CMV) promoter (GenBank Accession number: AF396260.1, bp 150-812, last updated on August 13, 2001), chimeric reduced version of the CMV and chicken beta-actin (CEB A) promoter (GenBank Accession number: AF396260.1, bp 160-526, last updated on August 13, 2001; GenBank Accession number: X00182.1, bp 268-571, last updated on November 14, 2006), human phosphoglycerate kinase (hPGK) promoter (GenBank Accession number: AH002938.2, bp 2-516, last updated on August 01, 2016), chimeric CMV enhanced and humanphosphoglycerate kinase (ePGK) promoter (GenBank Accession number: AF396260.1, pb 160- 500, last updated on August 13, 2001; GenBank Accession number AH002938.2, bp 2-516, last updated on August 01, 2016), EFl alpha promoter (GenBank Accession number: J04617.1, pb 378-1560, last updated on November 7, 1994), rous sarcoma virus RSV promoter (GenBank Accession number: J02025.1, pb 287-549, last updated on April 28, 1993) and human surfactant protein B (SPB) promoter (GenBank Accession number: NG 016967.1, pb 4924-5558, last updated on April 29, 2025).Preferably each polynucleotide is comprised within an expression vector, preferably a viral vector, preferably an adeno associated viral (AAV) vector, preferably said AAV vector comprises capsid protein of AAV selected from the group consisting of 1, 2, 3, 4, 5, 6, 6.2, 6.2FF, 7, 8, 9 or RhlO, preferably AAV5, AAV6, AAV6.2, AAV6.2FF, AAV8 or AAV9.In a preferred embodiment, said combination is administered in subject by a parenteral route, more preferably by intravenous, intraarterial, intramuscular, intranasal, or intratracheal, more preferably intratracheal.The present disclosure also relates to a kit comprising: i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’ : a N-terminal fragment of ABCA3 protein and N-split intein, fused directly or indirectly via a linker, ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C- terminal fragment of ABC A3 protein from residue 1023 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C- terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABC A3 protein up to residue 1255 and the C- terminal fragment of ABCA3 protein from residue 1256 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal ABCA3 fragment and the C-terminal ABCA3 fragment respectively consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, more preferably wherein the first and second fusion proteins comprise amino acid sequences selected from any one of the following pairs: SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, SEQ ID NO: 42 and 43, SEQ ID NO: 44 and 45, SEQ ID NO: 46 and 47, SEQ ID NO: 48 and 49, and SEQ ID NO: 50 and 51 or any functional variant thereof, preferably having at least 90 % identity to any one of sequences SEQ ID NO: 34-51.LEGEND FIGUREFigure 1: Splicing efficiency of ABCA3 protein by Western blot: ABC A3 protein was first split at one of the suitable positions (767-768) and the N-terminal fragment (AA1N) recombinantly fused to N-inteins, and the C-terminal fragment (AA1C) to the C-inteins. Cultured HEK293T cells were co-transfected with equimolar amounts of plasmids encoding for the N and C-terminal fragments and splicing efficiency monitored by Western blotting.Figure 2: Western Blot of transfected HEK293T cells lysates of negative controls, full length, fragments and protein trans-splicing (PTS) for ABCA3 Site 1 (767-768). The rectangle indicated the PTS- reconstituted ABCA3 full-length protein.Figure 3. Western Blot of transfected HEK293T cells lysates of negative controls, full length, fragments and protein trans-splicing (PTS) for ABCA3 from Site 2-7 (site 2: 743 / 744, site 3: 904 / 905; site 4:874 / 875; site 5: 1022 / 1023, site 6: 1065 / 1066, site 7: 935 / 936). A) Western blot sites 2, 3 and 4. B) Western blot Sites 5, 6 and 7. The rectangles indicated the PTS-reconstituted ABCA3 full-length protein.Figure 4. Western blot (WB) analysis of protein trans-splicing bands was quantified by calculating the ratio relative to (3-tubulin and the full-length ABCA3 protein. All protein trans-splicing (PTS) ratios were subsequently normalized to the 100% expression level of the full-length ABCA3 protein.DETAILED DESCRIPTIONCombination of polynucleotides encoding first and second fusion proteinsThe limited cargo capacity of the AAV vectors precludes its use for delivering large genes such as ABCA3 gene in gene therapy. To deliver ABCA3 gene in patients, the inventors took the advantage of the intrinsic ability of split inteins to mediate protein trans-splicing to reconstitute phospholipid-transporting ATPase ABCA3 encoded by large ABCA3 gene following their fragmentation into two split-intein flanked polypeptides. In particular, they determined the optimal split positions that allow to efficiently reconstitute ABCA3 protein.The present disclosure relates to a combination of polynucleotides comprising: i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’ : a N-terminal fragment of phospholipid-transporting ATPase ABCA3 and N-split intein, fused directly or indirectly via a linker, ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-split intein and a C-terminal fragment of phospholipid-transporting ATPase ABCA3 protein, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in a cell generates full length ABCA3 by protein splicing.According to the present disclosure, the combination of polynucleotides comprises a first polynucleotide encoding a first fusion protein and a second polynucleotide encoding a second fusion protein.According to the present disclosure, by the term “combination” is meant that the first and second polynucleotides according to the present disclosure can be formulated in a single or as separate formulations.According to the present disclosure, the term “nucleic acid sequence”, “nucleic acid molecule”, “nucleotide sequence” or “polynucleotide” may be used interchangeably to refer to any molecule composed of or comprising monomeric nucleotides. A polynucleotide may be a DNA or RNA.Herein, the terms "peptide", "oligopeptide", "polypeptide" and "protein" are employed interchangeably and refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain.The term "amino acid" refers to naturally occurring and unnatural amino acids (also referred to herein as "non-naturally occurring amino acids"), e.g., amino acid analogues and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogues refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogues can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid. The terms "amino acid" and "amino acid residue" are used interchangeably throughout.The term "fusion protein" as used herein, refers to a recombinant protein comprising two or more protein domains from at least two different proteins linked, preferably covalently. Said fusion protein is obtained or obtainable by genetic fusion, for example by genetic fusion of at least two gene fragments encoding separate domains of distinct proteins. In preferred embodiments, a fusion protein is a single chain polypeptide which may be fully encoded by a nucleic acid sequence and includes at least two protein domains directly covalently linked by peptidic bound or optionally covalently linked via a peptidic linker.According to the present disclosure, said first fusion protein comprises a N-terminal fragment of ABCA3 protein and N-split intein, fused directly or indirectly via a linker and said secondfusion protein comprises a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker.The term "linker" as used herein, refers to a chemical group or a molecule linking two adjacent molecules or moieties. In some embodiments, the polynucleotide encodes a linker selected from the group consisting of a (GGS)n , a (GGGGS)n (SEQ ID NO: 83), a (G)n , an (EAAAK)n (SEQ ID NO: 84), a XTEN-based linker, or an (XP)n motif , or a combination of any of these, wherein n is independently an integer between 1 and 50. In other embodiments, a linker is not used. Instead, e.g., the polynucleotide sequences comprise nucleic acids encoding a first and second protein domains and further comprise additional nucleic acids in at least one of their ends that make the function of linker.According to the present disclosure, the expression of first and second polynucleotides encoding said first and second fusion proteins in a cell generates ABCA3 protein by protein splicing.The term “protein trans-splicing” or “protein splicing” refers to the excision of a split-intein from a larger precursor polypeptide through the cleavage of two peptide bonds and, the concomitant ligation of the flanking protein fragments, also called exteins through the formation of a new peptide bond to form a mature protein and the free intein (Shah NH and Muir TW, Chem Sci. 2014; 5(1): 446-461. 2013).As used herein, the term “intein” refers to a protein that is capable of ligating the flanking sequences (exteins) into a new protein.As used herein, the term “split-inteins” or “trans-splicing inteins” means naturally occurring or engineered constructed protein fragments (i.e., N-intein and C-intein) which bind to form a catalytically competent enzyme capable of catalyzing a protein splicing reaction that excises the N- and C-intein sequences and joins flanking sequences (N- and C-exteins) with a peptide bond.As used herein, the term “peptide bond” refers to covalent chemical bond -CO-NH- formed between two molecules when the carboxy part of one molecule (carboxy component, C- component or C-terminal component) reacts with the amino part of another molecule (amino component, N-component or N-terminal component).According to the present disclosure, the term “N-split intein”, “N-intein”, or “N-terminal split intein” refers to any N-terminal amino acid sequence of a split intein that is capable of associating with a C-terminal amino acid sequence of said split intein to form a functional split intein that is capable of catalyzing a protein splicing reaction that excises the N- and C-intein sequences and joins flanking sequences (N- and C-exteins) with a peptide bond.According to the present disclosure, the term “C-intein”, “C-split intein” or “C-terminal split intein” refers to any C-terminal amino acid sequence of a split intein that is capable of associating with a N-terminal amino acid sequence of said split intein to form a functional split intein that is capable of catalyzing a protein splicing reaction that excises the N and C-intein sequences and joins flanking sequences (N- and C-exteins) with a peptide bond.In a particular embodiment, said N- and C-split inteins according to the present disclosure comprised in the first and second fusion proteins respectively can derive from the catalytic subunit of DNA polymerase III (DriaE) gene from different organisms such as cyanobacteria including Nostoc punctiforme (Npu), Synechocystis sp. Strain PCC6803 (Ssp), Fischerella sp. PCC 9605, Scytonema tolypothrichoides, Cyanobacteria bacterium SW 9 47-5, Nodularia spumigena, Nostoc flagelliforme, Crocosphaera watsonii WH 8502, Chroococcidiopsis cubana CCALA 043, or Trichodesuium erythraeum; preferably from Npu or Ssp.In another particular embodiment, the N- and C-split inteins can derive from the DnaB gene from Cyanobacteria including R. marinus (Rma), Synechocystis sp. PC6803 (Ssp), Porphyra purpurea chloroplast (Ppu), or can derive from gp41-l, gp41-8, NrdJ-1, or IMPDH-1, preferably Nrdj-1 (Carvajal- Vallejos P. et al. J Biol Chem. 2012 Aug 17; 287(34): 28686- 28696).In a preferred embodiment, N- and / or C- split inteins according to the present disclosure comprised in the first and second fusion proteins respectively can be engineered N- and / or C- split inteins. Said engineered N- and / or C- split inteins can be engineered by introducing mutations in natural N- and / or C- split intein sequences, in particular to enhance protein splicing activity.In a preferred embodiment, the N-split intein and / or C-split intein sequences comprised in the first and second fusion proteins respectively comprise or consist of amino acid sequences selected from any of N- and C-split inteins listed in Table 1 below:Table 1: Examples ofpairs ofN- and C-split inteins that can be used according to the present disclosure.Amino acids in bold can be replaced by GEP amino acids to improve extein tolerance.In a particular embodiment, the N- and C-split inteins comprised in the first and second fusion proteins respectively according to the present disclosure are N- and C-split inteins comprising or consisting of amino acid sequences of SEQ ID No: 1 (Cfa-N split intein) and SEQ ID No: 2 (Cfa-C-split intein), SEQ ID No: 3 (Npu-N) and 4 (Npu-C), SEQ ID No: 5 (Cat-N) and 6 (Cat- C), SEQ ID No: 7 (Gp41-N) and 8 (Gp41-C), SEQ ID No: 9 (ConN) and 10 (ConC) or SEQ ID No: 11 (Nrdj 1-N) and 12 (Nrdj 1-C) or any functional variant(s) thereof, preferably SEQ ID No:1 (Cfa-N split intein) and SEQ ID No: 2 (Cfa-C-split intein) or any functional variant(s) thereof.As used herein, the term "variant" or “functional variant” refers to a polypeptide sequence that is derived from N- and / or C-split inteins as described above and comprises an alteration, i.e., a substitution, insertion, and / or deletion, at one or more positions, but retain the capacity when bound to form a functional enzyme to catalyze a protein splicing reaction that excises the N and C-intein sequences and joins flanking sequences (N- and C-exteins) with a peptide bond.The variant may be obtained by various techniques well known in the art. Examples of techniques for altering the nucleotide sequence encoding the native protein, include, but are not limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction.The protein splicing efficiency of N- and / or C-split intein functional variants may be assessed for instance by measuring the protein reconstitution efficiency in a cell. In particular, the protein reconstitution efficiency can be measured by expressing in a cell a combination of polynucleotides, said first polynucleotide encodes a N-terminal fragment of a reporter protein (e.g., GFP) fused to N-split intein and a second polynucleotide encodes a C-terminal fragment of said gene reporter fused to the C-split intein. The reconstitution efficiency of the reporter protein can then be monitored by determining the level of expression of reconstituted protein.The expression level of reconstituted protein may be determined by any suitable methods known by skilled persons. The quantity of the protein may be measured, for example, by semi- quantitative Western blots, enzyme-labelled and mediated immunoassays, such as ELISAs, biotin / avidin type assays, radioimmunoassay, immunoelectrophoresis, mass spectrometry, or immunoprecipitation or by protein or antibody arrays. In a particular embodiment, when said reporter protein is a fluorescent protein, the quantity of protein may be measured by flow cytometry or fluorescence microscopy.The expression level can then be compared to a control value. According to a preferred embodiment, the term "control value " refers to the expression level of protein reconstituted with the native N- and C- split inteins in a cell expressing a combination of polynucleotides, said first polynucleotide encodes a N-terminal fragment of a reporter protein (e.g., GFP) fused to native N-split intein and a second polynucleotide encodes a C-terminal fragment of said gene reporter fused to the native C-split intein.The protein reconstitution efficiency of a functional variant is similar to that of native split intein in a cell when the expression level of said protein reconstituted with functional variants of N- and / or C-split intein(s) in a cell is similar than the control value (i.e., expression level ofsaid protein reconstituted with native N- and C-split inteins), in particular the expression level varies by less than 40%, 30%, 20% or 10% of the control value.As used herein, the term "variant" or “functional variant” may refer to a polypeptide having an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity any one of the N- and / or C-split intein(s) as described above, in particular in Table 1 and preferably retains protein splicing capacity of said polypeptide as described above.In a particular embodiment, said N-split intein and C-split intein according to the present disclosure comprised in the first and second fusion proteins respectively comprise or consist of SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10 or SEQ ID NO: 11 and 12 or any functional variant(s) thereof, preferably having 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1 to 12, preferably SEQ ID NO: 1 and 2 or any functional variant(s) thereof, preferably having 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to any one of the amino acid sequences consisting of SEQ ID NO: 1 and 2.As used herein, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (NEEDLEMAN, and Wunsch).The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk, Rice et al 2000 Trends Genet 16 :276-277). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary aminoacid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification.The term "variant" or “functional variant” may also refer to a polypeptide having an amino acid sequence that differs from a native sequence by less than 10, 9, 8, 7, 6, 5, 4 or 3 substitutions, insertions and / or deletions. In a preferred embodiment, the variant differs from the native sequence by one or more conservative substitutions, preferably by less than 10, 9, 8, 7, 6, 5, 4 or 3 conservative substitutions. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), Hydroxyl or sulfur / selenium-containing amino acids (serine, cysteine, threonine, methionine), polar amino acids (glutamine and asparagine), hydrophobic amino acids (methionine, leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), hydroxyl or sulfur / selenium-containing amino acids (serine, cysteine, threonine, methionine) and small amino acids (glycine, alanine, serine and threonine).The inventors previously engineered N- and C-split intein with superior protein trans-splicing properties. They showed that Cfa-N and Cfa-C-split inteins have a higher protein splicing efficiency than Npu split inteins (WO2017 / 132582 and WO2021 / 191447).In a preferred embodiment, the N- and C-split inteins according to the present disclosure comprised in the first and second fusion proteins respectively are Cfa-N- and Cfa-C-split inteins comprising or consisting of SEQ ID NO: 1 (Cfa-N split intein :) and SEQ ID NO: 2 (Cfa-C- split intein) or any functional variants thereof, preferably having 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 1 and / or 2.In a preferred embodiment, the functional variant of Cfa N- and / or Cfa C-Split intein retain the functional splicing activity of native Cfa split intein, more preferably have a protein splicing efficiency higher than Npu split-intein.The protein splicing efficiency of functional variants of split inteins (e.g., functional variants of Cfa-N- and / or Cfa- C-split inteins) may be assessed as described above. In a preferred embodiment, the expression level of reconstituted protein with functional variant(s) of Split inteins (e.g., functional variants of Cfa-N- and / or Cfa- C-split inteins) is then compared to a control value that refers to the expression level of protein reconstituted with Npu inteins.The protein reconstitution efficiency is higher than Npu split-intein in a cell when the expression level of said protein reconstituted with engineered split intein(s) (e.g., Cfa-N- and / orCfa-C-split intein(s)) in a cell is at least 1.5-fold higher, or 2, 3, 4, 5-fold higher or even more than in a control value (e.g., with Npu intein).In a more preferred embodiment, the functional variant of Cfa N- and / or Cfa C-Split intein can also splice faster than Npu split intein, preferably at least 1.5-fold higher, or 2, 3-fold higher or even more than Npu split intein. Protein trans-splicing activity can be measured by incubating N and C-split inteins individually in splicing buffer (e.g., lOOmM sodium phosphates, 150 mM NaCl, ImM EDTA, pH 7.2) with 2 mM tris(2-carboxyethyl)phosphine (TCEP) for 15 minutes. Splicing is initiated by mixing N- and C-split inteins and quenched by the addition of 8M guanidine hydrochloride, 4% Trifluoroacetic acid TFA (3: 1 v / v). Splicing reactions progress can be monitored by RP-HPLC or SDS-PAGE. When using RP-HPLC, each individual peak is normalized against the total area of all peaks combined and reaction curves are plotted (see detailed protocol, paragraphs

[0724] -

[0731] of WO2017 / 132580 application. When using SDS-PAGE quantification of splicing product is performed by densitometry using P-tubulin as a loading control.A major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site. Engineered versions of naturally split inteins, in particular C-split intein comprising GEP amino acids in positions 20, 21 and 22 wherein said residue is numbered according to SEQ ID NO: 2 possess improved extein tolerance.According to the present disclosure, the C-split intein derived from the catalytic subunit of DNA polymerase III (DnaE) gene may comprise GEP amino acids in positions 20, 21 and 22 wherein said residue is numbered according to SEQ ID NO: 2 (see Stevens et al., J Am Chem Soc. 2016 Feb 24; 138(7): 2162-2165, or Fig. 7A and B, C-intein of SEQ ID NO: 5 -358 of WO2017 / 132580), in particular instead of amino acid positions indicated in bold in the Table 1.In a preferred embodiment, the C-split intein is selected from any C-split inteins disclosed in Table 2.Table 2: Engineered C-split intein with improved extein tolerance.In a preferred embodiment, C-split intein is a mutated C-split intein having GEP amino acids in positions 20, 21 and 22 wherein said residue is numbered according to SEQ ID NO: 2 or any functional variant thereof, preferably retaining the functional splicing activity of split intein as described above, and more preferably having improved extein tolerance.In a particular embodiment, the C-split intein may be functional variants of the C-split inteins as described in Table 2, preferably comprising or consisting of amino acid sequence SEQ ID NO: 13 or 14, or any functional variants thereof having 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to any one of sequences SEQ ID NO: 13 or 14 and preferably retaining the functional splicing activity of C-split intein as described above, and more preferably having improved extein tolerance.The extein tolerance can be assessed for instance in kanamycine resistance assay as described in WO2017 / 132580 p. 60, paragraphs

[0735] -

[0738] in which a nucleic acid construct coding for a fragmented aminoglycoside phosphotransferase fused to a split intein with F, G, R or E present at the position +2 of the C-extein was transformed in DH5a competent cells and cultured at various concentrations of kanamycin. The cell density at 650 nm at 24 hours end point is measured and IC50 value is determined and compared with Cfa-Cmut. A functional variant having improved extein tolerance is a C-split intein having a similar IC50 than the split intein having GEP amino acids in positions 20, 21 and 22 wherein said residue is numbered according to SEQ ID NO: 2.In a preferred embodiment, the C-split intein is Cfa-Cmut split intein comprising or consisting of SEQ ID NO: 13 or any functional variant thereof having 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 13, preferably retaining the functional splicing activity of C- split intein as described above, and more preferably having improved extein tolerance, again more preferably having a similar IC50 than Cfa-Cmut comprising or consisting of SEQ ID NO: 2. In particular the IC50 varies by less than 40%, 30%, 20% or 10% of the positive control value (i.e., IC 50 of Cfa-C-mut comprising or consisting of SEQ ID NO: 13).In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N-terminal fragment of ABCA3 and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a Cfa C-split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of ABCA3, fused directly or indirectly via a linker.The inventors took the advantage of the intrinsic ability of split inteins as described above to mediate protein trans-splicing to reconstitute large ABCA3 following their fragmentation into two split-intein flanked polypeptides.Phospholipid-transporting ATPase ABCA3 (also named herein ABCA3 protein), encoded by ATP binding cassette subfamily A member 3 (ABCA3) gene is a phospholipid transporter located at the lysosomal-derived lamellar body limiting membrane that plays a critical role in surfactant assembly and lamellar body formation. The ABCA3 cDNA (NCBI reference sequence: NM_001089.3, updated on April 7, 2024) encodes a 1,704 amino acid polypeptide. Mature ABCA3 protein is folded in the endoplasmic reticulum and undergoes glycosylation in the Golgi. A second post-translational modification involves the N-terminal proteolytic cleavage of the 190 kD protein, shortening the protein to 150 kD.In a specific embodiment, ABCA3 protein is a human ATP binding cassette subfamily A member 3 (UniprotKB: Q99758, updated on 24 July 2024, SEQ ID NO: 15) encoded by ABCA3 gene (GENE ID: 21, updated on August 17, 2024), also known as ABC 3; ABC-C; SMDP3; LBM180; EST111653. According to the present disclosure, the term “phospholipid-transporting ATPase ABCA3” encompasses all known protein isoforms known of the phospholipidtransporting ATPase ABCA3.In a preferred embodiment, according to the present disclosure ABCA3 protein can be a human ABCA3 protein as disclosed above or any functional variant thereof.Preferably, as used herein, the term "variant" or “functional variant” refers to a polypeptide having an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 98 or 99% sequence identity to the native sequence and retain function of said polypeptide, herein ATP binding cassette subfamily A member 3, preferably human ABCA3 protein (SEQ ID NO: 15), in particular the catalyzation of ATP-dependent transport of phospholipids such as phosphatidylcholine and phosphoglycerol from the cytoplasm into the lumen side of lamellar bodies.In a preferred embodiment, ABCA3 is human ABCA3 protein comprising or consisting of SEQ ID NO: 15 or any functional variant thereof having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 15.More preferably, the term "variant" or “functional variant” refers to a polypeptide having an amino acid sequence that differs from a native sequence by less than 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10 or 5 substitutions, insertions and / or deletions. In a preferred embodiment, the functional variant differs from the native sequence by one or more conservative substitutions, preferably by less than 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10 or 5 conservative substitutions. In certain embodiment, the first methionine of the ABCA3 protein sequence can be removed.A number of different mammalians ABCA3 are known including, but being not limited to, human, pig, chimpanzee, dog, cow, mouse, rabbit or rat, and can be easily found in sequence databases. The coding sequence may be easily determined by the skilled person based on the polypeptide sequence.According to a specific embodiment of the present disclosure, a polynucleotide encoding the ABCA3 as described above is split into two nucleic acid sequences encoding N- and C-terminal ABCA3 fragments, each fragment being fused with at least N- and C-split inteins as described above to form a first and a second fusion proteins, respectively, in such a manner that following expression of said first and second fusion proteins, a protein splicing reaction can occur in a cell and induces the excision of the N and C-split intein sequences and the ligation of ABCA3 fragment flanking sequences (N- and C-terminal ABCA3 fragments, also named N- and C- exteins) with a peptide bond to reconstitute the full-length of ABCA3 protein as described above in a cell.According to the present disclosure, the ABCA3 or any functional variant thereof as described above can be split at any positions into a N- and C-terminal ABCA3 fragments. However, the inventors have shown that some specific split positions are particularly advantageous to increase the efficiency of ABCA3 reconstitution. In particular, the ABCA3 or any functional variant thereof as described above can be split into N- and C-terminal ABCA3 fragments at the split position between amino acids 447-448, 743-744, 767-768, 874-875, 904-905, 935-936, 1022-1023, 1065-1066 and 1255-1256 wherein said residue is numbered according to SEQ ID NO: 15. It will be easy for a person skilled in the art to determine the split positions in isoforms or functional variants of ABC A3, in particular by sequence alignment.In a particular embodiment, the N-terminal fragment of ABCA3 ends up to residue 447 and the C -terminal fragment of ABCA3 starts from residue 448 respectively wherein said residue is numbered according to SEQ ID NO: 15.In another particular embodiment, the N-terminal fragment of ABCA3 ends up to residue 743 and the C-terminal fragment of ABCA3 starts from residue 744 respectively wherein said residue is numbered according to SEQ ID NO: 15.In a preferred embodiment, the N-terminal fragment of ABCA3 fragment ends up to residue 767 and the C-terminal fragment of ABCA3 starts from residue 768 respectively wherein said residue is numbered according to SEQ ID NO: 15.In another preferred embodiment, the N-terminal fragment of ABCA3 fragment ends up to residue 874 and the C-terminal fragment of ABC A3 starts from residue 875 respectively wherein said residue is numbered according to SEQ ID NO: 15.In another preferred embodiment, the N-terminal fragment of ABCA3 fragment ends up to residue 904 and the C-terminal fragment of ABCA3 starts from residue 905 respectively wherein said residue is numbered according to SEQ ID NO: 15.In another particular embodiment, the N-terminal fragment of ABCA3 fragment ends up to residue 935 and the C-terminal fragment of ABC A3 starts from residue 936 respectively wherein said residue is numbered according to SEQ ID NO: 15.In another particular embodiment, the N-terminal fragment of ABCA3 fragment ends up to residue 1022 and the C-terminal fragment of ABC A3 starts from residue 1023 respectively wherein said residue is numbered according to SEQ ID NO: 15.In another particular embodiment, the N-terminal fragment of ABCA3 fragment ends up to residue 1065 and the C-terminal fragment of ABC A3 starts from residue 1066 respectively wherein said residue is numbered according to SEQ ID NO: 15.In another particular embodiment, the N-terminal fragment of ABCA3 fragment ends up to residue 1255 and the C-terminal fragment of ABCA3 starts from residue 1256 respectively wherein said residue is numbered according to SEQ ID NO: 15.In a particular embodiment, the first and second fusion proteins according to the present disclosure comprise a N-terminal ABCA3 fragment and a C-terminal ABCA3 fragment respectively comprising or consisting of amino acid sequences selected from the Table 3,preferably from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, or SEQ ID NO: 24 and 25, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of SEQ ID NO: 20 to 25 and preferably wherein reconstituted functional variant of ABC A3 retains the function of said native ABC A3, in particular catalyzation of ATP-dependent transport of phospholipids such as phosphatidylcholine and phosphoglycerol from the cytoplasm into the lumen side of lamellar bodies. The function of ABCA3 functional variant can be assessed for example by determining the phosphatidylcholine (PC) transport activity of ABCA3+vesicles, in particular by expressing ABCA3 in a cell (e.g., A549 cells) and incubating said cell with propagyl-choline and / or TopF- PC as described in Li Y et al. Biochim. Biophys. Acta (BBA)-Mol. Cell Biol. Lipids. 2019;1864: 158516, Yang X. et al. Int J Mol Sci. 2023 Apr; 24(8): 7554, Hoppner S., et al. Biochim. Biophys. Acta (BBA)-Mol. Cell Res. 2017;1864:2330-2335. The percentage of ABCA3+ vesicles colocalized with lysosomal compartment marker (e.g., CD63) is determined and compared with full-length ABCA3 protein.Table 3: Examples of N-and C-terminal ABCA3 fragments amino acid sequences. Highlighted amino acids in Full-length ABCA3 represent preferred split positions. Split positions are numbered according to ABCA3 full-length sequence with methionine. The first methionine of the ABCA-3 and N-terminal fragment sequences is indicated in the sequences listed in table 3. The first methionine can be maintained to initiate protein synthesis or removed in certain embodiments.In a particular embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABCA3 protein and N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,1- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C- terminal fragment of ABC A3 protein from residue 1023 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C- terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABC A3 protein up to residue 1255 and the C- terminal fragment of ABCA3 protein from residue 1256 respectively, preferably- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, more preferably- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively. wherein said residue is numbered according to SEQ ID NO: 15, more preferably wherein the N-terminal ABCA3 fragment and the C-terminal ABCA3 fragment respectively comprise or consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, or SEQ ID NO: 24 and 25 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25, more preferably SEQ ID NO: 22 and 23 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 22 and 23.In a more particular embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABC A3 protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-C-split intein of SEQ ID NO: 2 or Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of ABC A3 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C- terminal fragment of ABC A3 protein from residue 1023 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C- terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABC A3 protein up to residue 1255 and the C- terminal fragment of ABCA3 protein from residue 1256 respectively, preferably- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, more preferably- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively. wherein said residue is numbered according to SEQ ID NO: 15, more preferably wherein the N-terminal ABCA3 fragment and the C-terminal ABCA3 fragment respectively comprise or consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19 and SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, or SEQ ID NO: 24 and 25 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25, more preferably SEQ ID NO: 22 and 23 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 22 and 23.In a more preferred embodiment, the combination of polynucleotides according to the present disclosure comprises polynucleotides encoding a first fusion protein and a second fusion protein comprising amino acid sequences selected from the pairs disclosed in Table 4, preferably selected from the pairs consisting of: SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, SEQ ID NO: 42 and 43, SEQ ID NO: 44 and 45, SEQ ID NO: 46 and 47, SEQ ID NO: 48 and 49, and SEQ ID NO: 50 and 51 or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 34-51, preferably SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, or SEQ ID NO: 42 and 43, or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 38-43, more preferably SEQ ID NO: 40 and 41 or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 40 and 41.Table 4: Preferred first and second protein fusions according to the present disclosure.Cfa -N-Split intein and Cfa -C-mut-split inteins sequences are indicated in bold. First methionine of the fusion proteins is indicated and can be maintained or removed in certain embodiments.To prevent the accumulation of undesired starting materials and to eliminate the excised intein fragments, a degron can be fused directly or indirectly via a linker to the first and / or second fusion proteins to mediate degradation of the excised intein.A “protein degradation signal” or “degron” refers to a peptide fragment that induces degradation of the protein that contains the fragment. Protein degradation can happen through any of the many known protein degradation pathways, including but not limited to, ubiquitination, lysosomal degradation or autophagy. In a preferred embodiment, the degron targets protein to the ubiquitin-proteasome pathway. In a more preferred embodiment, the degron according to the present disclosure comprises amino acid sequence having less than 200, 190, 180, 170, 150, 130, 125, 110, 100, 95, 90, 85, 80, 75 amino acids.In a particular embodiment, said degron is fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, preferably said degron is located at the 3’-end of the N-Split intein or at the 5’end of the C-split intein.In a particular embodiment, said degron is fused, directly or indirectly via a linker, to the N- split intein and C-split intein comprised in the first and second fusion proteins, respectively, preferably said degron is located at the 3’-end of the N-Split intein and at the 5’end of the C- split intein.In a specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABCA3 protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker, wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein or at the 5’end of the C-split intein.In a specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABCA3 protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker, wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and at the 5’end of the C-split intein.In a specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABC A3 protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ a Cfa-C-Split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of ABC A3 protein, fused directly or indirectly via a linker, and wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein or at the 5’end of the C-split intein.In a specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABC A3 protein and Cfa-N-split intein of SEQ ID NO: 1 or anyfunctional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ a Cfa-C-Split intein of SEQ ID NO: 2 or a Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of ABC A3 protein, fused directly or indirectly via a linker, and wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and at the 5’end of the C-split intein.In a specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABCA3 protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C- terminal fragment of ABC A3 protein from residue 1023 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C- terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABC A3 protein up to residue 1255 and the C- terminal fragment of ABCA3 protein from residue 1256 respectively, preferably- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal ABCA3 fragment and the C-terminal ABCA3 fragment respectively comprise or consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23 or SEQ ID NO: 24 and 25 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25 and wherein said first or second fusion protein further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein or at the 5 ’-end of the C-split intein.In a specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABCA3 protein, N-split intein, fused directly or indirectly via a linker, anda second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C- terminal fragment of ABC A3 protein from residue 1023 respectively- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C- terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABC A3 protein up to residue 1255 and the C- terminal fragment of ABCA3 protein from residue 1256 respectively, preferably- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal ABCA3 fragment and the C-terminal ABCA3 fragment respectively comprise or consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30and 31, and SEQ ID NO: 32 and 33, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23 or SEQ ID NO: 24 and 25, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25 and wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and at the 5’end of the C-split intein.In a specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABC A3 protein and Cfa-N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-C-Split intein of SEQ ID NO: 2 or a Cfa-Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of ABC A3 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C- terminal fragment of ABC A3 protein from residue 1023 respectively- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C- terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABC A3 protein up to residue 1255 and the C- terminal fragment of ABCA3 protein from residue 1256 respectively; preferably- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal ABCA3 fragment and the C-terminal ABCA3 fragment respectively comprise or consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23 or SEQ ID NO: 24 and 25, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25 and wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein or at the 5 ’end of the C-split intein.In a specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABC A3 protein and Cfa-N-split intein of SEQ ID NO: 1 or anyfunctional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-C-Split intein of SEQ ID NO: 2 or a Cfa-Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of ABC A3 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C- terminal fragment of ABC A3 protein from residue 1023 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C- terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABC A3 protein up to residue 1255 and the C- terminal fragment of ABCA3 protein from residue 1256 respectively; preferably- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, wherein said residue is numbered according to SEQ ID NO: 15,preferably wherein the N-terminal ABCA3 fragment and the C-terminal ABCA3 fragment respectively comprise or consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23 or SEQ ID NO: 24 and 25, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25 and wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and at the 5 ’end of the C-split intein.In a more particular embodiment, the combination of polynucleotides according to the present disclosure encode a first fusion protein and a second fusion protein comprising amino acid sequences selected from the pairs consisting of: SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, SEQ ID NO: 42 and 43, SEQ ID NO: 44 and 45, SEQ ID NO: 46 and 47, SEQ ID NO: 48 and 49, and SEQ ID NO: 50 and 51; or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 34-51, preferably SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41 or SEQ ID NO: 42 and 43, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 38 to 43 and wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3’- end of the N-Split intein or at the 5’end of the C-split intein.In a more particular embodiment, the combination of polynucleotides according to the present disclosure encode a first fusion protein and a second fusion protein comprising amino acid sequences selected from the pairs consisting of: SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, SEQ ID NO: 42 and 43, SEQ ID NO: 44 and 45, SEQ ID NO: 46 and 47, SEQ ID NO: 48 and 49, and SEQ ID NO: 50 and 51; or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 34-51, preferably SEQ ID NO: 38 and 39, SEQ ID NO:40 and 41 or SEQ ID NO: 42 and 43, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 38 to 43 and wherein said first and second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3’- end of the N-Split intein and at the 5 ’end of the C-split intein.According to the present disclosure, said degron can be selected as non-limiting examples in the degrons listed in Table 5 below.Table 5: Examples of degrons and corresponding sequences.In a preferred embodiment, the degron comprised in the first and / or second fusion protein according to the present disclosure can be selected from the group consisting of: CL1 (SDD1) (SEQ ID NO: 52), Degl (SEQ ID NO: 53), PEST (SEQ ID NO: 54), DD1 (SEQ ID NO: 55), DD2 (SEQ ID NO: 56), DD3 (SEQ ID NO: 57), Ml (SEQ ID NO: 58), M2 (SEQ ID NO: 59), SopE (SEQ ID NO: 60), SopEl-78 (SEQ ID NO: 61), SopE 15-78 (SEQ ID NO: 62), SopE- 15-50 (SEQ ID NO: 63), L2 (SEQ ID NO: 64), L6 (SEQ ID NO: 65), L9 (SEQ ID NO: 66), LIO (SEQ ID NO: 67), Lil (SEQ ID NO: 68), L12 (SEQ ID NO: 69), LI 5 (SEQ ID NO: 70), L16 (SEQ ID NO: 71), M3 (SEQ ID NO: 72), M4 (SEQ ID NO: 73), M5 (SEQ ID NO: 74), V12 (SEQ ID NO: 75), DD4 (SEQ ID NO: 76), DD5 (SEQ ID NO: 77), DD6 (SEQ ID NO: 78), DD7 (SEQ ID NO: 79), T1 (SEQ ID NO: 80), T2 (SEQ ID NO: 81) and T3 (SEQ ID NO: 82), preferably DD1, DD3, PEST, SopE, V12, M4, L2, L9, more preferably SopE, L2, L9, M4 or VI 2 or any functional variant thereof that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of the excised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., ABCA3) preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 52 to 82.In a specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABCA3 protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker,wherein said first or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and / or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and / or at the 5’end of the C-split intein, and wherein the degron comprised in the first and / or second fusion protein according to the present disclosure is selected from the group consisting of SEQ ID NO: 52 to 82, preferably SEQ ID NO: 52, 54, 57, 60, 64, 66, 73 and 75, more preferably SEQ ID NO: 60, 64, 66, 73 or 75 or any functional variant thereof (e.g., that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of the excised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., ABCA3 protein), preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 52 to 82.In a more specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABC A3 protein and Cfa-N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ a Cfa-C-Split intein of SEQ ID NO: 2 or a Cfa-Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of ABC A3 protein, fused directly or indirectly via a linker, wherein said first and / or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and / or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and / or at the 5’end of the C-split intein, and wherein the degron comprised in the first and / or second fusion protein according to the present disclosure is selected from the group consisting of: preferably SEQ ID NO: 52, 54, 57, 60, 64, 66, 73 and 75, more preferably SEQ ID NO: 60, 64, 66, 73 or 75 or any functional variant thereof (e.g., that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of theexcised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., ABCA3 protein), preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 52 to 82.In a more specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABCA3 protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C- terminal fragment of ABC A3 protein from residue 1023 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C- terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABC A3 protein up to residue 1255 and the C- terminal fragment of ABCA3 protein from residue 1256 respectively, preferably- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal ABCA3 fragment and the C-terminal ABCA3 fragment respectively comprise or consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23 or SEQ ID NO: 24 and 25, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25 and wherein said first and / or second fusion protein further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and / or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and / or at the 5 ’end of the C-split intein, and wherein the degron comprised in the first and / or second fusion protein according to the present disclosure is selected from the group consisting of: SEQ ID NO: 52 to 82, preferably SEQ ID NO: 52, 54, 57, 60, 64, 66, 73 and 75, more preferably SEQ ID NO: 60, 64, 66, 73 or 75 or any functional variant thereof (e.g., that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of the excised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., ABCA3 protein), preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 52 to 82.In a more specific embodiment, the combination of polynucleotides according to the present disclosure comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABC A3 protein and Cfa-N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, anda second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a Cfa-C-Split intein of SEQ ID NO: 2 or a Cfa-Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of ABC A3 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 935 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C- terminal fragment of ABC A3 protein from residue 1023 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C- terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABC A3 protein up to residue 1255 and the C- terminal fragment of ABCA3 protein from residue 1256 respectively, preferably- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal ABCA3 fragment and the C-terminal ABCA3 fragment respectively comprise or consist of amino acid sequences selected from the pairs consisting of:SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23 or SEQ ID NO: 24 and 25, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25 and wherein said first and / or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein and / or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and / or at the 5 ’end of the C-split intein, and wherein the degron comprised in the first and / or second fusion protein according to the present disclosure is selected from the group consisting of: SEQ ID NO: 52-82, preferably SEQ ID NO: 52, 54, 57, 60, 64, 66, 73 and 75, more preferably SEQ ID NO: 60, 64, 66, 73 or 75 or any functional variant thereof (e.g., that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of the excised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., ABCA3 protein), preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 52 to 82.In a more specific embodiment, the combination of polynucleotides according to the present disclosure encode a first fusion protein and a second fusion protein comprising amino acid sequences selected from the pairs consisting of: SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, SEQ ID NO: 42 and 43, SEQ ID NO: 44 and 45, SEQ ID NO: 46 and 47, SEQ ID NO: 48 and 49, and SEQ ID NO: 50 and 51; or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 34-51, preferably SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41 or SEQ ID NO: 42 and 43, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 38 to 43 and wherein said first and / or second fusion proteins further comprises a degron, preferably fused directly or indirectly via a linker to the N-split intein or C-split intein comprised in the first and / or second fusion proteins, respectively, more preferably said degron is located at the 3’-end of the N-Split intein and / or at the 5’end of the C-split intein, and wherein the degron comprised in the first and / or second fusion protein according to the presentdisclosure is selected from the group consisting of: SEQ ID NO: 52-82, preferably SEQ ID NO: 52, 54, 57, 60, 64, 66, 73 and 75, more preferably SEQ ID NO: 60, 64, 66, 73 or 75 or any functional variant thereof (e.g., that induces degradation of the fusion protein comprising the protein of interest, the intein and the degron (i.e., starting material), and also induces degradation of the excised intein fused to the degron, preferably while maintaining the reconstitution of the protein of interest (i.e., ABCA3 protein), preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID Nos: 52 to 82.The functional variant of the degron as described above induces degradation of the excised intein that is fused to the degron, and also of the starting material fusion protein comprising the protein of interest, the intein and the degron and preferably does not interfere with the reconstitution of the protein of interest (i.e., ABCA3 protein).The degradation of the starting material consisting of the fusion protein comprising the protein of interest, the intein and the degron and the degradation of the excised intein that is fused to the degron can be tested according to Example 3 of WO2021181447, in particularly said degron is fused to a first fusion protein comprising a N-Split intein and a N-terminal fragment of a protein, said degron being fused to the 3’ end of N-Split intein and / or to a second fusion protein comprising a C-Split intein and a C-terminal fragment of a protein, said degron being fused to the 5 ’-end of the C-Split intein of a protein. The polynucleotides encoding said first and second protein is transfected in a cell, and the amount of starting material and excised intein is determined for example by Western blot. The degron induces degradation of starting material (i.e., the fusion protein comprising the protein of interest, the intein and the degron) and excised intein(s) when the amount of the starting material and excised intein is lower than the starting material and excised intein amount in a cell transfected with fusion proteins without degrons, preferably when the amount of intein is at least 1.2, 1.3, 1.4, 1.5, 1.8 or 2.0 fold lower than the starting material and intein amount in a cell transfected with fusion proteins without degrons. The expression level of reconstituted protein may be determined by any suitable methods known by skilled persons. The quantity of the reconstituted protein may be measured, for example, by semi-quantitative Western blots, enzyme-labelled and mediated immunoassays, such as ELISAs, biotin / avidin type assays, radioimmunoassay, immunoelectrophoresis, mass spectrometry, or immunoprecipitation or by protein or antibody arrays.In some embodiments, each first and second polynucleotides according to the present disclosure encoding the first and second fusion proteins, respectively, as described above may be a nucleic acid construct.In a particular embodiment said first and second polynucleotides may be an optimized sequence encoding the first and second fusion proteins. The term "codon optimized" means that a codon that expresses a bias for human (i.e. is common in human genes but uncommon in other mammalian genes or non-mammalian genes) is changed to a synonymous codon (a codon that codes for the same amino acid) that does not express a bias for human. Thus, the change in codon does not result in any amino acid change in the encoded protein.The term “nucleic acid construct” as used herein refers to a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleic acid molecule, either single- or double-stranded, which has been modified to contain segments of nucleic acids sequences, which are combined and juxtaposed in a manner, which would not otherwise exist in nature. A nucleic acid construct usually is a “vector”, i.e., a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell.Said nucleic acid construct comprises one or more control sequence required for expression of said coding sequence. Generally, the nucleic acid construct comprises a coding sequence and regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence that are required for expression of the selected gene product. Thus, a nucleic acid construct typically comprises a promoter sequence, a coding sequence and a 3' untranslated region that usually contains a polyadenylation site and / or transcription terminator. In a preferred embodiment, said polyadenylation site is a bovine growth hormone polyadenylation signal (bGH), SV401atepA, SV40flpA and / or a synthetic polyadenylation signal (SynpA).The nucleic acid construct may also comprise additional regulatory elements such as, for example, enhancer sequences, a polylinker sequence facilitating the insertion of a DNA fragment within a vector and / or splicing signal sequences.According to a preferred embodiment, said nucleic acid construct may comprise a SV40 intron.In one embodiment, the polynucleotide or nucleic acid construct according to the present disclosure comprises a promoter. Said promoter initiates transgene expression upon introduction into a host cell.In a preferred embodiment, the promoter according to the present disclosure can be selected from the group consisting of: Cytomegalovirus (CMV) promoter, chimeric reduced version of the CMV and chicken beta-actin (CEBA) promoter, human phosphoglycerate kinase (hPGK) promoter, chimeric CMV enhanced and human phosphoglycerate kinase (ePGK) promoter, EFl alpha promoter, rous sarcoma virus (RSV) promoter and human surfactant protein B (SPB) promoter. However, any suitable promoter known in the art may be used. As used herein, the term "promoter" refers to a regulatory element that directs the transcription of a nucleic acid to which it is operably linked. A promoter can regulate both rate and efficiency of transcription of an operably linked nucleic acid. A promoter may also be operably linked to other regulatory elements which enhance ("enhancers") or repress ("repressors") promoter-dependent transcription of a nucleic acid. These regulatory elements include, without limitation, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter, including e.g., attenuators, enhancers, and silencers. The promoter is located near the transcription start site of the gene or coding sequence to which it is operably linked, on the same strand and upstream of the DNA sequence (towards the 5' region of the sense strand). A promoter can be about 100-3000 base pairs long. Positions in a promoter are designated relative to the transcriptional start site for a particular gene (i.e., positions upstream are negative numbers counting back from -1, for example -100 is a position 100 base pairs upstream).As used herein, the term “operably linked” refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically but not necessarily contiguous; where it is necessary to join two protein encoding regions, they are contiguous and in reading frame.In a preferred embodiment, each polynucleotide or nucleic acid construct according to the present disclosure may be comprised in an expression vector.As used herein, the term "expression vector" refers to a nucleic acid molecule used as a vehicle to transfer genetic material, and in particular to deliver a nucleic acid into a host cell, either in vitro or in vivo. Expression vector also refers to a nucleic acid molecule capable of effectingexpression of a gene (transgene) in host cells or host organisms compatible with such sequences. Expression vectors typically include at least suitable transcription regulatory sequences and optionally 3 ’-transcription termination signals.Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements able to respond to a precise inductive signal (endogenous or chimeric transcription factors) or specific for certain cells, organs or tissues. Vectors include, but are not limited to, plasmids, phasmids, cosmids, transposable elements, viruses, and artificial chromosomes (e.g., YACs).Preferably, the vectors of the disclosure is vectors suitable for use in gene or cell therapy, and in particular is suitable to target lung cells.In some embodiments, the expression vector is a viral vector, such as vectors derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV or SNV, lentiviral vectors (e.g. derived from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) or equine infectious anemia virus (EIAV)), adenoviral (Ad) vectors, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors.As is known in the art, depending on the specific viral vector considered for use, suitable sequences should be introduced in the vector of the disclosure for obtaining a functional viral vector, such as AAV ITRs for an AAV vector, or LTRs for lentiviral vectors. In a particular embodiment, said vector is an AAV vector.AAV has arisen considerable interest as a potential vector for human gene therapy. Among the favorable properties of the virus are its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and the wide range of cell lines derived from different tissues that can be infected. The AAV genome is composed of a linear, single-stranded DNA molecule which contains 4681 bases (Berns and Bohenzky, 1987, Advances in Virus Research (Academic Press, Inc.) 32:243-307). The genome includes inverted terminal repeats (ITRs) at each end, which function in cis as origins of DNA replication and as packaging signals for the virus. The ITRs are approximately 145 bp in length. The internal non-repeated portion of the genome includes two large open reading frames, known as the AAV rep and cap genes, respectively. These genes code for the viral proteins involved in replication and packaging ofthe virion. In particular, at least four viral proteins are synthesized from the AAV rep gene, Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight. The AAV cap gene encodes at least three proteins, VP1, VP2 and VP3. For a detailed description of the AAV genome, see, e.g., Muzyczka, N. 1992 Current Topics in Microbiol, and Immunol. 158:97- 129.Thus, in one embodiment, the polynucleotides, nucleic acid constructs or expression vectors according to the present disclosure thereof further comprises a 5’ITR and a 3TTR sequences, preferably a 5’ITR and a 3’ ITR sequences of an adeno-associated virus.As used herein the term “inverted terminal repeat (ITR)” refers to a nucleotide sequence located at the 5’-end (5’ITR) and a nucleotide sequence located at the 3’-end (3’ITR) of a virus, that contain palindromic sequences and that can fold over to form T-shaped hairpin structures that function as primers during initiation of DNA replication. They are also needed for viral genome integration into the host genome; for the rescue from the host genome; and for the encapsidation of viral nucleic acid into mature virions. The ITRs are required in cis for the vector genome replication and its packaging into the viral particles.In one embodiment, the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure further comprises a 5’ITR and a 3’ITR of an AAV, preferably of a serotype AAV2.The polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins as described above may be packaged into a virus capsid to generate a "viral particle", also named “viral vector particle”. In a particular embodiment, the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure is packaged into an AAV-derived capsids to generate an "adeno- associated viral particles" or "AAV particles". The present disclosure relates to viral particles comprising the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure and preferably comprising capsid proteins of adeno-associated virus.The construction of recombinant AAV viral particles is generally known in the art and has been described for instance in US 5,173,414 and US5,139,941; WO 92 / 01070, WO 93 / 03769, Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (ColdSpring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533- 539; Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793-801.Thus, in AAV viral particle according to the present disclosure, the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins as described above including ITR(s) of a given AAV serotype can be packaged, for example, into: a) a viral particle constituted of capsid proteins derived from the same or different AAV serotype [e.g. AAV2 ITRs and AAV5 capsid proteins; AAV2 ITRs and AAV8 capsid proteins; AAV2 ITRs and Anc80 capsid proteins; AAV2 ITRs and AAV9 capsid proteins]; b) a mosaic viral particle constituted of a mixture of capsid proteins from different AAV serotypes or mutants [e.g. AAV2 ITRs with AAV1 and AAV5 capsid proteins]; c) a chimeric viral particle constituted of capsid proteins that have been truncated by domain swapping between different AAV serotypes or variants [e.g. AAV2 ITRs with AAV5 capsid proteins with AAV3 domains].The skilled person will appreciate that the AAV viral particle for use according to the present disclosure may comprise capsid proteins from any AAV serotype including AAV1, AAV2, AAV3 (including types 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, synthetic AAV variants such as NP40, NP59, NP84 (Paulk et al. Mol then 2018.26(l):289-303), LK03 (Wang L et al. Mol Then 2015. 23(12): 1877-87), AAV3-ST (Vercauteren et al. Mol Then 2016.24(6): 1042-1049), Anc80 (Zinn E et al., Cell Rep. 2015;12(6): 1056-68), AAVrhlO and any other AAV serotype now known or later discovered.Thus, in a further aspect, the present disclosure relates to a viral particle comprising the polynucleotides, nucleic acid constructs or expression vectors comprising nucleic acid sequences encoding the first and second fusion proteins as described above and preferably comprising capsid proteins of adeno-associated virus such as capsid proteins from AAV1, AAV2, AAV4, AAV5, AAV6, AAV6.1, AAV6.2FF, AAV8, AAV9 or AAVRhlO.Therapeutic useAccording to the present disclosure, the combination of polynucleotides, nucleic acid constructs, expression vectors or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins as described above is administered in a subject in need thereof for use in gene therapy, preferably for the treatment of ABC A3 -associated disease.As used herein, "gene therapy" refers to the administration of a gene-therapy vector to treat a disease caused by a change in the subject DNA sequence, in particular a disease caused by a mutation in at least one gene (i.e., genetic disease) such as ABCA3 into a subject in need thereof.As used herein, the term "treatment", "treat" or "treating" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease. In certain embodiments, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease. According to the present disclosure, examples of symptoms associated with ABC A3 -associated disease are symptoms associated with surfactant dysfunction, in particular breathing problems, rapid breathing (tachypnea), low concentrations of oxygen in the blood (hypoxemia); and an inability to grow or gain weight at the expected rate (failure to thrive).The term “subject” or “patient” as used herein, refers to mammals. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, humans, nonhuman primates such as apes, chimpanzees, monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like. In particular embodiment, said subject is a human patient, preferably neonates, infant or adult human patient.The combination of polynucleotides, nucleic acid constructs, expression vectors or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure will be typically included in a pharmaceutical composition or medicament, optionally in combination with a pharmaceutical carrier, diluent and / or adjuvant. Such composition or medicinal product comprises the product of the disclosure in an effective amount, sufficient to provide a desired therapeutic effect, and a pharmaceutically acceptable carrier or excipient.As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency or recognized pharmacopeia such as European Pharmacopeia, for use in animals and / or humans. The term "excipient" refers to a diluent, adjuvant, carrier, or vehicle with which the therapeutic agent is administered. As is well known in the art, pharmaceutically acceptable excipients are relatively inert substances that facilitate administration of a pharmacologically effective substance and can be supplied as liquid solutions or suspensions, as emulsions, or as solid formssuitable for dissolution or suspension in liquid prior to use. For example, an excipient can give form or consistency, or act as a diluent. Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolality, encapsulating agents, pH buffering substances, and buffers.In one embodiment, the pharmaceutical composition is a parenteral pharmaceutical composition, including a composition suitable for intravenous, intraarterial, intramuscular, intranasal, or intratracheal administration. These pharmaceutical compositions are exemplary only and do not limit the pharmaceutical compositions suitable for other parenteral and non- parenteral administration routes. The pharmaceutical compositions described herein can be packaged in single unit dosage or in multidosage forms.Surfactant dysfunction due to mutations in ABCA3 gene (also called ABC A3 -related disease) can cause severe, often fatal breathing problems in newborns or gradual onset of milder breathing problems in children or adults. More than 100 ABCA3 gene mutations that cause surfactant dysfunction have been identified. ABCA3 gene mutations result in abnormal surfactant composition and function. The loss of functional surfactant raises surface tension in the alveoli, causing difficulty breathing and collapse of the lungs. Mutations that eliminate ABCA3 protein function cause severe forms of surfactant dysfunction, and mutations that leave some residual ABCA3 activity cause milder forms of the condition.The signs and symptoms of surfactant dysfunction can vary in severity. The most severe form of this condition causes respiratory distress syndrome (RDS) in newborns. Affected babies have extreme difficulty breathing and are unable to get enough oxygen. The lack of oxygen can damage the baby's brain and other organs. This syndrome leads to respiratory failure, and most babies with this form of the condition do not survive more than a few months.Less severe forms of surfactant dysfunction cause gradual onset of breathing problems in children or adults. Signs and symptoms of these milder forms are abnormally rapid breathing (tachypnea); low concentrations of oxygen in the blood (hypoxemia); and an inability to grow or gain weight at the expected rate (failure to thrive).In a more preferred embodiment, the present disclosure relates to the combination of polynucleotides, nucleic acid constructs, expression vectors or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins as described above or pharmaceutical composition thereof for use in the treatment of a ABC A3 -associated disease, preferably surfactant dysfunction, more preferably selected from the group consisting of:respiratory distress syndrome (RDS), interstitial lung disease, or pulmonary fibrosis, preferably neonate RDS, childhood interstitial lung disease (chiLD) or adult pulmonary fibrosis.The present disclosure also relates to the use of the combination of polynucleotides, nucleic acid constructs, expression vectors or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins as described above or pharmaceutical composition thereof for the manufacture of a medicament for treating an ABC A3 -associated disease (i.e., surfactant dysfunction).The disclosure also provides a method for treating a ABC A3 -associated disease (i.e., surfactant dysfunction) as described above in a patient in need thereof comprising administering to said patient a therapeutically effective amount of the polynucleotides, nucleic acid constructs, expression vectors or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins as described above or pharmaceutical composition thereof.As used herein a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the products of the disclosure may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the product or pharmaceutical composition to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also typically one in which any toxic or detrimental effect of the product or pharmaceutical composition is outweighed by the therapeutically beneficial effects. According to the present disclosure, a therapeutically effective amount allows to reduce for example the breathing problem or hypoxemia.According to the present disclosure, said combination of polynucleotides can be administered into the subject in a single formulation or as separate formulations simultaneously, sequentially or separately. Such administration encompasses co-administration of the first and second polynucleotides in a substantially simultaneous manner, such as in a single formulation having a fixed ratio of the polynucleotides or in separate formulations for each polynucleotide. In addition, such administration also encompasses the use of each polynucleotide in a sequential or separate manner, either at approximately the same time or at different times. Regardless of whether the polynucleotides are administered as a single formulation or in separate formulations, the first and second polynucleotides are administered to the same subject as partof the same course of therapy. In any case, the treatment regimen will provide beneficial effects in treating the diseases described herein.In one embodiment, the combination of polynucleotides, nucleic acid constructs, expression vectors or viral particles comprising nucleic acid sequences encoding the first and second fusion proteins according to the present disclosure for its therapeutic use is administered to the subject or patient by a parenteral route, in particularly by intravenous, intraarterial, intramuscular, intranasal, or intratracheal route.The amount of product of the disclosure that is administered to the subject or patient may vary depending on the particular circumstances of the individual subject or patient including, age, sex, and weight of the individual; the nature and stage of the disease, the aggressiveness of the disease; the route of administration; and / or concomitant medication that has been prescribed to the subject or patient. Dosage regimens may be adjusted to provide the optimum therapeutic response.For any particular subject, specific dosage regimens may be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions. The dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.KitIn another aspect, the disclosure further relates to a kit, preferably for use in the treatment of ABC A3 -associated disease as described above, preferably surfactant disease, more preferably selected from the group consisting of: respiratory distress syndrome (RDS), interstitial lung disease, or pulmonary fibrosis, preferably neonate RDS, childhood interstitial lung disease (chiLD) or adult pulmonary fibrosis, said kit comprising a combination of polynucleotides as described above, comprising: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABCA3 protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker,optionally wherein said first and / or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and / or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3’-end of the N-Split intein and / or at the 5’end of the C-split intein, again more preferably selected from the group consisting of SEQ ID NO: 52 to 82 or any functional variant thereof that induces degradation of the protein that contains the fragment, preferably having at least at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 52 to 82.In a more specific embodiment, the kit comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABC A3 protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : Cfa C-split intein of SEQ ID NO: 2 or Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of ABC A3 protein, fused directly or indirectly via a linker, and optionally wherein said first and / or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and / or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3’-end of the N-Split intein and / or at the 5’end of the C-split intein, again more preferably selected from the group consisting of SEQ ID NO: 52 to 82 or any functional variant thereof that induces degradation of the protein that contains the fragment, preferably having at least at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 52 to 82.In a more specific embodiment, the kit comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABCA3 protein, N-split intein, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker,wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C- terminal fragment of ABC A3 protein from residue 1023 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C- terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABC A3 protein up to residue 1255 and the C- terminal fragment of ABCA3 protein from residue 1256 respectively, preferably- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal ABCA3 fragment and the C-terminal ABCA3 fragment respectively comprise or consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23 or SEQ ID NO: 24 and 25, orany functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25 and optionally wherein said first and / or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and / or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and / or at the 5 ’end of the C-split intein, again more preferably selected from the group consisting of SEQ ID NO: 52 to 82 or any functional variant thereof that induces degradation of the protein that contains the fragment, preferably having at least at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 52 to 82.In a more specific embodiment, the kit comprises: a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’: a N- terminal fragment of ABC A3 protein and Cfa N-split intein of SEQ ID NO: 1 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 1, fused directly or indirectly via a linker, and a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’: aCfa C-split intein of SEQ ID NO: 2 or Cfa Cmut-split intein of SEQ ID NO: 13 or any functional variant thereof having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 or 13 and a C-terminal fragment of ABC A3 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively,the N-terminal fragment of ABCA3 protein up to residue 1022 and the C-terminal fragment of ABC A3 protein from residue 1023 respectively, the N-terminal fragment of ABCA3 protein up to residue 1065 and the C-terminal fragment of ABC A3 protein from residue 1066 respectively, or the N-terminal fragment of ABCA3 protein up to residue 1255 and the C-terminal fragment of ABCA3 protein from residue 1256 respectively; preferably- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, wherein said residue is numbered according to SEQ ID NO: 15, preferably wherein the N-terminal ABCA3 fragment and the C-terminal ABCA3 fragment respectively comprise or consist of amino acid sequences selected from the pairs consisting of: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23 or SEQ ID NO: 24 and 25, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25, optionally wherein said first and / or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and / or C-split intein comprised in the first and second fusion proteins, respectively, more preferably said degron is located at the 3 ’-end of the N-Split intein and / or at the 5 ’end of the C-split intein, again more preferably selected from the group consisting of SEQ ID NO: 52 to 82 or any functional variant thereof that induces degradation of the protein that contains the fragment, preferably having at least at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 52 to 82.In a more preferred embodiment, the kit comprises a combination of polynucleotides encoding a first fusion protein and a second fusion protein comprising amino acid sequences selected from the groups of pairs consisting of SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, SEQ ID NO: 42 and 43, SEQ ID NO: 44 and 45, SEQID NO: 46 and 47, SEQ ID NO: 48 and 49, and SEQ ID NO: 50 and 51; or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 34-51, preferably SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41 or SEQ ID NO: 42 and 43, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 38 to 43, and optionally wherein said first and / or second fusion proteins further comprise a degron, preferably fused directly or indirectly via a linker to the N-split intein and / or C-split intein comprised in the first and / or second fusion proteins, respectively, more preferably said degron is located at the 3’-end of the N-Split intein and / or at the 5’end of the C-split intein, again more preferably selected from the group consisting of SEQ ID NO: 52 to 82 or any functional variant thereof that induces degradation of the protein that contains the fragment, preferably having at least at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 52 to 82.The kit may include instructions or packaging materials that describe how to administer the polynucleotides contained within the kit to a patient.Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In certain embodiments, the kits may include one or more ampoules or syringes that contain the products of the invention in a suitable liquid or solution form.The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.EXAMPLES1. Materials and Methods1.1 Materials:Oligonucleotides were purchased from Eurofins genomics (Ebersberg, Germany). Synthetic genes were purchased from GENEWIZ (South Plainfield, NJ). Pfu Ultra fusion polymerase for cloning and all restriction enzymes was purchased from Thermofisher Scientific (Pittsburgh, PA). High-competency cells used for cloning were generated from XLIO-Gold chemically competent E. coli. HEK293T cells were purchased from ATCC (Manassas, VA). DNApurification kits were purchased from Qiagen (Thermofisher Scientific (Pittsburgh, PA). All plasmids were sequenced by Macrogen (Seoul, Korea). Luria Bertani (LB) media, and all buffering salts were purchased from Thermofisher Scientific (Pittsburgh, PA). Coomassie brilliant blue, phenylmethane sulfonyl fluoride, fetal bovine serum and asolectin from soybean were purchased from Sigma-Aldrich (San Luis MI). EDTA-free complete protease inhibitors were purchased from Roche (Branchburg, NJ). Lipofectamine 2000 transfection reagent, DMEM high glucose GlutaMAX supplement, RPMI 1640 medium GlutaMAX supplement, RIPA lysis and extraction buffer, BCA protein assay kit, MES-SDS running buffer, pre-stained protein ladder and SDS-PAGE (Bis-tris and Tris-acetate gels) were purchased from Thermofisher Scientific (Pittsburgh, PA). The primary anti-flag tag mouse monoclonal antibody and anti-tubulin rabbit polyclonal antibody were purchased from Invitrogen (Carlsbad, CA). The secondary goat anti-mouse IgG (H+L) highly cross-adsorbed antibody alexa fluor plus 680 and goat anti-rabbit IgG (H+L) secondary antibody dylight 800 4X PEG were purchased from Invitrogen (Carlsbad, CA). Dodecyl maltoside (D310) and cholesteryl hemisuccinate (CH210) solution were purchased from Anatrace (Maumme, OH).1.2 Equipment:Gels and Western-blots were imaged with a LI-COR Odyssey Infrared Imager. Cell lysis was carried out using a S-450D Branson digital sonifier.1.3 Cloning of Recombinant DNASynthetic genes to prepare constructs were purchased and introduced into pEGFP-Nl expression vectors using Kpnl and Notl restriction enzymes :- Site 1 : ABCA3-l-767-CfaN-3FT (SEQ ID NO: 38), ABC A3- 768-1704-CfaCmut-3FT (SEQ ID NO: 39),- Site2: ABCA3-l-743-CfaN-3FT (SEQ ID NO: 36), ABC A3- 744-1704-CfaCmut-3FT (SEQ ID NO: 37),- Site 3: ABCA3-l-904-CfaN-3FT (SEQ ID NO: 42), ABCA3- 905-1704-CfaCmut-3FT (SEQ ID NO: 43),- Site 4: ABCA3-l-874-CfaN-3FT (SEQ ID NO: 40), ABCA3- 875-1704-CfaCmut-3FT (SEQ ID NO: 41),- Site 5: ABCA3-l-1022-CfaN-3FT (SEQ ID NO: 46), ABCA3 -1023-1704-CfaCmut- 3FT (SEQ ID NO: 47),- Site 6: ABCA3-l-1065-CfaN-3FT (SEQ ID NO: 48), ABCA3- 1066-1704-CfaCmut- 3FT (SEQ ID NO: 49),- Site 7: ABCA3-l-935-CfaN-3FT (SEQ ID NO: 44), ABCA3-936-1704-CfaCmut-3FT (SEQ ID NO: 45),1.4 Transfection of intein plasmids in HEK293T cells:HEK293T cells were maintained in DMEM with 10% FBS and antibiotics at 37°C in a 5% CO2 atmosphere. Cells were co-transfected at around 80% confluence using lipofectamine 2000 and 1.25 pg of each plasmid in 6-well plate format. For the experiments where the plasmid encoded the full-length gene was used, a scramble plasmid was co-transfected with the full-length plasmid to achieve the same amount of DNA transfected when two intein plasmid were used. Cells were harvested after 48 h post-transfection the amount of protein was analyzed by Western blot.1.5 Western blot analysis:ABC A3 samples (HEK293T) were lysed in dodecyl maltoside (D310) and cholesteryl hemisuccinate (CH210) solution in PBS (1 :1) supplemented with protease inhibitors and 1 mM phenylmethyl sulfonyl. After lysis, ABCA3 samples were quantified by BCA protein assay kit. Samples with 25 pg of total protein were denatured at 37°C for 15 minutes in IX Laemmli sample buffer containing 2.5 mg / ml of asolectin. Lysates were separated by 3-8% Tris-acetate SDS-PAGE gels for 1.5 h at 150V. The antibodies used for immuno-blotting were either antiflag tag to detect the ABCA3 protein as well as the different fragments and anti-P-tubulin as loading control. The quantification of ABCA3 bands detected by Western blot was performed using LLCOR Odyssey Infrared Imager.2. Results and discussionATP -binding cassette sub-family A member 3 (ABCA3) is a protein encoded by the ABCA3 gene in humans. The membrane-associated protein produced by this gene belongs to the ATP- binding cassette (ABC) transporter superfamily, which is responsible for transporting a variety of molecules across extracellular and intracellular membranes. Specifically, ABCA3 is part of the ABC1 subfamily, the only major ABC subfamily that is exclusively found in multicellular eukaryotes. Although the ABCA3 transporter is found in many tissues including stomach, intestine, liver, kidney, and brain, it is highly expressed in the lung in an AT2-cell specific manner. In AT2 cells, ABCA3 is preferentially trafficked to the limiting membrane of thelaminar bodies (LBs) and thus is optimally positioned to promote lipid transport across this membrane barrier, playing a critical role in the regulation of pulmonary surfactant homeostasis.The large size of the ABCA3 protein (1704 amino acids) exceeds the packaging capacity of a single adeno-associated virus (AAV). To address this challenge, the inventors set out to determine whether split intein-mediated protein trans-splicing can be used to efficiently reconstitute phospholipid-transporting ATPase ABCA3 protein encoded by ABCA3 gene in a cell.2.1 Splicing efficiency of ABC A3 protein at position 768Constructs were cloned and tested as shown above. Specifically, ABCA3-(l-767)-CfaN, CfaCmut- ABCA3 (768-1704) constructs were used to evaluate splicing ABCA3. Sites were selected based on the topological structure of ABCA3 and taking into consideration the presence of folded domains. Sites were selected outside of well-defined folded domains. Briefly, constructs were co-transfected into HEK293T cells. Cells were lysed and protein reconstitution yields determined by Western Blot. Results show that ABCA3 is efficiently reconstituted by trans-splicing with this construct (Figure 1).2.2 Selection of optimal split positions for high ABCA3 splicing efficiencyThe ABCA3 sequence was analyzed to identify appropriate positions for splitting the protein, allowing reassembly through protein splicing. Notably, several cysteine residues are present in the amino acid sequence, which may influence the splicing and functionality of the reconstituted proteinThis design ensured that each fragment could be individually packaged into separate AAV vectors while maintaining the potential for functional reconstitution within target cells. Splitting sites were strategically designed in various regions of the protein to evaluate the efficiency of reconstitution based on their localization.To assess the ability of the engineered Cfa intein to mediate the reconstitution of ABCA3 protein via protein trans-splicing (PTS), a study was performed using seven different split sites. Constructs encoding split versions of ABCA3 fused to the Cfa intein, alongside a full-length ABCA3 vector, were generated. These constructs, referred to as AAXN and AAXC (where X indicates the corresponding split site), were cloned into expression vectors for further analysis.First, the inventors transfected HEK293T cells with either the full-length ABCA3 plasmid (AA) or co-transfected with plasmids encoding the split ABCA3 (ABCA3-N-terminal fragment 1-767-N-intein and ABCA3-C-terminal fragment 768-1704-C-intein) corresponding to Site 1 (split position 767-768) (2 replicates were performed).Following transfection, cells were lysed, and protein expression levels were examined via western blot. Detection of ABC A3, both fragments or full-length protein, were carried out using a mouse anti-FLAG tag antibody and a rabbit anti-tubulin antibody was used as a loading control to confirm uniform protein loading across samples Negative controls for PTS, consisting of mutant AA1N-2 and AA1C-2 constructs, were also included in the experiment (Figure 2). Following site-directed mutagenesis of the N- and C-fragments from site 1 (AA1N and AA1C), the first cysteine of the intein N, which plays a critical role in the trans-splicing reaction (transthioesterification), was substituted with alanine (CIA), thereby eliminating the nucleophilic capacity required for transthioesterification. Simultaneously, the last asparagine of the C-fragment, essential for succinimide formation and subsequent S-N acyl transfer, was also mutated to alanine (N36A). By combining these mutated inteins, the inventors successfully inhibited trans-splicing activity, creating an ideal negative control.As shown in Figure 2, ABCA3 protein was successfully reconstituted through the PTS reaction following transfection with both plasmids, as indicated by the rectangle. The reconstituted ABCA3 exhibited the same molecular weight as the full-length ABCA3. In contrast, individual plasmids did not demonstrate ABCA3 reconstitution via the PTS reaction, consistent with the results from co-transfection experiments. Furthermore, in the negative control, the ABCA3-N and ABCA3-C complex could not be denatured, displaying a band with a higher molecular weight compared to the full-length or reconstituted protein.After successful completion of cloning from site 2 until 7 for all N- and C-fragments, the inventors transfected HEK293T cells with the full-length ABCA3 plasmid (AA) as positive control or co-transfected with plasmids encoding the different splitting sites of ABCA3 by performing two replicates (Figure 3 A and 3B).Unexpectedly, not all candidates showed efficient reconstitution rates. The optimal candidates demonstrating high reconstitution rates for the protein splicing reaction were sites 1, 3, and 4, with reconstitution efficiencies of 50%, 75%, and 75%, respectively. By contrary, sites 5, 6, and 7 displayed significantly lower yields of PTS-reconstituted ABCA3, approximately 25% (Figure 4).Unexpectedly, the ABCA3 protein reconstitution efficiency is very high, making split inteins a prime choice for therapeutic applications.

Claims

CLAIMS1. A combination of polynucleotides for use in the treatment of ABC A3 -associated disease in a subject in need thereof wherein the combination comprises:(i) a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’ : a N-terminal fragment of ABCA3 protein and N-split intein, fused directly or indirectly via a linker,(ii) a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker, wherein expression of first and second polynucleotides in said subject generates ABCA3 protein by protein splicing.

2. The combination for use according to claim 1 wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C- terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C- terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C- terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C- terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C- terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C- terminal fragment of ABC A3 protein from residue 936 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C- terminal fragment of ABC A3 protein from residue 1023 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C- terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABC A3 protein up to residue 1252 and the C- terminal fragment of ABCA3 protein from residue 1256 respectively, wherein said residue is numbered according to SEQ ID NO: 15.

3. The combination for use according to claim 2 wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, wherein said residue is numbered according to SEQ ID NO: 15.

4. The combination for use according to any one claims 1 to 3 wherein the first and second fusion proteins comprise amino acid sequences selected from any one of the following pairs: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23 or SEQ ID NO: 24 and 25, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25.

5. The combination for use according to any one of claims 1 to 4 wherein said ABC A3 protein is human ABCA3 protein, preferably comprising or consisting of SEQ ID NO: 15 or any functional variant thereof having at least 90% identity to SEQ ID NO: 15.

6. The combination of polynucleotides for use according to any one of claims 1 to 5 wherein said N-split intein is a N-Cfa-intein of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity to SEQ ID NO: 1; and said C-split intein is a C-Cfa intein of SEQ ID NO: 2 or any functional variant thereof having at least 90% identity to SEQ ID NO: 2.

7. The combination of polynucleotides for use according to claim 6 wherein amino acid residues 20 to 22 of SEQ ID NO: 2 are GEP, preferably wherein said C-split intein is C-Cfamut intein of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity to SEQ ID NO: 13.

8. The combination for use according to any one of claims 1 to 7 wherein the first and second fusion proteins comprise amino acid sequences selected from any one of the following pairs: SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, SEQ ID NO: 42 and 43, SEQ ID NO: 44 and 45, SEQ ID NO: 46 and 47, SEQ ID NO: 48 and 49, and SEQ ID NO: 50 and 51; or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 34-51, preferably SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41 or SEQ ID NO: 42 and 43, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 38 to 43.

9. The combination for use according to any one of claims 1 to 8 wherein the first fusion protein and / or second fusion protein further comprises a degron, preferably wherein: i) the first fusion protein further comprises a degron located at the 3 ’end of the N-split-intein, and / or ii) the second fusion protein further comprises a degron located at 5 ’end of the C-split-intein.

10. The combination for use of claim 9 wherein said degron is selected from the group consisting of: SEQ ID NO: 52 to 82 or any functional variant thereof having at least 90% identity to any one of sequences SEQ ID NO: 52 to 82.

11. The combination for use according to any one of claims 1 to 10, wherein each polynucleotide further comprises a promoter selected from the group consisting of: Cytomegalovirus (CMV) promoter, chimeric reduced version of the CMV and chicken beta-actin (CEBA) promoter, human phosphoglycerate kinase (hPGK) promoter, chimeric CMV enhanced and human phosphoglycerate kinase (ePGK)promoter, EFl alpha promoter, rous sarcoma virus (RSV) promoter, and human surfactant protein B (SPB) promoter..

12. The combination for use according to any one of claims 1 to 11, wherein each polynucleotide is comprised within an expression vector, preferably wherein said expression vector is a viral vector, preferably an adeno associated viral (AAV) vector, more preferably said AAV vector comprises capsid protein of AAV selected from the group consisting of 1, 2, 3, 4, 5, 6, 6.2, 6.2FF, 7, 8, 9 or RhlO, preferably AAV5, AAV6, AAV6.2, AAV6.2FF, AAV8 or AAV9.

13. The combination for use according to any one of claims 1 to 12 wherein said ABC A3 -associated disease is a surfactant disease, preferably selected from the group consisting of: respiratory distress syndrome (RDS), interstitial lung disease, or pulmonary fibrosis, more preferably neonate RDS, childhood interstitial lung disease (chiLD) or adult pulmonary fibrosis.

14. A kit comprising:- a first polynucleotide encoding a first fusion protein comprising from 5’ to 3’ : a N-terminal fragment of ABCA3 protein and N-split intein, fused directly or indirectly via a linker,- a second polynucleotide encoding a second fusion protein comprising from 5’ to 3’ : a C-split intein and a C-terminal fragment of ABCA3 protein, fused directly or indirectly via a linker, wherein the first and second fusion proteins comprise:- the N-terminal fragment of ABCA3 protein up to residue 447 and the C-terminal fragment of ABCA3 protein from residue 448 respectively,- the N-terminal fragment of ABCA3 protein up to residue 743 and the C-terminal fragment of ABCA3 protein from residue 744 respectively,- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively,- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively,- the N-terminal fragment of ABC A3 protein up to residue 935 and the C-terminal fragment of ABC A3 protein from residue 936 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1022 and the C-terminal fragment of ABC A3 protein from residue 1023 respectively,- the N-terminal fragment of ABC A3 protein up to residue 1065 and the C-terminal fragment of ABC A3 protein from residue 1066 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 1255 and the C-terminal fragment of ABCA3 protein from residue 1256 respectively, preferably- the N-terminal fragment of ABCA3 protein up to residue 767 and the C-terminal fragment of ABCA3 protein from residue 768 respectively,- the N-terminal fragment of ABCA3 protein up to residue 874 and the C-terminal fragment of ABC A3 protein from residue 875 respectively, or- the N-terminal fragment of ABCA3 protein up to residue 904 and the C-terminal fragment of ABCA3 protein from residue 905 respectively, wherein said residue is numbered according to SEQ ID NO: 15.

15. The kit of claim 14 wherein the first and second fusion proteins comprise amino acid sequences selected from any one of the following pairs: SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23, SEQ ID NO: 24 and 25, SEQ ID NO: 26 and 27, SEQ ID NO: 28 and 29, SEQ ID NO: 30 and 31, and SEQ ID NO: 32 and 33 or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 16 to 33, preferably SEQ ID NO: 20 and 21, SEQ ID NO: 22 and 23 or SEQ ID NO: 24 and 25, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 20 to 25.

16. The kit of claim 14 or 15 wherein said N-split intein is a N-Cfa-intein of SEQ ID NO: 1 or any functional variant thereof having at least 90% identity to SEQ ID NO: 1; and said C-split intein is a C-Cfa intein of SEQ ID NO: 2 or any functional variant thereof having at least 90% identity to SEQ ID NO: 2, preferably wherein aminoacid residues 20 to 22 of SEQ ID NO: 2 are GEP, more preferably wherein said C- split intein is C-Cfamut intein of SEQ ID NO: 13 or any functional variant thereof having at least 90% identity to SEQ ID NO: 13.

17. The kit according to anyone of claims 14 to 16 wherein the first and second fusion proteins comprise amino acid sequences selected from any one of the following pairs: SEQ ID NO: 34 and 35, SEQ ID NO: 36 and 37, SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41, SEQ ID NO: 42 and 43, SEQ ID NO: 44 and 45, SEQ ID NO: 46 and 47, SEQ ID NO: 48 and 49 and SEQ ID NO: 50 and 51; or any functional variant thereof, preferably having at least 70, 75, 80, 85, 90, 95, 98 or 99% identity to any one of sequences SEQ ID NO: 34-51, preferably SEQ ID NO: 38 and 39, SEQ ID NO: 40 and 41 or SEQ ID NO: 42 and 43, or any functional variant thereof, preferably having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to any one of sequences SEQ ID NO: 38 to 43.

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