Polynucleotide construct capable of expressing LMNA and use thereof in prevention and treatment of dcm

Abstract

The present invention belongs to the field of genetic engineering, and specifically relates to a polynucleotide construct capable of expressing LMNA and the use thereof in the prevention and treatment of DCM. A synthetic intron comprises: I) a nucleotide sequence as set forth in SEQ ID No. 8, II) a nucleotide sequence having at least one mutation at positions 1-8 of the sequence as set forth in SEQ ID No. 8, or a nucleotide sequence having at least 90% homology with the sequences as set forth in I) or II). By using the synthetic intron, two proteins can be simultaneously expressed by means of alternative splicing, and mutations in splicing signals can regulate the ratio of the two proteins or even enable the individual expression of either protein. Compared with the existing gene therapy method for dilated cardiomyopathy caused by LMNA mutations, the use of an expression vector simultaneously expressing Lamin A and Lamin C proteins has a stronger rescue effect.

Description

Polynucleotide constructs expressing LMNA and their application in the prevention and treatment of DCM

[0001] Cross-reference to related applications

[0002] This invention claims priority to Chinese Patent Application No. 2024118977236, filed on December 20, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of genetic engineering, specifically relating to polynucleotide constructs that can express LMNA and their application in the prevention and treatment of DCM. Background Technology

[0004] The LMNA gene is located on human chromosome 1. LMNA mutations are inherited in an autosomal dominant pattern, with the main mutation types being missense and frameshift mutations. Diseases caused by these mutations are called laminopathies. Clinically, laminopathies can manifest as muscular dystrophy, limb-girdle muscular dystrophy, peripheral neuropathy, premature aging, and dilated cardiomyopathy (DCM). Many patients with laminopathies exhibit clinical features of abnormal cardiac function. In patients carrying LMNA mutations, the penetrance of cardiac-related phenotypes is 7% in those under 20 years of age, 66% between 20 and 39 years of age, 86% between 40 and 59 years of age, and nearly 100% after age 60 [NE Hasselberg et al., Eur Heart J 7; 39(10):835-860(2018)]. Patients with LMNA mutation-related DCM often present with defects in the cardiac conduction system, including atrioventricular block, ventricular arrhythmias, and atrial fibrillation, which can easily lead to sudden death. Current drug treatments, including beta-blockers and ACE inhibitors, cannot stop the progression of the disease, and there is an urgent need to develop new therapeutic drugs.

[0005] The LMNA gene can produce different proteins through alternative splicing. In mammalian cells, it mainly produces two functional proteins, Lamin A and Lamin C, with comparable expression levels. Lamin A and Lamin C share the same 566 amino acids at their C-terminus, differing only in their C-terminal sequences, each possessing 98 or 6 different C-terminal sequences. Lamin A and Lamin C are frequently studied together and are believed to play important roles in regulating various biological processes, including nuclear membrane stability, DNA replication, chromatin homeostasis, cell differentiation, and apoptosis. The nuclear membrane localization of Lamin C depends on Lamin A. While Lamin A and Lamin C may have different functions, both are likely indispensable for maintaining normal cellular function. Summary of the Invention

[0006] To simultaneously restore both Lamin A and Lamin C proteins in cells lacking these proteins, this invention, for the first time, employs a variable splicing approach to mediate the simultaneous expression of both proteins. Compared to multi-promoter or P2A / T2A cleavage peptide approaches, this method offers the advantage of a smaller vector size, and the expression ratio of the two proteins can be regulated by splicing signals.

[0007] Based on the above findings, the present invention first provides a synthetic intron comprising: I) a nucleotide sequence as shown in SEQ ID No. 8, II) a nucleotide sequence having at least one mutation at the 1st to 8th sites of the sequence shown in SEQ ID No. 8, or a nucleotide sequence having at least 90% homology with the sequence shown in I) or II).

[0008] The present invention also provides a polynucleotide construct comprising: a synthetic intron as described above, wherein a motif for regulating variable splicing is provided at the junction of the synthetic intron and the exons on both sides.

[0009] The present invention also provides a method for simultaneously expressing two target peptides, comprising: expressing the target peptides using synthetic introns or polynucleotide constructs as described above.

[0010] The present invention also provides a polynucleotide construct comprising: an LMNA gene exon encoding Lamin A and Lamin C via variable splicing, and a synthetic intron as described above; wherein the synthetic intron is located between exon 10 and exon 11 of the LMNA gene, and a motif for regulating variable splicing is provided at the junction of the synthetic intron with the exons on both sides.

[0011] The present invention also provides a recombinant expression vector containing at least one copy of the polynucleotide construct as described above.

[0012] The present invention also provides a host cell containing at least one copy of the polynucleotide construct as described above.

[0013] The present invention also provides a pharmaceutical composition comprising the polynucleotide construct as described above or the recombinant expression vector as described above.

[0014] The present invention also provides the use of the polynucleotide constructs as described above, or the recombinant expression vectors as described above, or the host cells as described above in the prevention, treatment and / or alleviation of diseases, wherein the diseases are associated with deletion mutations or downregulation of Lamin A and / or Lamin C.

[0015] The present invention also provides the use of the polynucleotide constructs as described above, or the recombinant expression vectors as described above, or the host cells as described above in the preparation of medicaments for the prevention, treatment and / or alleviation of diseases, wherein the diseases are associated with deletion mutations or downregulation of Lamin A and / or Lamin C.

[0016] The present invention also provides a method for simultaneously expressing Lamin A and Lamin C, comprising: culturing host cells as described above; and optionally recovering the target proteins.

[0017] The synthetic intron of this invention enables the simultaneous expression of two proteins via alternative splicing. When used for the simultaneous expression of Lamin A and Lamin C proteins, it not only exhibits excellent expression efficiency but also achieves comparable expression levels for both. For dilated cardiomyopathy caused by LMNA mutations, compared to existing gene therapy methods using expression vectors containing DNSUN1, the expression vector of this invention, which simultaneously expresses Lamin A and Lamin C proteins, demonstrates a stronger rescue effect. Furthermore, by mutating the splicing signal without altering the intron sequence, alternative splicing of exons can be mediated, thereby regulating the expression levels of Lamin A and Lamin C proteins, and even enabling the expression of Lamin A or Lamin C individually, thus further broadening the application scope of this synthetic intron. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 is a schematic diagram of the mechanism by which the LMNA gene produces two proteins, Lamin A and Lamin C, through variable splicing.

[0020] Figure 2 shows the expression effects of plasmids containing different cleavage peptides on Lamin A and Lamin C in the embodiments of the present invention; in the figure, Figure A is a schematic diagram of the construction of different plasmids; Figures B and C are the expression results of Lamin A and Lamin C in different plasmids.

[0021] Figure 3 shows the expression effects of the variable splicing vector containing synthetic introns on Lamin A and Lamin C in the embodiments of the present invention; in the figure, Figure A is a schematic diagram of the construction of different plasmids; Figure B shows the expression results of Lamin A and Lamin C after different plasmids were transfected into human HEK293T cells; Figure C shows the expression results of Lamin A and Lamin C after different plasmids were transfected into mouse N2a cells.

[0022] Figure 4 is a schematic diagram illustrating the principle of using synthetic introns and their mutants to simultaneously express Lamin A and Lamin C in an embodiment of the present invention.

[0023] Figure 5 shows the results of simultaneously expressing Lamin A and Lamin C using other synthetic introns or optimized exon sequences in the embodiments of the present invention; in the figure, Figure A is a schematic diagram of the principle and expression results of plasmid 12, and Figure B is a schematic diagram of the principle and expression results of plasmid 13.

[0024] Figure 6 is a schematic diagram of the construction of different therapeutic AAV vectors in the embodiments of the present invention.

[0025] Figure 7 shows the expression of different AAV viruses in the heart of LMNA knockout mice in the embodiments of the present invention.

[0026] Figure 8 shows the survival of LMNA knockout mice after different AAV virus treatments in the embodiments of the present invention.

[0027] Figure 9 shows how LMNA-Intron1 treatment restored the nuclear membrane localization of Lamin A and Lamin C proteins in LMNA knockout mouse cardiomyocytes in an embodiment of the present invention.

[0028] Figure 10 shows the effects of different therapeutic AAV viruses on conditional knockout of LMNA (LMNA) in cardiomyocytes in embodiments of the present invention. CKO The therapeutic effect in mice; in the figure, A is a representative M-mode cardiac ultrasound image, and B is a statistical cardiac ultrasound image.

[0029] Figure 11 illustrates the use of different plasmids to treat LMNA in an embodiment of the present invention. CKO Electrocardiographic phenotypes of mice after surgery; in the figure, A is the representative electrocardiogram, and B and C are the statistical results of QTc and R wave amplitude, respectively.

[0030] Figure 12 illustrates the treatment of LMNA using different plasmids in an embodiment of the present invention. CKO The results showed that the mice had prolonged survival.

[0031] Figure 13 illustrates the use of different plasmids to treat LMNA in an embodiment of the present invention. CKO Cardiac dilation phenotype in mice.

[0032] Figure 14 is a schematic diagram of the construction of the therapeutic AAV vector in Embodiment 8 of the present invention.

[0033] Figure 15 illustrates the use of different plasmids to treat LMNA in an embodiment of the present invention. + / - The results showed that the mice had prolonged survival.

[0034] Figure 16 shows the effects of different therapeutic AAV viruses on LMNA monoallelic knockout (LMNA) in embodiments of the present invention. + / - The therapeutic effect in mice; in the figure, A is a representative M-mode cardiac ultrasound image, and B is a statistical cardiac ultrasound image.

[0035] Figure 17 illustrates the use of therapeutic AAV virus in LMNA monoallelic knockout (LMNA) in an embodiment of the present invention. + / - Statistical analysis of cardiac ultrasound in mice to evaluate the long-term therapeutic effect.

[0036] In the figures of this invention, “(N)” refers to plasmid N; as an example, in Figure 3, (5) refers to plasmid 5, and (6-11) refers to plasmid 6-11. Detailed Implementation

[0037] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0038] Terminology Explanation

[0039] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0040] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0041] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0042] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0043] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM may fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0044] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0045] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0046] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0047] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0048] In this invention, the term "variable splicing" refers to a mechanism of gene expression regulation that allows a gene to produce multiple different mature mRNA molecules during transcription. Variable splicing generates different transcripts by selectively splicing exons and introns of a gene, resulting in a variety of protein variants. This mechanism enables a single gene to encode multiple proteins with different functions, thereby increasing the functional diversity and complexity of genes.

[0049] In this invention, the term "motif regulating variable splicing," also known as "splicing signal," refers to specific sequences and structural elements present in pre-mRNA molecules that guide the occurrence of variable splicing. Splicing signals typically consist of a donor site, an acceptor site, and a branch point site. The donor site is located at the exon-intron boundary and is usually composed of a conserved GU dinucleotide sequence. The acceptor site is located at the intron-exon boundary and is usually composed of a conserved AG dinucleotide sequence. The branch point site is an A nucleotide located within an intron, forming a branch point connection with both the donor and acceptor sites. The sequence and structural characteristics of these splicing signals, as well as their interactions with splicing factors, determine the location and manner in which variable splicing occurs. The recognition and pairing of splicing signals are accomplished by the spliceosome, a complex protein-RNA complex. During splicing, it removes introns from the precursor mRNA and joins exons to form the mature mRNA molecule. The motifs regulating variable splicing mentioned in this article mainly refer to splicing sites located at the exon-intron junction, including the 5' and 3' splicing sites.

[0050] In this invention, nucleotide substitutions are named using the following method: initial nucleotide, position, substituted nucleotide. As an example, adenine (T) at the 6th position at the 5' end is substituted with cytosine (C), denoted as T6C.

[0051] In this invention, the term "multinucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule isolated from naturally occurring genes, or modified in a manner not otherwise found in nature to contain nucleic acid fragments, or synthesized, which contains one or more control sequences.

[0052] In this invention, the term "expression vector" refers to a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operatively linked to a control sequence for its expression. "operatively linked" means that the control sequence is positioned relative to the coding sequence of the polynucleotide at a suitable location, thereby guiding the expression of the coding sequence.

[0053] In this invention, the term "amino acid sequence" is synonymous with and interchangeable with the terms "polypeptide," "protein," and "peptide." Conventional single-letter or three-letter codes for amino acid residues are used, wherein the amino acid sequence is presented with a standard amino-to-carboxyl terminal orientation (i.e., N→C).

[0054] In this invention, the term "homology" refers to the correlation between two amino acid sequences or two nucleotide sequences. During alignment, algorithms are typically used to identify differences such as insertions, deletions, or substitutions between sequences. Common sequence alignment algorithms include BLAST, ClustalW, and MAFFT.

[0055] Synthetic introns

[0056] This invention first provides a synthetic intron comprising: I) a nucleotide sequence as shown in SEQ ID No. 8; II) a nucleotide sequence having at least one mutation at sites 1-8 of the sequence shown in SEQ ID No. 8; or, a nucleotide sequence having at least 90% homology with the sequence shown in I) or II). This invention unexpectedly discovered that, using a synthetic intron comprising the nucleotide sequence shown in SEQ ID No. 8, two proteins can be simultaneously expressed via alternative splicing. When used for the simultaneous expression of Lamin A and Lamin C proteins, not only is the expression effect excellent, but the expression levels of both are also comparable. Furthermore, by making at least one mutation at sites 1-8 of the sequence shown in SEQ ID No. 8, the expression levels of Lamin A and Lamin C proteins can be regulated, and even Lamin A or Lamin C can be expressed individually.

[0057] In some embodiments, the synthetic intron comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology to the sequence shown in I) or II), and still having the same or equivalent function as the sequence shown in I) or II).

[0058] In some embodiments, II) is a nucleotide sequence with a T6C mutation based on the sequence shown in SEQ ID No. 8. This can increase the proportion of Lamin C protein in the product while decreasing the proportion of Lamin A protein.

[0059] Polynucleotide constructs

[0060] The present invention also provides a polynucleotide construct comprising: a synthetic intron as described above, wherein a motif for regulating variable splicing is provided at the junction of the synthetic intron and the exons on both sides.

[0061] In some embodiments, the modulated motif for adjustable variable splicing contains the sequence: 5'-… / GT…AG / …–3', where / represents the cleavage site and … represents the intermediate sequences. Those skilled in the art can design different modulated motifs for adjustable variable splicing based on this conserved sequence to match the splicing requirements of different sequences.

[0062] In some preferred embodiments, the motif for regulating variable splicing comprises a 5' motif and a 3' motif; wherein the 5' motif contains any of the nucleotide sequences shown in SEQ ID No. 17-23, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology with any of the nucleotide sequences shown in SEQ ID No. 17-23; the 3' motif contains the nucleotide sequence described in SEQ ID No. 24, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology with the nucleotide sequence described in SEQ ID No. 24. The synthetic introns of the present invention, linked to the above motifs, can promote and regulate variable splicing.

[0063] In some embodiments, the polynucleotide construct is an empty vector. This allows those skilled in the art to construct different exogenous genes or sequences therein, and also serves as a control group to evaluate the effects caused by other expression vectors containing exogenous genes.

[0064] In some embodiments, the polynucleotide construct further comprises exons encoding two target polypeptides via variable splicing, wherein the synthetic introns are located between the cleavage sites of the exons.

[0065] In practice, those skilled in the art can identify the corresponding exon sequences and splicing sites based on specific genes with known alternative splicing mechanisms, thereby obtaining polynucleotide constructs expressing different target polypeptides. Alternatively, gene sequences can be manipulated to induce a splicing mechanism in the gene.

[0066] In some embodiments, the polynucleotide construct further contains splice enhancers and / or splice repressors. A splice enhancer is a sequence element that enhances the selectivity of a splice site; a splice repressor is a sequence element that inhibits the selectivity of a splice site. By cleverly designing and introducing these sequence elements, the splicing selectivity of genes can be regulated, thereby producing different mRNA isotypes.

[0067] Methods of expression

[0068] The present invention also provides a method for simultaneously expressing two target peptides, comprising: expressing the target peptides using synthetic introns or polynucleotide constructs as described above.

[0069] The aforementioned polynucleotide constructs can be used for variable splicing, thereby forming different mRNA subtypes and corresponding target peptides.

[0070] Polynucleotide construct for expressing LMNA

[0071] The present invention also provides a polynucleotide construct comprising: an exon of an LMNA gene encoding Lamin A and Lamin C via variable splicing, and a synthetic intron as described above; wherein the synthetic intron is located between exon 10 and exon 11 of the LMNA gene, and a motif for regulating variable splicing is provided at the junction of the synthetic intron with the exons on both sides.

[0072] In some embodiments, the motif for regulating variable splicing contains the following sequences: a 5' motif and a 3' motif; wherein the 5' motif contains any one of the nucleotide sequences shown in SEQ ID No. 17-23, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology with any one of the nucleotide sequences shown in SEQ ID No. 17-23; the 3' motif contains the nucleotide sequence described in SEQ ID No. 24, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology with the nucleotide sequence described in SEQ ID No. 24. This is more conducive to regulating the expression of Lamin A and Lamin C.

[0073] In some embodiments, the Lamin A contains an amino acid sequence as shown in SEQ ID No. 14, or an amino acid sequence having at least 90% homology with the sequence shown in SEQ ID No. 14; the Lamin C contains an amino acid sequence as shown in SEQ ID No. 15, or an amino acid sequence having at least 90% homology with the sequence shown in SEQ ID No. 15.

[0074] In some embodiments, the Lamin A contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology to the sequence shown in SEQ ID No. 14, and still has the same or equivalent function as the sequence shown in SEQ ID No. 14.

[0075] In some embodiments, the Lamin C contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology to the sequence shown in SEQ ID No. 15, and still has the same or equivalent function as the sequence shown in SEQ ID No. 15.

[0076] Those skilled in the art can identify the sequence of the corresponding exons of the LMNA gene encoding Lamin A and Lamin C via variable splicing based on the above-described amino acid sequence. Due to codon degeneracy, the exon sequences in the polynucleotide construct can be multiple different nucleotide sequences encoding the same amino acid.

[0077] As an example, in some specific embodiments, the nucleotide sequence encoding Lamin A comprises: the nucleotide sequence shown in SEQ ID No. 1, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology to the sequence shown in SEQ ID No. 1.

[0078] As an example, in some specific embodiments, the nucleotide sequence encoding Lamin C comprises: the nucleotide sequence shown in SEQ ID No. 2, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology to the sequence shown in SEQ ID No. 2.

[0079] In some embodiments, the polynucleotide construct comprises: a nucleotide sequence as shown in SEQ ID No. 10 or 13, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology to the sequence shown in SEQ ID No. 10 or 13.

[0080] In some embodiments, the polynucleotide construct comprises a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% homology to the sequence shown in SEQ ID No. 10 or 13, and still having the same or equivalent function as the sequence shown in SEQ ID No. 10 or 13.

[0081] In some implementations, the expression of Lamin A and Lamin C is driven by different promoters selected from CMV promoter, CAG promoter, EF1α promoter, cTnT promoter, αMHC promoter, Des promoter, MLC2v promoter, CK8e promoter, MHCK7 promoter, SM22α promoter, ACTA1 promoter, and Myog promoter.

[0082] Recombinant expression vector for expressing LMNA

[0083] The present invention also provides a recombinant expression vector containing at least one copy of the polynucleotide construct as described above.

[0084] In some embodiments, the control sequences in the recombinant expression vector include promoters and transcription and translation termination signals. In some embodiments, the recombinant expression vector may also contain other control sequences, such as enhancers and repressors. The individual control sequences may be endogenous (i.e., from the same gene) or exogenous (i.e., from different genes) for the polynucleotide encoding the polypeptide, or endogenous or exogenous for each other.

[0085] In some implementations, various nucleotides and control sequences can be linked together to produce a recombinant expression vector that may contain one or more convenient restriction sites that allow the insertion or substitution of polynucleotides of the target polypeptide at those sites. Alternatively, polynucleotides can be expressed by inserting polynucleotides or nucleic acid constructs containing polynucleotides into a suitable expression vector. During the construction of the expression vector, the coding sequence of the target polypeptide can be introduced into the vector, and the coding region sequence of the target polypeptide, the coding region sequence of a selective marker (or mutant), and the control sequence can be operatively linked to appropriately express the coding sequence via the control sequence.

[0086] In some implementations, the recombinant expression vector can be any vector (e.g., plasmid or virus) that allows for convenient recombinant DNA procedures and induces polynucleotide expression. The choice of vector typically depends on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid.

[0087] In some embodiments, the recombinant expression vector is adenovirus or adeno-associated virus. In some preferred embodiments, the recombinant expression vector is adeno-associated virus 9 (AAV9).

[0088] host cells

[0089] The present invention also provides a host cell containing at least one copy of the polynucleotide construct as described above.

[0090] The host cell can be any cell that can be used in the recombinant production of the polypeptides of the present invention, such as fungal cells.

[0091] The term "fungi" as used in this invention includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Oomycota, and all mitotic fungi. The host cell for the fungi can be a yeast cell. The term "yeast" as used in this invention includes Ascomycota, Basidioycota, and yeasts belonging to the Deuteromycetes.

[0092] In some embodiments, the fungal host cell can be a filamentous fungal cell. "Filamentous fungi" encompasses all filamentous forms within the phylum Fungi and subphylum Oomycetes. Filamentous fungi are typically characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal elongation, and carbon metabolism is obligate aerobic. In contrast, yeasts, such as *Saccharomyces cerevisiae*, exhibit vegetative growth through budding of single-celled cells, and carbon metabolism can be fermentative.

[0093] In some embodiments, the filamentous fungal host cell may be a genus such as *Acremonium*, *Aspergillus*, *Aureobasidium*, *Bjerkandera*, *Ceriporiopsis*, *Chrysosporium*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Humicola*, *Magnaporthe*, *Mucor*, or *Mycelioph*. Cells of the genera *Thora*, *Neocallimastix*, *Neurospora*, *Paecilomyces*, *Penicillium*, *Phanerochaete*, *Phlebia*, *Piromyces*, *Pleurotus*, *Schizophyllum*, *Talaromyces*, *Thermoascus*, *Thielavia*, *Tolypocladium*, *Trametes*, or *Trichoderma*.

[0094] In some embodiments, more than one copy of the polynucleotide of the invention can be inserted into the host cell to increase polypeptide production. The increase in polynucleotide copy number can be achieved by integrating at least one additional copy of the sequence into the host cell genome; or by including an amplifiable selection marker gene with the polynucleotide, thereby allowing selection of cells containing an amplified copy of the selection marker gene by culturing cells in the presence of a suitable selector, and thus selection of cells containing an additional copy of the polynucleotide.

[0095] Pharmaceutical Composition

[0096] The present invention also provides a pharmaceutical composition comprising the polynucleotide construct as described above or the recombinant expression vector as described above.

[0097] In some embodiments, the pharmaceutical composition further contains at least one auxiliary component selected from encapsulation materials (such as nanomaterials, liposomes, polymers, virus-like particles, etc.), delivery carriers (such as sponge carriers, etc.), and protectants (such as antioxidants, cryoprotectants, etc.) to enhance the stability, delivery efficiency, or biocompatibility of the drug.

[0098] application

[0099] The present invention also provides the use of the polynucleotide constructs as described above, or the recombinant expression vectors as described above, or the host cells as described above in the prevention, treatment and / or alleviation of diseases, wherein the diseases are associated with deletion mutations or downregulation of Lamin A and / or Lamin C.

[0100] The present invention also provides the use of the polynucleotide constructs as described above, or the recombinant expression vectors as described above, or the host cells as described above in the preparation of medicaments for the prevention, treatment and / or alleviation of diseases, wherein the diseases are associated with deletion mutations or downregulation of Lamin A and / or Lamin C.

[0101] The "deletion mutation" mentioned in this invention refers to a protein deletion mutation, specifically a type of mutation in a gene that causes the gene to lose its function. Generally, this type of mutation may lead to a reduction or complete loss of function of the protein encoded by the gene. This type of mutation may manifest as gene deletion, the appearance of an early stop codon, loss of function due to amino acid substitution, or changes in protein structure. These changes prevent the protein from performing its biological functions normally, thereby affecting the normal physiological activities of cells or organisms.

[0102] In this invention, "downregulation of expression" refers to a decrease in protein expression levels, particularly when protein expression levels fall below the normal threshold. This may be due to inhibition of transcription, increased RNA degradation, or post-translational regulation, leading to a reduction in the production of messenger RNA and proteins.

[0103] In some embodiments, the disease includes laminopathies.

[0104] In some embodiments, the diseases include muscular dystrophy, limb-girdle muscular dystrophy, peripheral neuropathy, progeria, and dilated cardiomyopathy (DCM).

[0105] In some implementations, the disease includes cardiac dysfunction associated with LMNA gene mutations.

[0106] In some embodiments, the disease includes dilated cardiomyopathy associated with LMNA gene mutations.

[0107] In some embodiments, the prevention, treatment, and / or alleviation of disease includes at least one of the following aspects: i) prolonging the survival of the drug recipient; ii) restoring the nuclear membrane localization of Lamin A and Lamin C in the cardiomyocytes of the drug recipient; iii) improving the cardiac function of the drug recipient; iv) improving the cardiac electrophysiology of the drug recipient; and v) inhibiting the cardiac dilatation phenotype of the drug recipient.

[0108] The subjects of drug administration described in this invention include humans and animals.

[0109] In some implementations, the recipient of the medication is an individual suffering from the disease.

[0110] In some implementations, the recipients of the medication are individuals carrying gene mutations associated with the disease, particularly those carrying LMNA mutations. These individuals may not exhibit clinical symptoms, but are deemed at risk of developing the disease based on their genotype.

[0111] Methods of expressing Lamin A and / or Lamin C

[0112] The present invention also provides a method for expressing Lamin A and / or Lamin C, comprising: culturing host cells as described above; and optionally recovering the target protein.

[0113] In some embodiments, host cells are cultured in a nutrient medium suitable for peptide production using methods known in the art. For example, cells can be cultured by shake-flask culture in a suitable medium under conditions allowing peptide expression and / or isolation, or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, fed-batch fermentation, or solid-state fermentation) in a laboratory or industrial fermenter. Culture is carried out in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts using steps known in the art. Suitable media are available from commercial suppliers or can be prepared according to a publicly disclosed composition. If the peptide is secreted into the nutrient medium, it can be recovered directly from the medium. If the peptide is not secreted, it can be recovered from cell lysates.

[0114] In some embodiments, the target protein can be detected using specific methods known in the art for proteins. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, enzyme assays can be used to determine the activity of the target protein.

[0115] In some embodiments, methods known in the art can be used to recover the target protein. For example, the target protein can be recovered from the nutrient medium by conventional steps, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0116] In some embodiments, the target protein can be purified to a substantially pure form by a variety of methods known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatographic focusing, and size exclusion), electrophoresis (e.g., preparative isoelectric focusing), differential dissolution (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction.

[0117] Example

[0118] The embodiments of the present invention will now be described in detail with reference to examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0119] For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the field, or other experimental methods known in the field, or follow the conditions recommended by the manufacturer.

[0120] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0121] The nucleic acid sequence information involved in the following examples is shown in Table 1 below:

[0122] Table 1

[0123] The plasmid protein sequence information involved in the following examples is shown in Table 2 below:

[0124] Table 2

[0125] Example 1: The P2AT2A cleavage peptide exhibits higher activity than P2A and T2A.

[0126] Lamin A and Lamin C are transcribed and translated from the same gene LMNA, but they differ in their amino acid sequences at the C-terminus (Figure 1). To simultaneously overexpress Lamin A and Lamin C, this example first tested the activity of different cleavage peptides. Plasmids containing different cleavage peptides were constructed, as shown in Figure 2(A). After transfection into 293T cells, their cleavage efficiency was detected by Western blot, showing that P2AT2A had the highest cleavage efficiency, as shown in Figure 2(B). However, P2AT2A, P2A, and T2A could not achieve equal expression of Lamin A and Lamin C. The results are shown in Figure 2(B).

[0127] Example 2: Constructing the LMNA-Intron plasmid to simultaneously express equal amounts of Lamin A and Lamin C proteins.

[0128] Since Lamin A and Lamin C are two different proteins produced by the LMNA gene through alternative splicing, this embodiment proposes to achieve alternative splicing of exogenous expression genes by adding an intron sequence between exons 10 and 11 of the LMNA gene. In this embodiment, an LMNA-Intron plasmid (plasmid 5, schematic diagram shown in Figure 3A) was first constructed, retaining the DNA sequence of exons 1-10 of the LMNA gene, adding an intron 1 containing a splicing signal, and then adding the sequence of exons 11-12. A Flag tag protein was added to the N-terminus; in this embodiment, the promoter is CMV. These plasmids were transfected into HEK293T cells, and Western blot experiments demonstrated that the synthetic intron 1 could mediate the simultaneous production of both Lamin A and Lamin C proteins (anti-Lamin A & C, CST; anti-Flag, CST) by the LMNA-Intron1 plasmid, and that the expression levels of both were comparable, as shown in Figure 3B and Figure 4. Mutating the splicing signal without altering the intron sequence mediates complete exon splicing, producing only the longer Lamin A protein, as shown in Figure 3B. Similarly, in mouse N2a cells, it was demonstrated that the combination of synthetic intron 1 and motif 1, which regulates variable splicing, mediates incomplete exon splicing, producing both Lamin A and Lamin C proteins, as shown in Figure 3C and Figure 4.

[0129] To further verify the ability of synthetic intron 1 to mediate alternative splicing, this embodiment also mutated parts of LMNA intron 1 and exon 10, constructing six LMNA-Intron1 mutants. The results showed that different variants could mediate the production of different proportions of Lamin A and Lamin C proteins. However, replacing intron 1 with intron 10 of wild-type LMNA (i.e., synthetic intron 2, corresponding to plasmid 12) could not mediate alternative splicing, and only Lamin C protein was produced (Figure 5A). Optimizing the coding sequence of LMNA exons 1-10 while retaining the synthetic intron sequence (i.e., optiLMNA-Intron1, corresponding to plasmid 13) could also mediate the simultaneous expression of Lamin A and Lamin C proteins (Figure 5B), indicating that synthetic introns can also mediate splicing of the codon-optimized sequence (LMNA-Intron1).

[0130] Example 3: Simultaneous expression of Lamin A and lamin C using LMNA-Intron1 significantly prolonged the survival of LMNA knockout mice.

[0131] LMNA knockout mice began dying at P11, with the longest survival time being 21 days after birth. The target genes Lamin A, Lamin C, LaminA-P2AT2A-LaminC, LMNA-Intron1, and SUN1 (patent number: US2020 / 0347408 A1) were constructed into an adeno-associated virus 9 (AAV9) expression vector and packaged with AAV virus. In this example, the promoter used was CMV, and the vector construction is shown in Figure 6. In this example, the DNSUN1 sequence underwent sequence optimization, and all AAV vectors contained chimeric introns and UTRs.

[0132] Genotyping of newborn mice was performed at P0, and LMNA was analyzed. - / - Mice were subcutaneously injected with AAV at P2 to overexpress the target protein, with an injection dose of 5*10^ 10The virus was successfully expressed in the mouse heart after injection (vg / g), as shown in Figure 7. The results showed that co-expression of Lamin A and Lamin C in LMNA-Intron1 using adeno-associated virus 9 (AAV9) extended the median survival of systemic knockout mice from 18 days to 21 days, with death occurring only at day 18 (P18), and the longest survival reaching 50 days after birth. Overexpression of DNSUN1L delayed the time to death in mice, with death first detected at day 16 (P16), but did not prolong survival. These results indicate that co-expression of Lamin A and Lamin C significantly prolongs the survival of systemic LMNA knockout mice, and its effect is stronger than that of DNSUN1, as shown in Figure 8.

[0133] Example 4: Overexpression of LMNA-Intron1 restored the expression of Lamin A and Lamin C on the cell membrane of cardiomyocytes.

[0134] In LMNA knockout mice, Lamin A and Lamin C were absent in cardiomyocytes. However, after overexpression of LMNA-Intron1, immunofluorescence staining revealed that Lamin A and Lamin C were restored in some cells and correctly located in the nuclear membrane. The results are shown in Figure 9.

[0135] Example 5: Simultaneous expression of Lamin A and Lamin C using LMNA-Intron1 significantly enhances LMNA expression. CKO cardiac function in mice

[0136] To investigate the effect of reintroduced Lamin protein on LMNA cardiomyocyte-specific knockout mice, at P7, MCM Cre+;LMNA... Flox / Flox (abbreviated as LMNA) CKO Mice were fed 5*10^ 10 Subcutaneous injection of adeno-associated virus type 9 (AAV9) at a dose of vg / g followed by tamoxifen (25 mg / kg, intraperitoneal injection, for 5 consecutive days) at P14 to induce specific knockout of LMNA in cardiomyocytes. Cardiac function was assessed by echocardiography at week 4 post-induction. Figures 10A and B show that, 4 weeks post-induction, compared to wild-type (WT) mice, LMNA knockout... CKO The mice exhibited significant cardiac dysfunction.

[0137] Comparison with LMNA CKOIn mice, simultaneous expression of Lamin A and Lamin C using LMNA-Intron1 significantly improved the dilated cardiomyopathy phenotype, as evidenced by an increase in ejection fraction (EF) from 20.17±3.235 to 35.36±7.366 (p=0.0004); an increase in left ventricular end-systolic posterior wall thickness from 0.6565±0.05699 mm to 0.8154±0.05344 mm (p=0.0039); and a decrease in left ventricular end-systolic diameter from 3.876±0.3045 mm to 3.234±0.3131 mm (p=0.0015).

[0138] Comparison with LMNA CKO In mice, overexpression of negatively regulated DNSUN1 increased the left ventricular ejection fraction (EF) from 20.17±3.235 to 24.46±7.968 (p=0.6052); the left ventricular end-systolic posterior wall thickness increased from 0.6565±0.05699 mm to 0.6700±0.07557 mm (p=0.9961), which was not statistically significant; and the left ventricular end-systolic diameter decreased from 3.876±0.3045 mm to 3.469±0.3585 mm (p=0.1019), which was also not statistically significant.

[0139] The above results indicate that the salvage effect of simultaneous expression of Lamin A and Lamin C is superior to that of DNSUN1L. LMNA-Intron1 treatment significantly inhibited LMNA. CKO Dilated cardiomyopathy phenotype in mice.

[0140] Example 6: Simultaneous expression of Lamin A and Lamin C using LMNA-Intron1 can improve electrophysiology in mice.

[0141] Four weeks after tamoxifen induction, LMNA CKO The mice exhibited significant electrophysiological abnormalities. Compared to wild-type mice, LMNA... CKO The QTc of mice increased from 4.975±0.5304 ms to 10.40±0.6554 ms (p<0.0001). Simultaneous expression of Lamin A and Lamin C using LMNA-Intron1 reduced the QTc of knockout mice to 7.850±2.162 (p=0.0061), while overexpression of DNSUN1 reduced it to 7.660±1.1643 (p=0.0053). Compared to wild-type (WT) mice, LMNA... CKOThe R-wave amplitude in mice decreased from 0.8935±0.1849 mV to 0.5102±0.07131 mV (p<0.0001). Simultaneous expression of Lamin A and Lamin C using LMNA-Intron1 increased the R-wave amplitude to 0.7039±0.04646 mV (p=0.0159), while overexpression of SUN1 restored it to 0.4412±0.1469 mV (p=0.6609). These results indicate that the rescue effect of simultaneous expression of Lamin A and Lamin C is superior to that of DNSUN1L, and LMNA-Intron1 significantly inhibits LMNA. CKO Abnormal electrocardiographic phenotypes in mice. The results are shown in Figures A and B in Figure 11.

[0142] Example 7: Simultaneous expression of Lamin A and Lamin C using LMNA-Intron1 significantly prolongs LMNA expression. CKO Survival time of mice and inhibition of cardiac dilation phenotype

[0143] LMNA induced by hemoxifen CKO Mice died within 10 days, with the longest survival being 48 days. Mice injected with negatively regulated SUN1 had a longer survival, but began to die at 46 days. Mice injected with LMNA-Intron1 remained viable 60 days after induction, significantly prolonging LMNA regulation. CKO Lifespan of the mice. The results are shown in Figure 12.

[0144] Four weeks after tamoxifen induction, mouse hearts were harvested and compared with wild-type mice; LMNA was found to be significantly lower in LMNA-containing mice. CKO The hearts of mice injected with negatively regulated SUN1 nearly doubled in size, while the hearts of mice injected with LMNA-Intron1 tended to decrease in size but still showed severe dilation. The hearts of mice injected with LMNA-Intron1 were comparable in size to those of wild-type mice. This indicates that LMNA-Intron1 injection blocks LMNA. CKO The heart of the mouse was dilated, and the results are shown in Figure 13.

[0145] Example 8: Simultaneous expression of Lamin A and Lamin C using LMNA-Intron1 significantly prolongs LMNA expression. + / - mouse lifespan

[0146] The target gene LMNA-Intron1 was constructed into the adeno-associated virus 9 (AAV9) expression vector and packaged with AAV virus. In this example, the promoter used was cTnT. The vector construction is shown in Figure 14.

[0147] LMNA monoallelic knockout (LMNA for short) + / -Mice in this group began to die at 401 days of age, with the longest survival time being 447 days. Regarding LMNA... + / - Adult mice were injected via tail vein at P270 to overexpress the target protein, with an injection dose of 3*10^ 13 vg / kg. Results showed that using adeno-associated virus 9 (AAV9) to simultaneously express Lamin A and Lamin C in LMNA-Intron1 resulted in LMNA... + / - The median survival time of mice was extended from 431 days to 559 days, and the mice did not die until 479 days after birth, with the longest survival reaching 596 days after birth. These results indicate that simultaneous expression of Lamin A and Lamin C can significantly prolong LMNA. + / - The survival time of the mice is shown in Figure 15.

[0148] Example 9: Simultaneous expression of Lamin A and Lamin C using LMNA-Intron1 can significantly enhance LMNA expression. + / - cardiac function in mice

[0149] LMNA monoallelic knockout mice exhibit typical pathogenesis of dilated cardiomyopathy (DCM) in adulthood, typically developing a stable DCM phenotype by 9 months of age. To investigate the effect of lamin protein reinjection on LMNA cardiomyocyte monoallelic knockout mice, LMNA mice that had already developed the phenotype at 9 months of age were subjected to... + / - Mice were 3*10^ 13 Mice were injected with adeno-associated virus type 9 (AAV9) via tail vein at a dose of vg / kg. Ultrasound examination was started two months after administration to assess cardiac function in mice.

[0150] Figures 16A and B show that at 9 months of age, compared to wild-type (WT) mice, LMNA... + / - The mice exhibited significant cardiac dysfunction. (Compared to LMNA) + / - In mice, simultaneous expression of Lamin A and Lamin C using LMNA-Intron1 improved the dilated cardiomyopathy phenotype at 9-12 months of age. This was manifested in increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), as well as increased left ventricular end-systolic posterior wall thickness (LVPW;s) and left ventricular end-systolic diameter (LVID;s). The rescue effect of simultaneous expression of Lamin A and Lamin C effectively prevented the development of the dilated cardiomyopathy phenotype. At 3 months post-drug administration, cardiac function was close to that of wild-type mice, with no significant ventricular dilation or systolic dysfunction, and LMNA expression was consistently inhibited. + / - Dilated cardiomyopathy phenotype in mice.

[0151] Figure 17 shows the comparison of LMNA at three months of age after administration at 9 months of age. + / - In mice, simultaneous expression of Lamin A and Lamin C using LMNA-Intron1 significantly improved the phenotype of dilated cardiomyopathy. Specifically, the left ventricular ejection fraction increased from 33.67±2.643 to 46.48±2.955 (p=0.0062), showing a significant difference; the left ventricular fractional shortening increased from 15.79±1.537 to 23.04±1.719 (p=0.0073), showing a significant difference; the left ventricular end-systolic diameter decreased from 3.442±0.2146 mm to 3.103±0.2399 mm (p=0.3797), showing no significant difference; and the left ventricular end-systolic posterior wall thickness increased from 0.8329±0.05063 mm to 1.053±0.05661 mm (p=0.0114), showing a significant difference.

[0152] The above results demonstrate that, compared to LMNA + / - In mice, simultaneous expression of Lamin A and Lamin C using LMNA-Intron1 significantly regulated the development of DCM phenotype and persistently inhibited LMNA expression. + / - Dilated cardiomyopathy phenotype in mice.

[0153] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A synthetic intron comprising: I) The nucleotide sequence shown in SEQ ID No. 8, II) A nucleotide sequence having at least one mutation at sites 1 to 8 of the sequence shown in SEQ ID No.

8. Alternatively, III) is a nucleotide sequence that has at least 90% homology with the sequence shown in I) or II).

2. The synthetic intron according to claim 1, wherein, The second part (II) is: a nucleotide sequence with a T6C mutation based on the sequence shown in SEQ ID No.

8.

3. A polynucleotide construct comprising: The synthetic intron as described in claim 1 or 2; A motif for adjusting variable splicing is provided at the junction of the synthetic intron and the two exons.

4. The polynucleotide construct according to claim 3, wherein, The motif of the modulated variable splicing includes a 5' motif and a 3' motif; Wherein, the 5' motif contains any one of the nucleotide sequences shown in SEQ ID No. 17 to 23, or a nucleotide sequence having at least 90% homology with any one of the nucleotide sequences shown in SEQ ID No. 17 to 23; The 3' motif contains the nucleotide sequence described in SEQ ID No. 24, or a nucleotide sequence that has at least 90% homology with the nucleotide sequence described in SEQ ID No.

24.

5. The polynucleotide construct according to claim 3 or 4, wherein, The polynucleotide construct also includes exons encoding two target polypeptides via variable splicing, with the synthetic introns located between the cleavage sites of the exons.

6. A method for simultaneously expressing two target peptides, comprising: The target polypeptide is expressed using the synthetic intron as described in claim 1 or 2 or the polynucleotide construct as described in any one of claims 3 to 5.

7. A polynucleotide construct comprising: The LMNA gene exons encoding Lamin A and Lamin C are spliced ​​using variable splicing. And, the synthetic intron as described in claim 1 or 2; The synthetic intron is located between exon 10 and exon 11 of the LMNA gene, and a motif for regulating variable splicing is provided at the junction of the synthetic intron and the two exons.

8. The polynucleotide construct according to claim 7, wherein, The motif of the modulated variable splicing includes a 5' motif and a 3' motif; Wherein, the 5' motif contains any one of the nucleotide sequences shown in SEQ ID No. 17 to 23, or a nucleotide sequence having at least 90% homology with any one of the nucleotide sequences shown in SEQ ID No. 17 to 23; The 3' motif contains the nucleotide sequence described in SEQ ID No. 24, or a nucleotide sequence that has at least 90% homology with the nucleotide sequence described in SEQ ID No.

24.

9. The polynucleotide construct according to claim 7 or 8, wherein, The LaminA contains an amino acid sequence as shown in SEQ ID No. 14, or an amino acid sequence having at least 90% homology with the sequence shown in SEQ ID No. 14; The LaminC contains an amino acid sequence as shown in SEQ ID No. 15, or an amino acid sequence that has at least 90% homology with the sequence shown in SEQ ID No.

15.

10. The polynucleotide construct according to claim 7 or 8, wherein, The polynucleotide construct comprises: Nucleotide sequences as shown in SEQ ID No. 10 or 13, Alternatively, a nucleotide sequence that has at least 90% homology with the sequence shown in SEQ ID No. 10 or 13.

11. The polynucleotide construct according to claim 7, wherein, The expression of Lamin A and Lamin C is driven by different promoters selected from CMV promoter, CAG promoter, EF1α promoter, cTnT promoter, αMHC promoter, Des promoter, MLC2v promoter, CK8e promoter, MHCK7 promoter, SM22α promoter, ACTA1 promoter and Myog promoter.

12. A recombinant expression vector containing at least one copy of the polynucleotide construct as described in any one of claims 7 to 11.

13. The recombinant expression vector according to claim 12, wherein, The recombinant expression vector is an adenovirus or adeno-associated virus; optionally, the recombinant expression vector is AAV9.

14. A host cell containing at least one copy of the polynucleotide construct as described in any one of claims 7 to 11.

15. A pharmaceutical composition comprising the polynucleotide construct as described in any one of claims 7 to 11 or the recombinant expression vector as described in claim 12 or 13.

16. The use of the polynucleotide construct according to any one of claims 7 to 11, or the recombinant expression vector according to claim 12 or 13, or the host cell according to claim 14, in any of the following aspects: 1) Prevention, treatment and / or relief of disease; 2) To prepare medicines for the prevention, treatment and / or relief of diseases; in, The disease is associated with deletion mutations or downregulation of Lamin A and / or Lamin C expression.

17. The application according to claim 16, wherein, The disease includes laminosis, preferably including at least one of muscular dystrophy, limb-girdle muscular dystrophy, peripheral neuropathy, progeria, and dilated cardiomyopathy.

18. The application according to claim 16, wherein, The prevention, treatment, and / or alleviation of the disease includes at least one of the following aspects: i) Extend the survival time of the treated organisms; ii) Restore the nuclear membrane localization of Lamin A and Lamin C in the cardiomyocytes of the drug-treated patients; iii) Improve the cardiac function of the recipient; iv) Improve the cardiac electrophysiological condition of the drug recipient; v) Inhibits the cardiac dilation phenotype in the drug recipient.

19. A method for expressing Lamin A and / or Lamin C, comprising: Cultivate the host cells as described in claim 14; And optionally, the target protein can be recovered.

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