Polynucleotide constructs, adeno-associated virus vector, and methods for inducing production of SMN protein

Abstract

The present disclosure provides a polynucleotide for expressing survival of motor neuron (SMN) protein. Also provided are a vector and a recombinant Adeno- associated virus (AAV) comprising the polynucleotide that are capable of expressing the SMN protein, methods for preparing the recombinant AAV, and methods for inducing production of SMN protein in a cell.

Description

POLYNUCLEOTIDE CONSTRUCTS, ADENO-ASSOCIATED VIRUS VECTOR, AND METHODS FOR INDUCING PRODUCTION OF SMN PROTEIN FIELD OF INVENTION

[0001] The present disclosure broadly relates to polynucleotide constructs and Adeno-associated virus (AAV) vectors, and compositions thereof. In particular, the present invention relates to constructs and vectors for inducing production of a survival of motor neuron (SMN) protein, and methods of preparation and implementations thereof.BACKGROUND OF INVENTION

[0002] Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by loss or mutation of the telomeric copy of the survival of motor neuron (SMN1) gene located on chromosome 5. The disease is one of the most common genetic diseases leading to infant mortality.

[0003] The SMN1 gene normally produce a SMN protein (necessary for normal motor neuron function) which is full-length and functional. Mutation or deletion of SMN1 gene leads to the disease. In most individuals suffering from the SMA disease, deletion of the SMN1 gene has been found as the cause leading to the disease, while mutation of the SMN1 gene has been observed in some patients. Muscle-controlling nerve cells (motor neurons) being mostly located in the spinal cord, SMN protein deficiency results in the SMA disease with symptoms like progressive loss of motor neurons in the spinal cord, muscle weakness, and atrophy.

[0004] SMA is clinically classified into 5 types (SMA Type 0, Type I - IV) based on the severity of the symptoms, age of onset and survival of the patient wherein SMA Type 0 being the most severe form of the disease, while SMA Type IV being the mildest form of the disease.

[0005] The deficiency of SMN protein can partially be compensated by the presence of neighbouring SMN2 gene, which is an identical copy to SMN1 gene. However,most of the proteins produced from SMN2 genes are short and not functional while only a few are full-length and functional.

[0006] Conventionally, the SMA disease remains incurable as there is no effective method to deliver therapeutically active molecules to the spinal cord that may compensate for the SMN protein deficiency. Recently, several disease-modifying therapies including oral and intrathecal administration of antisense oligonucleotides have been introduced in clinical practice. These antisense oligonucleotides enhance the production of fully functional SMN protein by binding to the pre-mRNA of the SMN2 gene. However, these therapeutic approaches are expensive, less effective, and cause adverse effects such as pyrexia, headache, vomiting, and back pain in patients. Therefore, there exists a need for improved, cost-effective and efficient therapies for treatment of spinal muscular atrophy.SUMMARY OF THE INVENTION

[0007] In an initial aspect of the present disclosure, there is provided a polynucleotide comprising: (a) a nucleic acid insert having a transgene encoding Survival of Motor Neuron (SMN) protein operably linked to a promoter, and an intron; and (b) at least one 5'-inverted terminal repeat (5'-ITR) and at least one 3'-inverted terminal repeat (3'-ITR), heterologous to the transgene, wherein the 5'-ITR is positioned at the 5' end of the nucleic acid insert, and the 3'-ITR is positioned at the 3' end of the nucleic acid insert.

[0008] In another aspect of the present disclosure, there is provided a self-complementary polynucleotide, preferably self-complementary DNA (scDNA), comprising the polynucleotide as disclosed herein.

[0009] In another aspect of the present disclosure, there is provided a vector comprising the polynucleotide as disclosed herein.

[0010] In another aspect of the present disclosure, there is provided a recombinant AAV comprising the polynucleotide, the self-complementary polynucleotide, or the vector as disclosed herein.

[0011] In an aspect of the present disclosure, there is provided a composition comprising the polynucleotide, the self-complementary polynucleotide, the vector, or the recombinant AAV as disclosed herein.

[0012] In an aspect of the present disclosure, there is provided a host cell comprising the polynucleotide, the self-complementary polynucleotide, or the vector as disclosed herein.

[0013] In an aspect of the present disclosure, there is provided a method of preparing recombinant AAV, comprising: (a) transfecting a producer cell with a vector as disclosed herein, and providing the producer cell with AAV2 replication genes (AAV2-Rep) and AAV9 capsid genes (AAV9-Cap); culturing the producer cell under conditions to allow replication and packaging of recombinant AAV particles; and harvesting the recombinant AAV particles by lysing the producer cell.

[0014] In an aspect of the present disclosure, there is provided a method for treating and / or preventing spinal muscular atrophy in a subject, comprising administering the polynucleotide, the self-complementary polynucleotide, the vector, or the recombinant AAV as disclosed herein.

[0015] In an aspect of the present disclosure, there is provided a method for treating and / or preventing spinal muscular atrophy in a subject comprising, producing a recombinant AAV comprising the polynucleotide as disclosed herein; and administering the recombinant AAV to the subject.

[0016] In an aspect of the present disclosure, there is provided a method for inducing production of SMN protein in a cell, comprising providing the polynucleotide, the self-complementary polynucleotide, the vector, or the recombinant AAV, as disclosed herein, to the cell and expressing the protein.

[0017] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essentialfeatures of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0018] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0019] Figures 1(a) and 1(b) depict schematic representations of exemplary polynucleotides, in accordance with the embodiments herein.

[0020] Figure 2 depicts a flowchart of method for preparing the recombinant AAV, in accordance with the embodiments herein.

[0021] Figure 3(a) and 3(b) shows the transfection efficiency of the EGFP construct by fluorescence microscopy in (a) adherent HEK293 cells (b) and suspension HEK293 cells, in accordance with the embodiments herein.

[0022] Figures 4(a) and 4(b) show the quantitative analysis of transfection efficiency of different EGFP constructs by flow cytometry analysis. Data are presented as percentage (%) of transfected cells relative to total viable cell population, in accordance with the embodiments herein.

[0023] Figures 5(a) and 5(b) show the transduction efficiency of EGFP Construct 2 by flow cytometry analysis, at different multiplicity of infection (MOI) in U87-MG cells. Data are presented as percentage (%) EGFP-positive cells relative to untreated control, in accordance with the embodiments herein (2.5 * 104viral particles per cell (25K virus), 5.0 x 104viral particles per cell (50K virus), 7.5 x 104viral particles per cell (75K virus), and 1.0 x 105viral particles per cell (100K virus))

[0024] Figures 6(a) and 6(b) show transduction efficiency of EGFP Constructs 2 and 3 in U87-MG cells at 1 x 105viral particles per cell. Data are expressed as percentage (%) EGFP -positive cells, in accordance with the embodiments herein.

[0025] Figures 7(a) and 7(b) show the transduction efficiency of EGFP Construct 2 in U87-MG and SH-SY5Y cells at varying multiplicities of infection (MOIs). Data are presented as percentage (%) EGFP-positive cells relative to untreated controls, in accordance with the embodiments herein.

[0026] Figures 8(a), 8(b), and 8(c) show transduction efficiency of EGFP Construct 2 in U87-MG and SHSY-5Y cells as determined by cDNA expression and endpoint PCR analysis. Figures 8(a) and 8(b) show quantitative analysis of EGFP cDNA expression levels normalized to an internal housekeeping gene (GAPDH) and expressed relative to untransduced (OK) control cells of (a) U87-MG and (b) SHSY-5Y. Figure 8(c) shows agarose gel electrophoresis image of end-point PCR products amplified from cDNA derived from OK control cells and 75K (7.5 x 104viral particles per cell) and 100K (1.0 x io5viral particles per cell) transduced cells. The housekeeping gene (GAPDH) amplification serves as an internal control for cDNA quality and loading consistency, in accordance with the embodiments herein.

[0027] Figures 9(a) and 9(b) show the transduction efficiency of SMN1 Construct 2 in U87-MG and SHSY-5Y cells at 1.0 xlO5viral particles per cell, in accordance with the embodiments herein.

[0028] Figures 10(a) and 10(b) show the dose-dependent transduction efficiency of SMN1 Construct 2 as evaluated by Western blot analysis in U87-MG and SHSY-5Y cells. P-tubulin was used as a loading control to confirm equal protein loading across samples. Densitometric analysis of SMN1 band intensity was normalized to the loading control and is presented as relative expression compared to untransduced cells, in accordance with the embodiments herein.

[0029] Figures 11(a), 11(b), and 11(c) show the in vivo effect on the lifespan, body weight, and motor functions of the SMN1 construct in mice. Control mice (untreated, n=4; and formulation buffer, n=4), and mice injected with a low dose ofthe construct (SMN1 construct (L), n=5), and medium dose of the construct (SMN1 construct (M), n=4), in accordance with the embodiments herein.

[0030] Figures 12(a), 12(b), and 12(c) show the expression of the SMN1 construct in the neuromuscular system of the mice, in accordance with the embodiments herein.DETAILED DESCRIPTION OF THE INVENTION

[0031] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

[0032] Sequences listed in the disclosure:

[0033] Table 1:Definitions

[0034] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0035] The articles “a”, “an” and “the” are used to refer to one or more than one (i.e., to at least one) of the grammatical object of the article.

[0036] The terms “comprise” or “contain” and “comprising” or “containing” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0037] The term "at least one" is used to mean one or more and thus includes individual components as well as mixtures / combinations.

[0038] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0039] The term “including” is used to mean “including but not limited to”. The terms “including” and “including but not limited to” are used interchangeably.

[0040] The terms “nucleic acid”, “polynucleotide”, or “nucleotide fragment”, used interchangeably herein, refers to a chain of nucleotides. It includes deoxyribose nucleic acid (DNA) and ribose nucleic acid (RNA).

[0041] The term “vector”, as used herein, broadly refers to a nucleic acid molecule, such as DNA or RNA, that is capable of transferring a passenger nucleic acid sequence (i.e., DNA or RNA) into a cell. The term vector includes plasmids, viruses, and phages. In an embodiment, the vector is a plasmid. In another embodiment, the vector is a virus, also referred to as a viral vector. In an embodiment, the vector or viral vector is a recombinant adeno-associated virus (AAV) comprising a transgene. The viral vector may comprise some or all the viral gene required for viral replication, and packaging of viral particles. In another embodiment, the vector is a recombinant adeno-associated virus comprising a transgene, AAV-Rep genes encoding AAV replication proteins (for e.g.: Rep78, Rep68, Rep52, and Rep40), and AAV-Cap genes encoding AAV capsid proteins (for e.g.:VPl, VP2, and VP3). In some embodiments, the vector is a plasmid having a nucleotide sequence of at least 90% sequence identity to a sequence selected from SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, and SEQ ID No. 23.

[0042] As used herein, a first sequence is considered to “comprise a sequence with at least X % identity to” a second sequence if an alignment of the first sequence to the second sequence shows that X % or more of the positions of the second sequence in its entirety are matched by the first sequence. The differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogues do not contribute to differences in identity or complementarity among polynucleotides. Exemplary alignment algorithms that may be used to determine the percentage identity are the Smith -Waterman and Needleman-Wunsch algorithms, which are well-known in the art. A person of skill in the art would understand the choice of settings or parameters of said algorithms to determine the percentage identity, however in general, the default settings of said algorithms are generally appropriate.

[0043] Embodiments herein provide a polynucleotide for expressing survival of motor neuron (SMN) protein. Accordingly, the polynucleotide, according to embodiments herein, comprises a nucleic acid insert comprising a transgene encoding a SMN protein. Further embodiments herein provide a self-complementary polynucleotide and a vector comprising the polynucleotide as disclosed herein. Also disclosed herein are recombinant Adeno-associated virus comprising the polynucleotide or self-complementary polynucleotide as disclosed herein, capable of expressing SMN protein. The polynucleotides, self-complementary polynucleotide, vectors, and recombinant AAV, as disclosed herein may be used for expressing SMN protein in subjects. Deletion or mutations in the SMN1 gene in a subject causes a deficiency of functional SMN protein in the subject which may further result in spinal muscular atrophy (SMA). Providing the polynucleotides, self-complementary polynucleotides, vectors, and recombinant AAVs of the present disclosure to a subject reduces any deficiency of native SMN protein in the subject, and facilitates treatment of SMA and alleviates symptoms associated with conditions such as spinal muscular atrophy (SMA). Accordingly,embodiments herein provide a method for treating and / or preventing SMA or symptoms thereof. Further embodiments herein also provide a recombinant AAV and methods for preparing the recombinant AAV.Polynucleotide

[0044] Embodiments herein provide a polynucleotide for expressing survival of motor neuron (SMN) protein. Accordingly, the polynucleotide, according to embodiments herein, comprises a nucleic acid insert comprising a transgene encoding a survival of motor neuron (SMN) protein. The polynucleotide may be in the form of a single stranded structure, for e.g.: single stranded DNA (ssDNA) molecule, or a double stranded structure, for e.g. : double stranded DNA (dsDNA) molecule. In a preferred embodiment, the polynucleotide is a single stranded DNA molecule that folds itself to form double stranded DNA molecule. The polynucleotide, according to embodiments herein, comprises a nucleic acid insert comprising self-complementary sequences that facilitate formation of dsDNA.

[0045] The term “complementary” or “complement” in the context of nucleic acids refers to the ability of a nucleic acid or nucleotide base to form base pairs with another nucleic acid or nucleotide base. The term “self-complementary”, as used herein, refers to the ability of a single strand of nucleic acid to form base pairs with itself. A single stranded nucleic acid (for e.g. ssDNA) can fold back on itself to form a duplex, double-stranded structure (for e.g. dsDNA) which can serve as a template for nucleic acid synthesis. The term self-complementary, according to embodiments herein, refers to the ability of a single strand of nucleic acid to fold back on itself to form a double stranded structure. The term “self-complementary DNA”, as used herein, refers to a single stranded DNA (i.e. ssDNA) molecule capable of folding back on itself to form a double stranded structure (i.e. dsDNA). In an embodiment, the single stranded nucleic acid or ssDNA comprises complementary fragments or complementary nucleotide sequences that facilitate folding itself. Complementarity may be complete or partial complementarity (i.e.100% or lesser). For example, the term “complementary sequence” includes anucleotide sequence that is partially or completely complementary to another sequence. Typically, a complementary sequence is non-covalently attached to another sequence, wherein the complementarity of the two strands is defined by the complementarity of the bases. For example, the base A in one sequence pairs with the base T or U on the other, and the base G in one sequence pairs with the base C on the other. In some embodiments, the complementary sequence is partially complementary so as to hybridize or bind with another sequence. The term “partially complementary” as used herein encompasses the term “substantially complementary” and refers to complementarity wherein the nucleic acid is capable of hybridizing to another. The complementary sequence or complementary fragment or complementary nucleic acid refers to a sequence or fragment or nucleic acid, respectively, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85, at least 90%, at least 95%, or 100% complementarity to another.

[0046] In an embodiment, the polynucleotide comprises (a) a nucleic acid insert having a transgene encoding a SMN protein operably linked to a promoter; and (b) inverted terminal repeats (ITR). The SMN protein, according to embodiments herein, include, but is not limited to, the human SMN protein as set out in NCBI protein database number NP 000335.1 (SEQ ID NO. 13). In an embodiment, the polynucleotide comprises (a) a nucleic acid insert having a transgene encoding a SMN protein having at least 95%, at least 97%, at least 98%, or 100% identity to an amino acid sequence as set forth in SEQ ID NO. 13, operably linked to a promoter; and (b) at least two inverted terminal repeats (ITR).

[0047] In an embodiment, the polynucleotide comprises a 5'-inverted terminal repeat (5'-ITR) positioned at the 5' end; and at least one 3'-inverted terminal repeat (3 '-ITR) positioned at the 3' end.

[0048] In an embodiment, the polynucleotide comprises (a) a nucleic acid insert having a transgene encoding SMN protein having at least 95% identity to an amino acid sequence as set forth in SEQ ID NO. 13, operably linked to a promoter, and anintron; and (b) at least one 5'-inverted terminal repeat (5'-ITR) and at least one 3'-inverted terminal repeat (3'-ITR), wherein the 5'-ITR is positioned at the 5' end of the nucleic acid insert, and the 3'-ITR is positioned at the 3' end of the nucleic acid insert.

[0049] In an embodiment, the polynucleotide comprises (a) a nucleic acid insert having a transgene encoding SMN protein having an amino acid sequence as set forth in SEQ ID NO. 13, operably linked to at least one promoter, and an intron; and (b) at least one 5'-inverted terminal repeat (5'-ITR) and at least one 3'-inverted terminal repeat (3'-ITR), heterologous to the transgene, wherein the 5'-ITR is positioned at the 5' end of the nucleic acid insert, and the 3'-ITR is positioned at the 3' end of the nucleic acid insert.Nucleic acid insert

[0050] The polynucleotide, according to embodiments herein, comprises a nucleic acid insert. The nucleic acid insert comprises a transgene encoding a SMN protein. In an embodiment, the nucleic acid insert further comprises at least one promoter; and an intron, such that the transgene is operably linked to the promoter. In an embodiment, the intron is positioned upstream to the transgene. Accordingly, in an embodiment, there is provided a polynucleotide comprising a nucleic acid insert having a transgene encoding the SMN protein operably linked to the promoter, and an intron, preferably the intron is positioned upstream to the transgene. In another embodiment, the nucleic acid insert comprises a transgene encoding the SMN protein operably linked to at least one promoter, an intron, an enhancer, and a polyadenylation signal. Accordingly, in an embodiment, there is provided a polynucleotide comprising a nucleic acid insert, wherein the nucleic acid insert further comprises an enhancer, and a polyadenylation signal.

[0051] The transgene, according to embodiments herein, is a nucleotide encoding the SMN protein, preferably human SMN protein. The transgene may be codon optimized to facilitate expression in a suitable expression system. In anembodiment, the transgene has a nucleotide sequence of at least 90%, at least 95% or at least 98% identity to a sequence as set forth in SEQ ID NO. 6.

[0052] In an embodiment, the polynucleotide comprises (a) a nucleic acid insert comprising a transgene having a nucleotide sequence of at least 90% identity to a sequence as set forth in SEQ ID NO. 6, operably linked to at least one promoter, and an intron; and (b) at least one 5'-inverted terminal repeat (5'-ITR) and at least one 3'-inverted terminal repeat (3'-ITR), heterologous to the transgene, wherein the 5'-ITR is positioned at the 5' end of the nucleic acid insert, and the 3'-ITR is positioned at the 3' end of the nucleic acid insert.

[0053] In an embodiment, the intron is from simian virus 40 (SV40) and has a nucleotide sequence of at least 90% sequence identity to a sequence as set forth in SEQ ID NO: 5.

[0054] The promoter, according to embodiments herein, may be suitable for tissue specific expression of the transgene in subjects. In an embodiment, the promoter is selected from a chicken beta-actin (CBA) promoter, chicken beta-actin hybrid (CBh) promoter, or combination thereof. In an embodiment, the nucleic acid insert comprises a promoter, wherein the promoter is selected from a chicken beta-actin (CBA) promoter having at least 90% identity to a nucleotide sequence as set forth in SEQ ID No. 3, a portion thereof; and a chicken beta-actin hybrid (CBh) promoter having at least 90% sequence identity to a nucleotide sequence as set forth in SEQ ID No. 4, or combination thereof. The promoter regulates the expression of the transgene and may also be referred to herein as the regulatory element. The regulatory element may further comprise an enhancer, preferably positioned upstream of the promoter. Accordingly, in an embodiment, the regulatory element comprises a promoter and enhancer, preferably having at least 90% identity to a nucleotide sequence as set forth in SEQ ID No. 3.

[0055] In another embodiment, the regulatory element comprises two promoters, and an enhancer. In an embodiment, the nucleic acid insert comprises two promoters and an enhancer, wherein the promoters are selected from a chicken beta-actin (CBA) promoter, chicken beta-actin hybrid (CBh) promoter, or combination thereof, preferably flanking the enhancer. In an embodiment, the nucleic acid insert comprises a promoter having at least 90% identity to a nucleotide sequence as set forth in SEQ ID No. 3; and a CBh promoter having at least 90% identity to a nucleotide sequence as set forth in SEQ ID No. 4, preferably the CBh promoter is upstream to the CBA promoter.

[0056] The nucleic acid insert, as disclosed herein, comprises a transgene operably linked to a promoter, an intron, an enhancer, and a polyadenylation signal. As described herein above, in an embodiment, the enhancer is positioned upstream to the promoter to form the regulatory element that regulates the expression of the transgene positioned downstream. In an embodiment, the nucleic acid insert comprises an enhancer, wherein the enhancer is cytomegalovirus (CMV) enhancer having a nucleotide sequence of at least 90% sequence identity to a sequence as set forth in SEQ ID No. 24, or portion thereof.

[0057] The polyadenylation signal, according to embodiments herein, is operably linked to the transgene, preferably downstream to the transgene. In an embodiment, the nucleic acid insert comprises a polyadenylation signal, preferably the polyadenylation signal is selected from polyadenylation signal of bovine growth hormone (bGH) gene or polyadenylation signal of Simian virus 40 (S V40). A strong polyadenylation signal stabilizes mRNA and enhances transcription termination. It also increases production, nuclear export and / or translation of vector encoded mRNA. This in turn increases the mRNA levels of the transgene and results in higher productivity by the producer cells. The polyadenylation signals are termination signals for transcription which are required for polyadenylation of the transcribed mRNA. The polyadenylation signals of bGH and SV40 are termination signals derived from the 3' untranslated region of bovine growth hormone gene and SV40 gene, respectively.

[0058] Embodiments herein include inverted terminal repeats (ITRs). The ITRs, according to embodiments herein, refer to the inverted terminal repeats present at5' and 3' ends of the polynucleotide and / or the nucleic acid insert. The ITRs have nucleotide sequences that are complementary to each other and facilitate in base pairing the end regions of the polynucleotide, and / or duplex (also referred to as hairpin) formation of the single stranded polynucleotide. In an embodiment, there is provided a polynucleotide comprising at least two ITRs. In an embodiment, there is provided a polynucleotide comprising at least one 5'-inverted terminal repeat (5'-ITR) and at least one 3 '-inverted terminal repeat (3'-ITR). In an embodiment, there is provided a polynucleotide comprising at least one 5'-inverted terminal repeat (5'-ITR) and at least one 3'-inverted terminal repeat (3'-ITR), wherein the 5'-ITR is positioned at the 5' end of the nucleic acid insert, and the 3'-ITR is positioned at the 3' end of the nucleic acid insert.

[0059] In an embodiment, the ITRs are heterologous to the transgene. In an embodiment, the ITRs are derived from AAV. In another embodiment, the ITRs comprise a 5'-ITR and a 3'-ITR, wherein the 5'-ITR and 3'-ITR are derived from AAV. The ITRs may be modified ITRs or unmodified ITRs. In an embodiment, the 5'-ITR and / or a 3'-ITR are ITRs derived from AAV. In another, 5'-ITR and / or a 3'-ITR are modified ITRs comprising at least one modification (also referred to herein as “hybrid ITR”).

[0060] The 5'-ITR and 3'-ITR may or may not be completely complementary to each other. In an embodiment, the 5'-ITR and 3'-ITR may not be completely complementary to each other, i.e., they may include one or more mis-matched base pairs. The mis-matched base pairs of the ITRs helps in improving the packaging efficiency of the polynucleotide into AAV particles during preparation of recombinant AAV. The ITRs, according to embodiments herein, allows the polynucleotide to fold into its dsDNA structure (as shown in Figure 1(a) and (b)), speeding up the expression of the transgene by providing immediate dsDNA for transcription and minimizing recombination events that could compromise the stability of the polynucleotide. The dsDNA structure of the polynucleotide provides faster and efficient gene expression of the transgene compared to, for example, a single stranded structure of a polynucleotide. In a preferred embodiment, there isprovided a polynucleotide comprising at least one 5'-ITR having a nucleotide sequence of at least 98% sequence identity to a sequence as set forth in SEQ ID No.1 or SEQ ID No. 15; and 3'-ITR having a nucleotide sequence of at least 98% sequence identity to a sequence as set forth in SEQ ID No. 2 or SEQ ID No.: 16.

[0061] In an embodiment, the polynucleotide is a single stranded or double stranded DNA. In another embodiment, there is provided a polynucleotide, wherein the polynucleotide is a double stranded DNA comprising the nucleic acid insert, and a complementary fragment of the nucleic acid insert covalently attached at the 3' end, allowing formation of the double stranded DNA (dsDNA).

[0062] The polynucleotide, according to embodiments herein, may comprise one or more copies of the nucleic acid insert and / or complementary fragments, thereof preferably positioned to facilitate self-folding of the polynucleotide. In another embodiment, the polynucleotide comprises the nucleic acid insert comprising self-complementary sequences / fragments, and folds itself to form a self-complementary polynucleotide (scP), preferably a self-complementary DNA (scDNA). In another embodiment, the polynucleotide forms a self-complementary polynucleotide (scP), preferably a self-complementary DNA (scDNA). In an embodiment, the polynucleotide folds itself to form a double stranded structure, preferably a dsDNA.

[0063] In an embodiment, there is provided a self-complementary polynucleotide comprising the polynucleotide as disclosed herein.

[0064] Embodiments herein provide a composition for therapeutic application. In an embodiment, the composition comprises the polynucleotide, the self-complementary polynucleotide, vector, and / or recombinant AAV as disclosed herein. In another embodiment, the composition further comprises at least one pharmaceutically acceptable excipient. Excipients include carrier, diluent, buffer, sugars, or salt. Various excipients are known in the art and may be used in the composition according to embodiments herein.

[0065] Figures 1(a) and 1(b) depict schematic representations of exemplary polynucleotides, in accordance with the embodiments herein. Figure 1(a) depicts aportion of an scDNA molecule, for incorporation in an Adeno-associated virus particle (or particle). The scDNA molecule extends between a 5’ end and a 3’ end. The scDNA molecule includes a plurality of pre-defined regions arranged along the length of the scDNA molecule. The regions include, but are not limited to, inverted terminal repeats (ITRs), chicken beta-actin (CBA) promoter, at least one intron, SMN1 transgene (or transgene), polyadenylation signal. Additionally or optionally, an enhancer may be disposed upstream of the promoter. The enhancer is encoded by a portion of the promoter CBA.

[0066] Figure 1(b) depicts a portion of an scDNA molecule, wherein the scDNA molecule extends between a 5’ end and a 3’ end, and includes inverted terminal repeats (ITRs), the promoters: chicken beta-actin (CBA) promoter and chicken beta-actin hybrid (CBh) promoter, an intron, SMN1 transgene (or transgene), a one polyadenylation signal. Additionally or optionally, an enhancer may be disposed upstream of the promoter. The enhancer is encoded by a portion of the promoter CBA. In an embodiment, the enhancer has a nucleotide sequence as set forth in SEQ ID NO: 24.Methods

[0067] Embodiments herein provide a method for preparing recombinant AAV as disclosed herein. The polynucleotide, as disclosed herein, may be introduced into a vector which is then introduced into a producer cell. The producer cell may then be cultured / incubated to obtain recombinant AAV particles. In an embodiment, the method for preparing recombinant AAV comprises (a) transfecting a producer cell with a vector comprising the polynucleotide as disclosed herein; (b) culturing the producer cells (i.e. transfected cells) under conditions to allow replication and packaging of recombinant AAV particles; and (c) harvesting the recombinant AAV particles by lysing the producer cells. The term “producer cell”, as used herein, refers to cells that are capable of replicating and packaging AAV particles. In an embodiment, the producer cells comprise AAV2 replication genes (AAV2-Rep) and AAV9 capsid gene (AAV9-Cap) for replicating and packaging AAV particlesin the cell. In an example, the producer cells may comprise AAV-Rep genes encoding AAV replication proteins (for e.g.: Rep78, Rep68, Rep52, and Rep40), and AAV-Cap genes encoding AAV capsid proteins (for e.g.:VPl, VP2, and VP3).

[0068] In an embodiment, the method for preparing recombinant AAV comprises (a) transfecting a producer cell with a vector comprising the polynucleotide as disclosed herein; (b) culturing the producer cells (i.e. transfected producer cells), preferably for a period of 40 to 72 hours, 40 to 65 hours, or 45 to 50 hours, more preferably for 48 hours, preferably at 35 °C to 38°C, more preferably 37°C, preferably inside 7-10%, more preferably 8%, CO2 environment, to allow replication and packaging of recombinant AAV particles; and (c) harvesting the recombinant AAV particles by lysing the producer cells.

[0069] In an embodiment, the method for preparing recombinant AAV comprises (a) transfecting a producer cell with a vector comprising the polynucleotide as disclosed herein, and providing the producer cell with a AAV2 replication genes (AAV2-Rep) and AAV9 capsid gene (AAV9-Cap); (b) culturing the producer cells under conditions to allow replication and packaging of recombinant AAV particles, preferably wherein the recombinant AAV particles are produced in the cell; and (c) optionally, harvesting the recombinant AAV particles by lysing the producer cells.

[0070] In an embodiment the conditions, that allow replication and packaging of recombinant AAV particles, include a period of 40 to 72 hours, 40 to 65 hours, or 45 to 50 hours, more preferably for 48 hours, preferably at 35°C to 38°C, more preferably 37°C, preferably inside 7-10%, more preferably 8%, CO2 environment.

[0071] In an embodiment, the method for preparing recombinant AAV comprises (a) transfecting a producer cell with a vector comprising the polynucleotide as disclosed herein, and providing the producer cell with AAV2 replication genes (AAV2-Rep) and AAV9 capsid genes (AAV9-Cap); (b) culturing the producer cells (i.e. transfected producer cells), preferably for a period of 40 to 72 hours, 40 to 65 hours, or 45 to 50 hours, more preferably for 48 hours, preferably at 35°C to 38°C, more preferably 37°C, preferably inside 7-10%, more preferably 8%, CO2environment, to allow the producer cell to produce the recombinant AAV; and (c) harvesting the recombinant AAV by lysing the producer cells.

[0072] In an exemplary embodiment, the method for preparing recombinant AAV may be performed by suspension-based cell culture systems that that enable high-yield production of particles of the recombinant AAV. Alternatively, adherentbased cell culture systems may also be used to produce the particles of the recombinant AAV.

[0073] The method, according to the present disclosure, includes transfecting a producer cell with a vector comprising the polynucleotide, having the transgene encoding SMN protein, as disclosed herein. In an embodiment, the vector is a plasmid. The plasmid comprises the polynucleotide having the transgene encoding SMN protein, according to embodiments herein. Transfection may be performed by methods generally known in the field including electroporation, gene gun method, using transfection agents such as Polyethylenimine (PEI), calcium phosphate, etc. In an embodiment, the transfection is performed by contacting a pre-defined amount of producer cells with a pre-defined amount of the vector / plasmid having the transgene; and providing the producer cells with AAV2 replication genes (AAV2-Rep) and AAV9 capsid genes (AAV9-Cap), in the presence of a transfection agent. In an embodiment, the pre-defined amount of producer cells ranges from 3.0 x 106cells / mL to 3.5 x 106cells / mL. The AAV2-Rep genes and the AAV9-Cap genes may be provided by way of one or more plasmids. In an embodiment, the AAV2-Rep genes and the AAV9-Cap genes are provided in a single plasmid. In an embodiment, the vector having the polynucleotide as disclosed herein; and the plasmid having the AAV2-Rep genes and the AAV9-Cap genes may be in a mole ratio of 1 : 1.

[0074] In an embodiment, the AAV9-Cap genes may be derived from Adeno-associated virus serotype 9, preferably having a nucleotide sequence as set forth in SEQ ID No. 12. The AAV2-Cap genes encode a plurality of proteins including, but not limited to, VP1, VP2, and VP3. The proteins encoded by the AAV9-Cap genesmake the structural proteins of the capsid (i.e., the outer body / shell of the recombinant AAV particle). Accordingly, the proteins define the serotype of the particles which influences the affinity of the particles to specific tissues (for example, central nervous system, muscle, liver, and heart cells).

[0075] The AAV2-Rep genes may be derived from Adeno-associated virus serotype 2. The AAV2-Rep genes encode for a plurality of proteins including, but not limited to, Rep78, Rep68, Rep52, and Rep40. The proteins encoded by AAV2-Rep identifies the ITRs of the polynucleotide, as disclosed herein, and helps in replication and packaging of the polynucleotide.

[0076] The producer cells may further be provided with AAV helper plasmid. The AAV helper plasmid may be derived from helper genes of the Adeno-associated virus. The AAV helper plasmid includes a plurality of genes including, but not limited to, E2A, E4, and VA (Virus-associated RNA genes). These genes of the AAV helper plasmid helps the producer cell in replication and packaging of the polynucleotide. In an embodiment, the vector or plasmid having the polynucleotide as disclosed herein; the plasmid having the AAV2-Rep genes and the AAV9-Cap genes; and the AAV helper plasmid may be in a mole ratio range of 05-1 : 05- 1 :0.5-1, preferably 1:1:1.

[0077] In an embodiment, the producer cells are human embryonic kidney 293 (HEK293) cells.

[0078] In an embodiment, transfection is performed in the presence of a transfection agent selected from Polyethylenimine (PEI), calcium phosphate, or combination thereof. In an embodiment, the plasmids and the transfection agent are at a ratio range by weight of 0.5-1 :2.0-2.5; preferably at 1:2 or 1:2.5.

[0079] The transfection may be performed using a transfection mix comprising the plasmids and transfection agent. In an embodiment, transfection is performed by providing a transfection mix comprising the plasmids and transfection agent and the producer cells, in a first pre-defined medium, and incubating. In an embodiment, said transfecting is by providing a transfection mix comprising the plasmids andtransfection agent; and producer cells, in a first pre-defined medium, and incubating, preferably at 22 °C to 25 °C for a pre-defined time period ranging from 10 mins to 20 mins with periodic mixing. In an embodiment, the first pre-defined medium is chemically defined serum-free Dulbecco's Modified Eagle Medium (DMEM) (for e.g.: procured from Thermofisher Scientific) supplemented with 6 g / L of glucose, 4mM of L-Glutamine, 2.20 g / L of Sodium Bicarbonate, and 2 mL / L Anti-Clumping Agent.

[0080] In an embodiment, said transfecting is by providing a transfection mix comprising the plasmids and transfection agent; and producer cells, in a first predefined medium, and incubating at 25 °C for 20 mins with periodically vortex mixing after every 5 mins to 7 mins. Transfection may be performed in a tissue culture flask. The mixture comprising the transfection mix comprising the plasmids and transfection agent; and producer cells, is also referred to herein as “first mixture”.

[0081] The first mixture, post-transfection, having the transfected cells is then incubated under conditions to allow replication and packaging of recombinant AAV particles. The recombinant AAV particles are produced in the cell, which is then harvested.

[0082] The method, according to the present disclosure, includes the step of harvesting. Harvesting may be performed by methods known in the art.

[0083] In an embodiment, harvesting comprises centrifuging at pre-defined speed ranging from 4000 g to 4300 g, at pre-defined temperature ranging from 4 °C to 10 °C for a pre-defined time period ranging from 10 mins to 15 mins, to obtain a pellet; treating the pellet with a lysis buffer to obtain a lysate; treating the lysate with at least one endonuclease at a pre-defined temperature for a pre-defined time period to obtain a treated lysate; centrifuging the treated lysate to collect the supernatant; and subjecting the supernatant to one or more filtration steps to obtain a filtrate comprising recombinant AAV particles.

[0084] In an exemplary embodiment, the first mixture is centrifuged at 4300 g, 4 °C for 15 mins, to obtain a pellet.

[0085] In an embodiment, the pellet is treated with a lysis buffer comprising 50mM Tris HC1, 2 mM MgC12, 150 mM NaCl and 0.5% Triton X-100.

[0086] In an embodiment, the lysate is treated with at least one of Salt Active Nuclease High Quality (SAN-HQ) or Benzonase, at pre-defined temperature ranging from 30 °C to 37 °C for a pre-defined time period ranging from 45 mins to 60 mins, wherein the concentration of the endonucleases is in the range of 50U / mL to 100 U / mL. In a preferred embodiment, the lysate is treated with 100 U / mL of SAN-HQ at 37 °C for 60 mins.

[0087] In an embodiment, the treated lysate is centrifuged at a pre-defined speed ranging from 4000 g to 4300 g, at pre-defined temperature ranging from 4 °C to 10 °C for pre-defined temperature ranging from 4 °C to 10 °C, to collect the supernatant. In a preferred embodiment, the treated lysate is centrifuged at 4300 g, 4 °C for 20 mins. The pellet obtained is discarded.

[0088] Filtration may be performed using methods generally known in the art. In an embodiment, the supernatant is passed through a depth filter clarification to remove large particulate matter. Additionally or optionally, the filtrate may then be passed through a secondary filter. The secondary filter, in an embodiment, has a pore size ranging from 0.1 pm to 0.8 pm. The secondary filter may be made of resin, cellulose, diatomaceous earth (DE), etc. In an exemplary embodiment, the filtrate is passed through the secondary filter made of DE having pore size of 0.45 pm. The secondary filter reduces the turbidity of the filtrate to less than 40-50 NTU.

[0089] The filtrate may also be subjected to diafiltration to concentrate the particles per unit volume by one or more techniques. In an embodiment, the filtrate is concentrated by 5 to 10 fold using tangential flow filtration (TFF), to obtain the recombinant AAV particles. The TFF is performed using a filter having a molecular weight cut-off (MWCO) ranging from 100 KDa to 300 KDa. The filter has a predefined area ranging from 0.02 m2to 3.0 m2. In an exemplary embodiment, the TFFhas a filter having area of 0.1 m2with MWCO of 300 KDa. The filtrate is passed through the filter at a pre-defined flow rate and a pre-defined transmembrane pressure (TMP). The pre-defined flow rate ranges from 300 mL / min to 600 mL / min. The pre-defined TMP ranges from 0.2 bar to 0.6 bar. In an exemplary embodiment, the filtrate is passed through the filter at 400 mL / min, 0.4 bar.

[0090] In an alternate embodiment, the filtrate may be concentrated and purified using one of affinity column chromatography, CsCl ultracentrifugation or iodixanol gradient ultracentrifugation. In an exemplary embodiment, the filtrate is subjected to affinity column chromatography to obtain the recombinant AAV particles. The filtrate is loaded on a column (having POROS CaptureSelect AAVX resin column, procured from Thermofisher Scientific) having a pre-defined volume, i.e., column volume (CV). Before loading the filtrate, the column is equilibrated with 5-10 CV of IX phosphate buffer saline (PBS) having pH 7.4. The absorbance of an eluant exiting the column is monitored using an Akta pure 150 chromatography system (procured from Cytiva). After loading the filtrate in the column, the column retains the particles. The column is washed with IX PBS (ph 7.4) until a stable baseline absorbance is established. Thereafter, the column is first flushed with 5-8 CV of IM NaCl (sodium chloride) in lx PBS (ph 7.4). Then, the column is again flushed with 5-8 CV of lOOmM of sodium acetate buffer (pH about 5.0). Flushing the column washes away all the adulterants / impurities from the column and only the particles is retained within the column. After flushing the column, the particles are eluted from the column by adding 3-5 CV of lOOmM Glycine (ph 2.7) followed by stripping the column with 5-8 CV of 200mM Phosphoric acid (pH 2.0) to remove any tightly bound impurity.

[0091] The recombinant AAV particles, according to embodiments herein, may be filtered and suspended in a pre-defined medium to obtain a composition. In an exemplary embodiment, the particles are filtered through a 0.22 pm Polyethersulfone (PES) filter and suspended in a formulation buffer. In an embodiment, the formulation buffer may include one or more components including, but not limited to, surfactants, stabilizes, buffering agents such asphosphate based buffers, tonicity adjusting agents, wetting agents. For instance, the surfactants may be selected from polysorbate 20, polysorbate 80, pluronic acid, or combinations thereof; the buffers may be selected from acetate, succinate, citrate, histidine, phosphate buffered saline (PBS), Tris buffer, or combinations thereof; the tonicity adjusting agents and wetting agents may be selected from potassium chloride, glycerol, sorbitol, sodium acetate, sodium lactate, sodium chloride, magnesium chloride, calcium chloride, sorbitan monolaurate, triethanolomine oleate, or combinations thereof. In some embodiments, the formulation buffer comprises ImM- 5mM magnesium chloride, and 0.005% to 0.05% Pluronic acid, and one or more buffers selected from phosphate buffered saline (PBS), Tris buffer. The buffer may comprise from 10-20 mM Tris buffer (pH 8.0); 25mM to 40mM PBS buffer (pH 7.4), or combinations thereof.

[0092] Embodiments herein provide a method for expressing SMN protein. In an embodiment, the method for expressing SMN protein comprises: preparing the recombinant AAV particle, preferably by a method as disclosed herein; and transducing a host cell and incubating to allow expression of the SMN protein. In an exemplary embodiment, the method for expressing SMN protein comprises transducing host cell, preferably U87MG and SH-SY 5 Y cell lines, with the particles of the AAV vector of the present disclosure. The term “host cell”, as used herein refers to cells or cell lines where expression of SMN protein is intended. The term “host cells” includes cells having deficiency of functional SMN protein, cells having / expressing mutated SMN protein, or cells having deleted or mutated SMN1 and / or SMN2 gene.

[0093] Embodiments herein provide a method for treating and / or preventing spinal muscular atrophy in a subject. In an embodiment, the method for treating and / or preventing spinal muscular atrophy comprises administering the polynucleotide, the self-complementary polynucleotide, the vector, the recombinant AAV, or the composition, as disclosed herein, to said subject. In an embodiment, the host cells are selected from central nervous system, muscle, liver, and heart cells. The term “subject”, as used herein refers to human and non-human mammals. In anembodiment, subject is a human having or suspected of having deletion or mutation in the SMN1 gene. In an embodiment, subject is a patient having or suspected of having SMA or symptoms thereof.

[0094] In an embodiment, the method for treating and / or preventing spinal muscular atrophy comprises: (a) producing a recombinant AAV comprising the polynucleotide as disclosed herein; and administering the recombinant AAV to the subject.

[0095] In an embodiment, the method for inducing production of SMN protein in a cell comprises providing the polynucleotide, the self-complementary polynucleotide, the vector, the recombinant AAV, or the composition, as disclosed herein, to the cell and expressing the protein.

[0096] Figure 2 depicts a flowchart of method for preparing the recombinant AAV, in accordance with the embodiments herein.

[0097] In an exemplary embodiment, the method for preparing of the recombinant AAV comprises transfecting; incubating; harvesting; and filtration.

[0098] In an exemplary embodiment, there is provided a method comprising transfecting producer cells, wherein transfecting comprises by preparation of a transfection mix. A transfection mix may be prepared by mixing the plasmids, viz. plasmids having the AAV2-Rep genes & AAV9-Cap genes (procured from GenScript), AAV helper plasmid (procured from GenScript) and plasmid having the polynucleotide transgene (SEQ ID NO. 7 to 11) at a ratio by mole of 1 : 1 : 1; and adding a transfection agent (i.e. Polyethylenimine (PEI), such that the plasmids and the transfection agent are at a ratio by weight of 1:2.5. The transfecting step may further include incubation and contacting the producer cells and transfection mix. The transfection mix may be incubated at 25 °C for 10 mins, and the HEK293 cells (producer cells) at 3.5 million cells / mL is suspended in a tissue culture flask (or the like) having the first pre-defined medium (i.e. DMEM procured from Thermofisher Scientific, supplemented with 6 g / L of glucose, 4mM of L-Glutamine, 2.20 g / L of Sodium Bicarbonate and 2 mL / L Anti -Clumping Agent). The transfection mix maythen be added to the tissue culture flask having the producer cells (also referred to herein as the first mixture). 1 part by volume of transfection mix was added to 2.5 part by volume of producer cells (1:2.5). This may then be incubated at a predefined temperature of 25 °C for a pre-defined time period of 20 mins with periodically vortex mixing after every 5 mins to 7 mins.

[0099] In an exemplary embodiment, there is provided a method further comprises incubation. The tissue culture flask having the first mixture (i.e. the transfection mix and producer cells) may be incubated at 37 °C inside 8% CO2 environment for 48 hours with mixing after every 5 mins to 7 mins to allow replication and packaging of the recombinant AAV particles. The recombinant AAV particles are produced in the producer cell which may then be harvested.

[0100] In an exemplary embodiment, there is provided a method further comprises harvesting. After incubation for 48 hours, the contents from the tissue culture flask may be centrifuged at 4300 g, 4 °C for 15 mins to obtain a pellet. The pellet obtained comprises the producer cells. The pellet may be treated with 50mM Tris HC1, 2 mMMgC12, 150 mMNaCl and 0.5% Triton X-100 to lyse the producer cells and obtain a lysate. The producer cells in the pellet may be lysed using chemical lysis buffers. In an exemplary embodiment, the pellet is lysed using 50mM Tris HC1, 2 mM MgC12, 150 mM NaCl and 0.5% Triton X-100. The lysate may be treated with 100 U / mL of SAN-HQ at 37 °C for 60 mins. The treated lysate may be centrifuged at 4300 g, 4 °C for 20 mins, to collect the supernatant. The supernatant may be passed through a depth filter clarification to remove large particulate matter. The filtrate may be passed through a secondary filter made of diatomaceous earth (DE) having pore size of 0.45 pm, preferably to reduce the turbidity of the filtrate to less than 40-50 NTU.

[0101] In an exemplary embodiment, there is provided a method further comprises filtration. The filtrate obtained may be subjected to diafiltration. The filtrate may be concentrated by 5 to 10 fold using tangential flow filtration (TFF). The TFF may be performed using a filter having area of 0.1 m2 with MWCO of300 KDa, at a pre-defined flow rate and a pre-defined transmembrane pressure (TMP) of 400 mL / min, 0.4 bar, respectively, to obtain recombinant AAV particles.

[0102] Alternatively, the filtrate may be filtered using affinity column chromatography. The filtrated may be loaded on a column (having POROS CaptureSelect AAVX resin column, procured from Thermofisher Scientific) having a pre-defined volume, i.e., column volume (CV). Before loading the filtrate, the column may be equilibrated with 5-10 CV of IX phosphate buffer saline (PBS) having pH 7.4. The absorbance of an eluant exiting the column is monitored using an Akta pure 150 chromatography system (procured from Cytiva). After loading the filtrate in the column, the column retains the particles. The column may be washed with IX PBS (ph 7.4) until a stable baseline absorbance is established. Thereafter, the column is first flushed with 5-8 CV of IM NaCl (sodium chloride) in lx PBS (ph 7.4). Then, the column is again flushed with 5-8 CV of lOOmM of sodium acetate buffer (pH about 5.0). Flushing the column washes away all the adulterants / impurities from the column and only the particles is retained within the column. After flushing the column, the recombinant AAV particles are eluted from the column by adding 3-5 CV of lOOmM Glycine (ph 2.7) followed by stripping the column with 5-8 CV of 200mM Phosphoric acid (pH 2.0) to remove any tightly bound impurity.

[0103] In an exemplary embodiment, there is provided a method for preparation of recombinant AAV composition. The recombinant AAV particles may be filtered using 0.22 pm Polyethersulfone (PES) filter, and mixed with an excipient to obtain a composition. The recombinant AAV particles may be suspended in a formulation buffer comprising a buffer selected from 15-20 mM Tris buffer (pH 8.0) or 37mM PBS buffer (pH 7.4); ImM MgC12; and 0.005% Pluronic acid.

[0104] In an exemplary embodiment, there is provided a method for expressing SMN protein using the recombinant AAV. The recombinant AAV particles may be transduced into U87MG and SH-SY5Y cell lines and the SMN proteins expressed in the cell lines.

[0105] Although the present disclosure has been described in considerable detail with reference to certain embodiments and implementations thereof, other embodiments are possible to cover the modifications and variations of the present disclosure.Examples

[0106] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.Example 1Methods

[0107] Transfection, incubation, and harvest of cells: A transfection mix was prepared by (1) mixing the plasmids having the AAV2-Rep genes & AAV9-Cap genes (Rep2 / Cap9 plasmid GenScript Cat no: U1596MWJG0-4), AAV helper plasmid (procured from GenScript Cat no: U1596MWJG0-2) and plasmid having the target polynucleotide transgene (enhanced green fluorescent protein (EGFP) or SEQ ID NOs. 7 to 11) at a ratio by mole of 1 : 1 : 1; and (2) adding the transfection agent Polyethylenimine (PEI), such that the plasmids and the transfection agent were at a ratio by weight of 1:2.5. The transfection mix was incubated at 25 °C for 10 mins. HEK293 cells and virus production cells (Virus Production Cells 2.0 (VPC 2.0, procured from Gibco, Cat no #A49784) (referred to as producer cells) were suspended in a tissue culture flask at 3.5 million cells / mL in (DMEM procured from Thermofisher Scientific) supplemented with 6 g / L of glucose, 4mM of L-Glutamine, 2.20 g / L of Sodium Bicarbonate and 2 mL / L Anti-Clumping Agent. The transfection mix was then added to the tissue culture flask having the producer cells. 1 part by volume of transfection mix was added to 2.5 part by volume of producer cells (1 :2.5). This was then incubated at temperature of 25 °C for 20 mins with periodically vortex mixing after every 5 mins to 7 mins.

[0108] For incubation, the tissue culture flask having the transfection mix and producer cells was then incubated at 37 °C inside 8% CO2 environment for 72 hours with mixing after every 5 mins to 7 mins to allow replication and packaging of the recombinant AAV particles.

[0109] For harvesting of transfected cells, 48 hours-72 hours post-incubation the contents from the tissue culture flask were centrifuged at 4300 g, 4 °C for 15 mins to obtain a pellet.

[0110] Lysis of transfected cells: The pellet obtained after harvesting the cells were lysed using 50mM Tris HC1, 2 mM MgCh, 150 mM NaCl and 0.5% Triton X-100. The lysate were treated with 100 U / mL of SAN-HQ at 37 °C for 60 mins. The treated lysate was centrifuged at 4300 g, 4 °C for 20 mins, to collect the supernatant. The supernatant was passed through a depth filter clarification to remove large particulate matter. The filtrate was passed through a secondary filter made of diatomaceous earth (DE) having pore size of 0.45 pm, preferably to reduce the turbidity of the filtrate to less than 40-50 NTU.Example 2: Assessment of transfection efficiency of EGFP constructs.

[0111] Enhanced green fluorescent protein (EGFP) recombinant constructs were developed using the vector sequences provided in SEQ ID NOs: 7, 8, and 9 (without the SMN-1 sequences). The EGFP constructs (Constructs 1, 2, and 3) were transfected into adherent (Figure 3(a)) and suspension (Figure 3(b)) HEK293 cells using the transfection, incubation, and harvesting method as described in Example 1. 72 hours post-transfection, the cells were observed under a fluorescence microscope to detect the expression of the fluorescent reporter protein, EGFP. As shown in Figure 3(a) and 3(b) successful uptake and expression of the vector intransfected cells was observed in both the adherent and suspension HEK293 cells, indicating efficient cellular delivery and potential for EGFP protein expression.

[0112] Transfection efficiency of the EGFP-expressing constructs was also evaluated in two independent experimental sets using flow cytometric analysis. Flow cytometric analysis was performed using the BD Accuri C6 Plus Flow Cytometer (BD Biosciences). Data acquisition was carried out using standard instrument settings for EGFP detection (488 nm excitation, 533 / 30 nm emission filter, FL1 channel). A minimum of 10,000 events per sample was recorded. Cells were resuspended in phosphate-buffered saline (PBS) and analyzed using standard 12 x 75mmpolystyrene round-bottom tubes as the sample carrier. Forward and side scatter parameters were used to gate viable cells, and doublets were excluded during analysis. 72 hours post-transfection, HEK293 cells were harvested and analysed for EGFP expression. The percentage of EGFP-positive cells was determined to quantify cellular uptake and expression efficiency of each construct. As shown in Figures 4(a) and 4(b), effective delivery and expression of EGFP in the candidate vector systems was observed. In particular, comparative analysis between constructs in both experimental sets demonstrated reproducible and differential transfection efficiencies.Example 3: Determination of optimal multiplicity of infection (MOI) for EGFP Constructs in U87-MG cells and SHSY-5Y cells.

[0113] U87-MG cells were transduced with EGFP Construct 2 according to the procedure as described in Example 1, at increasing multiplicities of infection (MOIs) of 0 (control), 2.5 x 104, 5.0 x 104, 7.5 x 104, and 1.0 x 105viral particles per cell. At 48 hours post-transduction, the cells were harvested and analysed by flow cytometry to quantify enhanced green fluorescent protein (EGFP) expression. Transduction efficiency was determined as the percentage of EGFP-positive cells within the total viable cell population. As shown in Figures 5(a) and 5(b), dosedependent increase in EGFP expression was observed with increasing MOI with 7.5 x io4, and 1.0 x 105viral particles per cell showing the highest transductionefficiency. This enabled identification of an optimal viral dose that achieves maximal transgene expression while maintaining acceptable cellular viability.

[0114] Once the optimal viral dose was identified, U87-MG cells were transduced with EGFP Construct 2 and EGFP Construct 3 at a multiplicity of infection (MOI) of 1 x 105viral particles per cell according to the method of Example 1. At 48 hours post-transduction, cells were harvested and analysed by flow cytometry to quantify EGFP expression. Transduction efficiency was determined as the percentage of EGFP-positive cells within the total viable cell population. As shown in Figures 6(a) and 6(b), the comparative flow cytometric analysis showed efficient expression in both EGFP Construct 2 and EGFP Construct 3 confirming functional vector-mediated transgene expression at the tested viral dose.Example 4: Comparative evaluation of transduction efficiency of EGFP Construct 2 in U87-MG and SH-SY5Y cells.

[0115] U87-MG and SH-SY5Y cells were transduced with EGFP Construct 2 at (a) multiplicity of infection (MOI) of 0 (Control) and 1.0 x 105, and (b) MOI of 0 (Control)and 2.0 * 105viral particles per cell according to the method of Example 1. At 48 hours post-transduction, cells were harvested and analyzed by flow cytometry to quantify EGFP expression. Transduction efficiency was determined as the percentage of EGFP-positive cells within the total viable cell population. As shown in Figures 7(a) and 7(b), the optimal MOI in both cells was identified as 1.0 x 105viral particles per cell.

[0116] Viable expression of EGFP was also assessed by cDNA expression and end-point PCR analysis. U87-MG and SH-SY5Y cells were transduced with EGFP Construct 2 at 1 x 105(100K) and 7.5 x 104(75K) viral particles per cell. Total RNA was isolated from the cells at 48 hours post-transduction using the TRIzol Reagent (Invitrogen, Cat no # 15596018) according to the manufacturer’s protocol. Briefly, cells were lysed in TRIzol reagent, followed by phase separation using chloroform, RNA precipitation with isopropanol, and washing with 75% ethanol prior to resuspension in RNase-free water. The isolated RNA was reverse transcribed tocDNA and analyzed for EGFP expression. Quantitative analysis of EGFP cDNA expression levels were normalized to an internal housekeeping gene (GAPDH) and expressed relative to untransduced (OK) control cells of U87-MG and SHSY-5Y cells. As shown in Figure 8(c), a distinct EGFP-specific band of the expected size was observed in both 75K and 100K transduced samples, whereas no corresponding band is detected in OK control cells. The results as shown in Figures 8(a) and 8(b), however, confirmed efficient vector-mediated gene delivery and transcriptional expression in both cell lines at MOI 1 x 105viral particles per cell.Example 5: Transduction efficiency of SMN1 construct in U87-MG and SHSY-5Y cells.

[0117] Human glioblastoma cells (U87-MG) and human neuroblastoma cells (SHSY-5Y) were transduced with the SMN1 construct (corresponding to SEQ ID NO:7). Cloning was carried out as per manufacturer’s instructions (by Vector Builider Inc.). The cells were transduced at a multiplicity of infection (MOI) of IxlO5viral particles per cell. At 48 hours post-transduction, cells were harvested and analyzed by flow cytometry to determine transduction efficiency. The percentage of SMN1 -positive cells was quantified as a marker of vector delivery and expression, representing the functional activity of the corresponding SMN1 construct. As shown in 9(a) and 9(b), efficient cellular transduction of the SMN1 construct at high MOI in both the cell lines was observed, supporting the suitability of the AAV vector system for therapeutic SMN1 gene delivery.Example 6: Dose-dependent transduction efficiency of SMN1 construct as evaluated by Western blot analysis.

[0118] Human glioblastoma cells (U87-MG) and human neuroblastoma cells (SHSY-5Y) were transduced with the SMN1 construct corresponding to EGFP Construct 2 at 1 xlO5viral particles per cell (100K), 1.5 xio5viral particles per cell (150K), and 2xl05viral particles per cell (200K). The cells were transduced and whole-cell lysates were collected at 48 hours post-transduction according to the method of Example 1. The cell lysates were subjected to Western blotting using ananti-SMNl antibody. P-tubulin was used as a loading control to confirm equal protein loading across samples. As shown in Figures 10(a) and 10(b), western blot analysis demonstrated detectable SMN1 protein levels in both cell lines following transduction. SMN1 protein expression was observed to be saturated beyond 100K MOI. The results also showed detectable protein levels in both U87-MG and SHSY-5Y at 5.0 x 104(50K) viral particles per cell (Figure 10(b)). The results identify an effective MOI range for robust SMN1 protein expression in these cell lines, supporting optimization of vector dosing for therapeutic applications.Example 7: In vivo effect of SMN1 construct.

[0119] SMA mice models (FVB.SMNA7; SMN2; Smn- / -) were treated with different doses of the SMN1 construct (selected from SEQ ID No. 17, SEQ ID No.18, SEQ ID No. 19, SEQ ID No. 20, and SEQ ID No. 21) to test the in vivo effect of the construct. Referring to Figure 11 (a, b, and c), control mice (untreated, n=4; and formulation buffer, n=4), and mice injected with a low dose of the construct (SMN1 construct (L=0.5E+14 vg / kg), n=5), and medium dose of the construct (SMN1 construct (M=1.1E+14 vg / kg), n=4), were monitored until 15-days post injection for their body weight, reflexes (righting reflex test), and survival probability. As shown in Figure 11(a), 60% of low dose injected mice were alive at day 15, while the control mice and medium dose injected mice showed poor rates of survival. Further, low dose injected mice also showed an increase in body weight and better reflexes (Figures 11(b) and (c), respectively). Further, as shown in Figure 12 (a), (b), and (c), the construct also demonstrated high rates of expression in brain, spinal and skeletal muscle tissue of mice, confirming in vivo activity and mutant phenotype rescue.Advantages of the present disclosure

[0120] The present disclosure discloses a polynucleotide for expressing survival of motor neuron (SMN) protein. Also disclosed herein are recombinant Adeno-associated virus comprising the polynucleotide as disclosed herein, capable of expressing SMN protein. The polynucleotides, vectors, and recombinant AAV, asdisclosed herein may be used for expressing SMN protein in subjects. The embodiments herein also provide a self-complementary polynucleotide and a vector comprising the polynucleotide as disclosed herein. The polynucleotides and vectors disclosed herein enable high expression levels of soluble SMN protein which are detectable in vitro. These constructs may be used to facilitate treatment of spinal muscular atrophy (SMA) by delivering therapeutically active molecules to the spinal cord that may compensate for the SMN protein deficiency.

Claims

I / We Claim1. A polynucleotide comprising:(a) a nucleic acid insert having a transgene encoding Survival of Motor Neuron (SMN) protein having at least 95% identity to an amino acid sequence as set forth in SEQ ID NO. 13, operably linked to a promoter, and an intron; and(b) at least one 5'-inverted terminal repeat (5'-ITR) and at least one 3'- inverted terminal repeat (3'-ITR), heterologous to the transgene, wherein the 5'-ITR is positioned at the 5' end of the nucleic acid insert, and the 3'-ITR is positioned at the 3' end of the nucleic acid insert.

2. The polynucleotide as claimed in claim 1, wherein the transgene has a nucleotide sequence of at least 90% identity to a sequence as set forth in SEQ ID NO. 6.

3. The polynucleotide as claimed in claim 1, wherein the 5'-ITR has a nucleotide sequence of at least 98% sequence identity to a sequence as set forth in SEQ ID No. 1 or SEQ ID No. 15; and 3'-ITR has a nucleotide sequence of at least 98% sequence identity to a sequence as set forth in SEQ ID No. 2 or SEQ ID No. 16.

4. The polynucleotide as claimed in claim 1, wherein the intron is from simian virus 40 (SV40), and has a nucleotide sequence of at least 90% sequence identity to a sequence as set forth in SEQ ID NO: 5.

5. The polynucleotide as claimed in claim 1, wherein the promoter is selected from a chicken beta-actin (CBA) promoter, or portion thereof, a chicken beta-actin hybrid (CBh) promoter, or combinations thereof, wherein the chicken beta-actin (CBA) promoter has a nucleotide sequence of at least 90% sequence identity to a sequence as set forth in SEQ ID No. 3, and chicken beta-actin hybrid (CBh)promoter has a nucleotide sequence of at least 90% sequence identity to a sequence as set forth in SEQ ID No. 4.

6. The polynucleotide as claimed in claim 1, wherein the nucleic acid insert further comprises:an enhancer operably linked to the transgene; anda polyadenylation signal.

7. The polynucleotide as claimed in claim 6, wherein the enhancer is cytomegalovirus (CMV) enhancer having a nucleotide sequence of at least 90% sequence identity to a sequence as set forth in SEQ ID No. 24, or portion thereof.

8. The polynucleotide as claimed in claim 1, wherein the polynucleotide is a single stranded or double stranded DNA.

9. The polynucleotide as claimed in claim 8, wherein the polynucleotide is a double stranded DNA comprising a complementary fragment of the nucleic acid insert covalently attached at the 3' end, allowing formation of the double stranded DNA (dsDNA).

10. A self-complementary polynucleotide, preferably self-complementary DNA (scDNA), comprising the polynucleotide as claimed in claim 1.

11. A vector comprising the polynucleotide as claimed in claim 1 or the self- complementary polynucleotide as claimed in claim 10.

12. The vector as claimed in claim 11, wherein the vector is an Adeno-associated vector (AAV) comprising AAV2-Rep gene derived from Adeno-associated virus serotype 2 and AAV9-Cap gene derived from Adeno-associated virus serotype 9.

13. The vector as claimed in claim 12, wherein the AAV9-Cap gene has a nucleotide sequence of at least 90% sequence identity to a sequence selected from SEQ ID No. 12.

14. The vector as claimed in claim 11, wherein the vector is a plasmid having a nucleotide sequence selected from SEQ ID No. 7, SEQ ID No. 8, SEQ ID No.9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, and SEQ ID No. 21.

15. A recombinant AAV comprising the polynucleotide as claimed in claim 1, the self-complementary polynucleotide as claimed in claim 10, or the vector as claimed in any one of claims 11-14.

16. A composition comprising the polynucleotide as claimed in claim 1, the self- complementary polynucleotide as claimed in claim 10, the vector as claimed in any one of claims 11-14, or the recombinant AAV as claimed in claim 15.

17. A host cell comprising the polynucleotide as claimed in claim 1, the self- complementary polynucleotide as claimed in claim 10, or the vector as claimed in any one of claims 11-14.

18. A method of preparing a recombinant AAV, comprising:(a) transfecting a producer cell with a vector as claimed in any one of claims 11-14, and providing the producer cell with AAV2 replication genes (AAV2-Rep) and AAV9 capsid genes (AAV9-Cap);(b) culturing the producer cells under conditions to allow replication and packaging of recombinant AAV particles; and(c) harvesting the recombinant AAV particles by lysing the producer cells.

19. A method for treating and / or preventing spinal muscular atrophy in a subject, comprising administering the polynucleotide as claimed in claim 1, the self-complementary polynucleotide as claimed in claim 10, the vector as claimed in any one of claims 11-14, or the recombinant AAV as claimed in claim 15.

20. A method for treating and / or preventing spinal muscular atrophy in a subject comprising,(a) producing a recombinant AAV comprising the polynucleotide as claimed in claim 1; and(b) administering the recombinant AAV to the subject.

21. A method for inducing production of SMN protein in a cell, comprising providing the polynucleotide as claimed in claim 1, the self-complementary polynucleotide as claimed in claim 10, the vector as claimed in any one of claims 11-14, or the recombinant AAV as claimed in claim 15 to the cell and expressing the protein.

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