Gene replacement therapy for alpk3 cardiomyopathy

Abstract

The present disclosure relates, in part, to gene replacement to treat cardiomyopathy. In embodiments, the invention provides compositions and methods of use to treat ALPK3 cardiomyopathy. In embodiments, invention provides a composition comprising a nucleic acid encoding all or a portion of an ALPK3 protein.

Description

GENE REPLACEMENT THERAPY FOR ALPK3 CARDIOMYOPATHYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 512,200 filed on July 6, 2023, which is incorporated herein by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 28, 2024, is named 24978-0916, SL.xml and is 35,220 bytes in size.GOVERNMENT SPONSORSHIP

[0003] This invention was made with government support under grant HL 146759 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0004] The present invention relates to treatments for cardiomyopathy.BACKGROUND

[0005] Alpha Protein Kinase 3 (ALPK3) is an aty pical protein kinase that is a member of the alpha (a)-kinase family. Individuals carrying homozygous or compound heterozygous loss-of-function pathogenic ALPK3 mutations often exhibit a range of cardiomyopathy phenotypes. These frequently present at birth or early childhood as severe early-onset dilated cardiomyopathy (DCM) and subsequently progress towards a hypertrophic cardiomyopathy (HCM) phenotype over time. In contrast, individuals carrying heterozygous truncated variants in ALPK3 typically exhibit a late onset of hypertrophiccardiomyopathy (HCM). This is often accompanied by significant myocardial fibrosis, leading to the progression of heart failure. According to the genoAD database, loss-of- function ALPK3 variants are found in approximately 1 in 2,500 individuals. In two separate cohorts of HCM patients, comprising 770 and 2,047 patients respectively, it was found that 1.56% of HCM patients carried heterozy gous loss-of-function ALPK3 variants. Currently, no specific treatments are available for patients diagnosed with ALPK3 cardiomyopathy.SUMMARY OF THE INVENTION

[0006] The present disclosure relates, in part, to gene replacement to treat cardiomyopathy. In embodiments, the invention provides compositions and methods of use to treat ALPK3 cardiomyopathy. In embodiments, invention provides a composition comprising a nucleic acid of an ALPK3 gene, encoding ALPK3 protein.

[0007] In embodiments, the invention provides that the nucleic acid is a mini ALPK.3 gene comprising a sequence that encodes at least 700, 800, 900, 1000, 1100, 1200, 1300 or 1400 amino acids of a protein corresponding to the ALKP3 gene. In embodiments, the invention provides that the nucleic acid is a mini ALPK3 gene comprising a sequence that encodes about 1308 amino acids of a protein corresponding to the ALKP3 gene. In embodiments, the invention provides that the nucleic acid is not found in nature and contains fewer nucleotides that a naturally occurring ALPK3 gene.

[0008] In embodiments, the invention provides that the nucleic acid is a mini ALPK3 gene having at least a deletion of a portion of exon 6 sequence. In embodiments, the invention provides that the nucleic acid is a mini ALPK3 gene having at least a deletion of a portion of exons 5 and / or 6.

[0009] In embodiments, the invention provides that the composition further comprises a vector operationally linked to the portion of ALPK3 gene. In embodiments, invention provides that the vector is a muscle adeno-associated virus 2A (MyoAAV 2A). In embodiments, invention provides that the nucleic acid is cDNA or RNA.

[0010] In embodiments, the invention provides a composition comprising a tagged ALPK3 gene. In embodiments, the tag is a 3xFLAG tag. In embodiments, the tag is incorporated at the C-terminus of ALPK3 gene. In embodiments, the protein expressed by the tagged ALPK3 tag can be isolated with an antibody against the tag.

[0011] In embodiments, the invention provides means for expressing a portion of an ALPK3 gene and method of use. In embodiments, the invention provides means for isolating a portion of an ALPK3 gene and method of use.

[0012] In embodiments, invention provides methods for treating cardiomyopathy, comprising administering to a subject in need thereof an effective amount of the compositions or means described herein. In embodiments, the invention provides that the cardiomyopathy is ALPK3 cardiomyopathy. In embodiments, the ALKP3 gene is human. In embodiments, the subject is human.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1. Loss of ALPK3 in mice induces premature death. (Fig. 1 Panel A) RT-qPCR shows that ALPK3 mRNA is significantly downregulated in ALPK3 global knockout (gKO) mice. (Fig. 1 Panel B) Survival curve showed that ALPK3 gKO mice starts to die after one week of birth, half of gKO mice died before wean age.

[0014] Figure 2. Loss of ALPK3 in mice induces cardiac dysfunction. Fraction shortening (FS) is used for measurement of contractile function. Left ventricle internal dimension at diastole (LVIDd) and left ventricle internal dimension at systole (LVIDs) are used for measurement of chamber size.

[0015] Figure 3. Loss of ALPK3 in mice induces left ventricular hypertrophy. The left ventricular posterior wall end diastole (LVPWd) and interventricular septum thickness end diastole (IVSd) are used for measurement of left ventricular hypertrophy.

[0016] Figure 4. Generation of miniALPK3 mouse model. (Fig. 4 Panel A) Schematics shows the ALPK3 Exon-6 was cleaved by CRISPR complex. SEQ ID Nos: 9 and 10 show the sgRNA binding sites. This two-cut strategy generates 1119 bp in-frame deletion (373 aa) of miniALPK3 allele. (Fig. 4 Panel B) PCR of mouse genomic DNA from tails using P1 / P2 primers in Fig. 4 Panel A amplifies the miniALPK3 allele DNA fragment. (Fig. 4 Panel C) E Sanger sequencing confirming 1119 bp in-frame deletion (373 aa) in miniALPK3 mice. SEQ ID No: 11 shows the amino acid cleavage site, and SEQ ID No: 12 shows the corresponding nucleic acid sequence.

[0017] Figure 5. Echocardiography assessment the control and homozy gous miniALPK.3 mice. Fraction shortening (FS) is used for measurement of contractile function. Left ventricle internal dimension at diastole (LVIDd) and left ventricle internal dimension at systole (LVIDs) are used for measurement of chamber size. The left ventricular posterior wall end diastole (LVPWd) is used for measurement of left ventricular hypertrophy.

[0018] Figure 6. Generation of ALPK3-3xFLAG tagged knock-in mouse model. (Fig. 6 Panel A) Schematic representation of the CRISPR / Cas9 strategy7used to generate ALPK3- 3xFLAG tagged knock-in mice. (Fig. 6 Panel B) Western blot analysis of FLAG-tagged ALPK3 in control (Ctrl) and ALPK3-3xFLAG tagged (FLAG / FLAG) knock-in hearts. Histone H3 was used as a loading control. (Fig. 6 Panel C) Echocardiography measurements of fractional shortening (FS), left ventricular (LV) internal dimensions at end-diastole (LVIDd) and end-systole (LVIDs), as well as LV posterior wall thickness at end-diastole (LVPWd) in ALPK3-3xFLAG tagged knock-in (n=7) and Ctrl mice (n=6) at 3 months of age. NS, not significant.

[0019] Figure 7. Injecting MyoAAV-2A-cTnT-miniALPK3-3xFLAG into ALPK3 gene knockout (GKO) mice restores their heart function. Echocardiography measurements of fractional shortening (FS) in Ctrl (ALPK3-3xFLAG tagged knock-in) (n=3). GKO mice, GKO with MyoAAV-2A-cTnT-miniALPK3-3xFLAG (n=3) at 6 weeks of age.DETAILED DESCRIPTION

[0020] The present disclosure relates, in part, to gene replacement to treat cardiomyopathy. In embodiments, the invention provides pharmaceutical compositions and methods of use to treat ALPK3 cardiomyopathy. In embodiments, invention provides a pharmaceutical composition comprising a nucleic acid encoding all or a portion of a functional ALPK3 protein.

[0021] The present disclosure provides multiple embodiments for gene replacement in the treatment of ALPK3 cardiomyopathy. In an embodiment, the invention provides delivering a treatment effective amount of a pharmaceutical composition comprising the full- length or shortened wild-type or modified ALPK3 cDNA or mRNA to cardiomyocytes of a subject in need thereof, using either a viral or non-viral vector method, to mitigate cardiac symptoms in ALPK3 cardiomyopathy. In an embodiment, the invention provides deliveringa functional miniALPK3 cDNA or modified mRNA, such as that encoding about 1308 amino acids of ALPK.3 missing about 373 amino acids encoded by exon 6, to cardiomyocytes, using adeno-associated viruses (AAV) or other viral or non-viral vector constructs and gene delivery methods, to mitigate cardiac symptoms in ALPK3 cardiomyopathy. In an embodiment, the invention provides delivering other smaller ALPK3 cDNAs or modified mRNA, in the absence of a portion of sequence in exons 5 and / or 6, to cardiomyocytes, using AAV or other viral or non-viral vector constructs, to mitigate cardiac symptoms in ALPK.3 cardiomyopathy.

[0022] In embodiments, the invention provides that the nucleic acid is a mini ALPK3 nucleic acid comprising a sequence that encodes at least 700, 800. 900, 1000. 1100, 1200, 1300 or 1400 amino acids of a functional ALPK3 protein corresponding to the ALKP3 nucleic acid. In embodiments, the invention provides that the nucleic acid is a mini ALPK3 nucleic acid comprising a sequence that encodes about 1308 amino acids of a protein corresponding to the ALKP3 gene missing a portion of wild-type sequence in exons 5 and / or 6. In embodiments, the invention provides that the nucleic acid is not found in nature and contains fewer nucleotides that a naturally occurring ALPK.3 cDNA.

[0023] In embodiments, the invention provides that the nucleic acid is a mini ALPK3 protein having at least a deletion of a portion encoding exon 6 sequence which encodes 373 amino acids of a sequence corresponding to the ALKP3 gene. In embodiments, the invention provides that the nucleic acid is a mini ALPK3 gene having at least a deletion of a portion of exons 5 and / or 6. In embodiments, the invention provides that the miniALPK.3 is a human gene and encoded protein such as shown in SEQ ID Nos: 7 and 8 respectively.

[0024] In embodiments, the invention provides that the composition further comprises a vector operationally linked to the portion of ALPK3 gene. In embodiments, invention provides that the vector is a muscle adeno-associated virus 2A (MyoAAV 2A). In embodiments, invention provides that the nucleic acid is cDNA or RNA. In embodiments, invention provides methods of treating cardiomyopathy, comprising administering to a subject in need thereof an effective amount of the compositions described herein. In embodiments, the invention provides that the cardiomyopathy is ALPK3 cardiomyopathy. In embodiments, the invention provides that the cardiomyopathy is ALPK3 cardiomyopathy. In embodiments, the invention provides that the cardiomyopathy exhibits left ventricularhypertrophy. In embodiments, the invention provides that the cardiomyopathy is dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM).

[0025] In embodiments, the invention provides a composition comprising a tagged ALPK3 encoding nucleic acid. In embodiments, the tag is a 3xFLAG tag. In embodiments, the tag is incorporated at the C-terminus of ALPK3. In embodiments, the protein expressed by the tagged ALPK3 tag can be isolated with an antibody against the tag.

[0026] In embodiments, the invention provides means for expressing a portion of an ALPK3 gene and method of use. In embodiments, the invention provides means for isolating a portion of an ALPK3 gene and method of use.

[0027] In embodiments, invention provides methods for treating cardiomyopathy, comprising administering to a subject in need thereof an effective amount of the compositions or means described herein. In embodiments, the invention provides that the cardiomyopathy is ALPK3 cardiomyopathy. In embodiments, the ALKP3 gene is human. In embodiments, the subject is human.

[0028] Various further aspects and embodiments of the disclosure are provided by the following description. Before further describing various embodiments of the presently disclosed inventive concepts in more detail by way of exemplary description, examples, and results, it is to be understood that the presently disclosed inventive concepts are not limited in application to the details of methods and compositions as set forth in the following description. The presently disclosed inventive concepts are capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary, not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting unless otherwise indicated as so. Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the disclosure. However, it will be apparent to a person having ordinary skill in the art that the presently disclosed inventive concepts may be practiced without these specific details. In other instances, features which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description. All of thecompositions and methods of production and application and use thereof disclosed herein can be made and executed without undue experimentation in light of the present disclosure.

[0029] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0030] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein.

[0031] The practice of the present invention may employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ. Gait, ed.. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds.. 1993- 1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D .M. Weir and CC. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F .M. Ausubel et al . eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al, eds., 1994); Current Protocols in Immunology (J.E. Coligan et al, eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999). Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein. For the purposes of the present disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

[0032] ALPK3 and its gene are well-known in the art. ALPK3 has 14 exons along with two Ig-like and one alpha-kinase domains. Journal of the American College of Cardiology, 2016 Feb 9;67(5):515-25; European Heart Journal, 2021 Jul 15; ehab424. Detailed nucleotide information on the ALPK3 gene for humans and mice for example can also be found in the Ensembl database. The latest version of full length human ALPK3 protein (NP_065829.4) is 1705 amino acids (ncbi.nlm.nih.gov / protein / NP_065829)(SEQ ID No: 5 herein). For reference, the mouse full length ALPK3 amino acid sequence is shown in SEQ ID No: 1, and the encoding nucleic acid sequence is shown in SEQ ID No: 2. The mouse miniALPK3 amino acid sequence is shown in SEQ ID No: 3, and the encoding nucleic acid sequence is shown in SEQ ID No: 4. The human full length ALPK3 amino acid sequence is shown in SEQ ID No: 5, and the encoding nucleic acid sequence is shown in SEQ ID No: 6. The human miniALPK3 amino acid sequence is shown in SEQ ID No: 7, and the encoding nucleic acid sequence is shown in SEQ ID No: 8.

[0033] As used herein, the terms "comprises.” "comprising.” ‘"includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by,” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, nucleic acid sequence, a pharmaceutical composition, and / or a method that “comprises” a list of elements (e.g., components, features, or steps) is not necessarily limited to only those elements (or components or steps), but may include other elements (or components or steps) not expressly listed or inherent to the nucleic acid sequence, pharmaceutical composition and / or method. Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment.” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0034] As used herein, the transitional phrases “consists of’ and “consisting of exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or stepsspecifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase "‘consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0035] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a fusion protein, pharmaceutical composition, and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising’’ and “consisting of’. It is understood that aspects and embodiments of the invention described herein include “consisting” and / or “consisting essentially of’ aspects and embodiments.

[0036] When introducing elements of the present invention or the preferred embodiment(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0037] The term “and / or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression “A and / or B” is intended to mean either or both of A and B, i.e. A alone, B alone or A and B in combination. The expression “A, B and / or C” is intended to mean A alone. B alone. C alone. A and B in combination. A and C in combination, B and C in combination or A, B, and C in combination.

[0038] It should be understood that the description in range format is merely for convenience and brevity and should not be constmed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should beconsidered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Values or ranges may be also be expressed herein as “about,” from “about” one particular value, and / or to “about” another particular value. When such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0039] It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount. weight or length. In various embodiments, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0040] Variations in the nucleic acid and amino acid sequences, and regions thereof, are contemplated as being encompassed by the present disclosure, providing that the variations in the sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% identity. Certain percentages in between are included, such as 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate: (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family: (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family: and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional antibody can readily be determined by assaying the specific activity of the antibody derivative. Fragments or analogs of antibodies can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains.

[0041] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such antibodies. Antibodies can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).

[0042] As used herein any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.

[0043] Genome editing tools may be used to engineer and / or manipulate cells. In some embodiments, the nucleic acids of the disclosure may be engineered with either CRISPR, TALEN, or ZFN genome editing tools.

[0044] Genome editing tools such as the clustered regularly interspaced short palindromic repeats (CRISPR) system may be used to genetically modify cells. CRISPR can be used in a wide variety of organisms (e.g., used to add, disrupt, or change the sequence of specific genes). “CRISPR’' or “CRISPR gene editing’" as used herein refers to a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats. “Cas”, as used herein, refers to a CRISPR-associated protein. A “CRISPR / Cas” system refers to a system derived from CRISPR and Cas which can be used to silence, knock out, or mutate a target gene.

[0045] The CRISPR / Cas system is based on two elements. The first element is an endonuclease, or Cas, (e.g., Cas9 and MAD7) that has a binding site for the second element, which is the guide polynucleotide (e.g., guide RNA or gRNA). The guide polynucleotide (e.g., guide RNA) directs the Cas protein to double stranded DNA templates based on sequence homology. The Cas protein then cleaves that DNA template. By delivering the Cas protein and appropriate guide polynucleotides (e.g., guide RNAs) into a cell, the organism’s genome is cut at a desired location. Following cleavage of a targeted genomic sequence by a Cas / gRNA complex, one of two alternative DNA repair mechanisms can restore chromosomal integrity: 1) non-homologous end joining (NHEJ) which generates insertions and / or deletions of a few base-pairs (bp) of DNA at the gRNA cut site, or 2) homology- directed repair (HDR) which can correct the lesion via an additional “bridging” DNA template that spans the gRNA cut site. CRISPR / Cas systems are classified by class and by type. Class 2 systems currently represent a single interference protein that is categorized into three distinct types (types II, V and VI). Any class 2 CRISPR / Cas system suitable for gene editing, for example a type II, a type V or a type VI system, is envisaged as within the scope of the instant disclosure. Exemplary Class 2 ty pe II CRISPR sy stems include Cas9, Csn2 andCas4. Exemplary Class 2, pe V CRISPR systems include, Casl2, Casl2a (Cpfl), Casl2b (C2cl). Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f, Casl2g. Casl2h, Casl2i and Casl2k (C2c5). Exemplary' Class 2 Type VI systems include Casl3, Casl3a (C2c2) Casl3b, Casl3c and Casl3d.

[0046] The CRISPR sequence, sometimes called a CRISPR locus, comprises alternating repeats and spacers. In a naturally-occurring CRISPR, the spacers usually comprise sequences foreign to the bacterium such as a plasmid or phage sequence. As described herein, spacer sequences may also be referred to as “targeting sequences." In CRISPR / Cas systems for a genetic engineering, the spacers are derived from the target gene sequence (the gNA).

[0047] The targeting sequence can be designed or chosen using computer programs known to persons of ordinary skill in the art. The computer program can use variables, such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, % GC, frequency of genomic occurrence (e.g., of sequences that are identical or are similar but vary in one or more spots as a result of mismatch, insertion or deletion), methylation status, presence of SNPs, and the like. Available computer programs can take as input NCBI gene IDs, official gene symbols, Ensembl Gene IDs, genomic coordinates, or DNA sequences, and create an output file containing sgRNAs targeting the appropriate genomic regions designated as input. The computer program may also provide a summary' of statistics and scores indicating on- and off-target binding of the sgRNA for the target gene (Doench et al. Nat Biotechnol.34: 184-191 (2016)).

[0048] The target sequence is complementary to, and hybridizes with, the targeting sequence of the gRNA. The target nucleic acid sequence can comprise 20 nucleotides. The target nucleic acid can comprise less than 20 nucleotides. The target nucleic acid can comprise more than 20 nucleotides. The target nucleic acid can comprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The target nucleic acid can comprise at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22. 23, 24, 25, 30 or more nucleotides.

[0049] The CRISPR / Cas system can thus be used to edit a target gene, such as a gene targeted for editing in the cells described herein, by adding or deleting a base pair, introducing a premature stop codon, or introducing a frame-shift mutation which thusdecreases expression of the target, in part or completely. The CRISPR / Cas system can alternatively be used like RNA interference, turning off a target gene in a reversible fashion. In a mammalian cell, for example, the RNA can guide the Cas protein to a target gene promoter, sterically blocking RNA polymerases.

[0050] Further aspects of the CRISPR / Cas system known to those of ordinary skill are described in PCT Publication Nos. WO 2017 / 049266 and WO 2017 / 223538, the entire contents of which are hereby incorporated by reference. These and other well-known and new techniques, such as TALEN and Zinc Finger Nucleases, for generating nucleic acids of the present disclosure are contemplated by the present invention.

[0051] In some embodiments, the nucleic acid sequences described herein are edited using TALEN gene editing. “TALEN’' or “TALEN gene editing” refers to a transcription activator-like effector nuclease, which is an artificial nuclease used to edit a target gene. TALENs are produced artificially by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be engineered to bind any desired DNA sequence, including a portion of target genes such as TCR subunits, MHC class I complex components, or CD52. By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme can be produced which is specific to any desired DNA sequence, including a target gene sequence. These can then be introduced into a cell, wherein they can be used for genome editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscou el a / . (2009) Science 326: 3501.

[0052] In some embodiments, the nucleic acids described herein are edited using ZFN gene editing. “ZFN” or “Zinc Finger Nuclease” or “ZFN gene editing” refer to a zinc finger nuclease, an artificial nuclease which can be used to edit a target gene. Like a TALEN, a ZFN comprises a Fold nuclease domain (or derivative thereof) fused to a DNA-binding domain. In the case of a ZFN. the DNA-binding domain comprises one or more zinc fingers. Carroll et al. (201 1) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160.

[0053] The nucleic acid sequences described herein can be modified using methods known in the art. The various gene editing systems described herein may be used to modify the nucleic acid sequences to delete, inactivate, reduce expression, or otherwise inhibit function of a target gene or a target gene product.

[0054] The term “nucleic acid” or “polynucleotide”, includes DNA and RNA such as genomic DNA, cDNA and mRNA, or combinations thereof. The nucleic acid may comprise, in addition to the sequence enabling the genetic modifications of the disclosure, further sequences such as those required for the transcription and / or translation of the nucleic acid enabling said genetic modifications. This may include a promoter, enhancer, transcription and / or translation initiation and / or termination sequences, selection markers, sequences protecting or directing the RNA and / or enabling the genetic modifications within the cell. The selection and combination of these sequences is within the knowledge of the person skilled in the art and may be selected in accordance with the cell the nucleic acid is intended for.

[0055] Polynucleotides enabling the genetic modifications of the disclosure may be delivered to cells as an isolated nucleic acid or in a vector. The isolated nucleic acid or the vector may be delivered in lipid- or lipid-based delivery' system, such as a liposome. Alternatively, the vector may comprise viral proteins, such as when the vector is a viral vector. The term “vector” as used herein refers to a construction comprised of genetic material designed to direct transformation or transductions of a targeted cell. A vector contains multiple genetic elements positionally and sequentially oriented with other necessaryelements such that the nucleic acid in a nucleic acid cassette can be transcribed and when necessary translated in the transfected cells. The term vector as used herein can refer to nucleic acid, e.g., DNA derived from a plasmid, cosmid, phagemid, bacteriophage, virus, retrovirus, adenovirus, adeno-associated virus, lentivirus, or other type of virus into which one or more fragments of nucleic acid may be inserted or cloned which encode for particular proteins. The term “plasmid” as used herein refers to a construction comprised of extrachromosomal genetic material, usually of a circular duplex of DNA which can replicate independently of chromosomal DNA. The plasmid does not necessarily replicate.

[0056] Any suitable vectors are envisaged as within the scope of the instant disclosure. The polynucleotides enabling the genetic modifications of the disclosure can be cloned into a number of ty pes of vectors. For example, the polynucleotides enabling the genetic modifications of the disclosure may be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. Expression vectors may be provided to cells, such as immune cells,in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0057] The purpose of the vector is to provide a nucleic acid sequence in cells or tissue. Expression includes the efficient transcription of an inserted gene or nucleic acid sequence. Expression products may be proteins, polypeptides, or RNA. The nucleic acid sequence can be contained in a nucleic acid cassette. Expression of the nucleic acid can be continuous, constitutive, or regulated. The vector can also be used as a prokaryotic element for replication of plasmid in bacteria and selection for maintenance of plasmid in bacteria.

[0058] Methods of introducing and expressing genes into a cell are known in the art.In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0059] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, hpofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory', New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0060] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I,adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0061] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0062] In some embodiments, transducing comprises either calcium phosphate- mediated gene transfer, DEAE-dextran-mediated gene transfer, liposome-mediated gene transfer, electroporation-mediated gene transfer, viral vector-mediated gene transfer, or nucleofection-mediated gene transfer. In some embodiments, transducing is accomplished by calcium phosphate-mediated gene transfer. In some embodiments, transducing is accomplished by liposome-mediated gene transfer. In some embodiments, transducing is accomplished by electroporation-mediated gene transfer. In some embodiments, transducing is accomplished by viral vector-mediated gene transfer. In some embodiments, transducing is accomplished by nucleofection-mediated gene transfer.

[0063] Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant DNA sequence in the host cell, or confirm effect of genomic modulation, a variety of assays may be performed. Such assays include, for example, “molecular biological’" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or other assays.

[0064] “Amplification” refers to any known procedure for obtaining multiple copies of a target nucleic acid or its complement, or fragments thereof. The multiple copies may be referred to as amplicons or amplification products. Amplification, in the context of fragments, refers to production of an amplified nucleic acid that contains less than the complete target nucleic acid or its complement, e.g.. produced by using an amplification oligonucleotide that hybridizes to, and initiates polymerization from, an internal position of the target nucleic acid. Known amplification methods include, for example, replicase- mediated amplification, polymerase chain reaction (PCR). reverse transcription polymerasechain reaction (RT-PCR). ligase chain reaction (LCR), strand-displacement amplification (SDA). and transcription-mediated or transcription-associated amplification. Amplification is not limited to the strict duplication of the starting molecule. For example, the generation of multiple cDNA molecules from RNA in a sample using reverse transcription (RT)-PCR is a form of amplification. Furthermore, the generation of multiple RNA molecules from a single DNA molecule during the process of transcription is also a form of amplification. During amplification, the amplified products can be labeled using, for example, labeled primers or by incorporating labeled nucleotides.

[0065] “Codon” refers to a sequence of three nucleotides that together form a unit of genetic code in a nucleic acid. “Codon of interest” refers to a specific codon in a target nucleic acid that has diagnostic or therapeutic significance (e.g. an allele associated with viral genotype / subtype or drug resistance).

[0066] “Region” refers to a portion of a nucleic acid wherein said portion is smaller than the entire nucleic acid. “Region of interest” refers to a specific sequence of a target nucleic acid that includes all codon positions having at least one single nucleotide substitution mutation associated with a genotype and / or subtype that are to be amplified and detected, and all marker positions that are to be amplified and detected, if any.

[0067] A “sequence” of a nucleic acid refers to the order and identity7of nucleotides in the nucleic acid. A sequence is typically read in the 5' to 3' direction. The terms “identical” or percent “identity” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, e.g., as measured using one of the sequence comparison algorithms available to persons of skill or by visual inspection. Exemplary algorithms that are suitable for determining percent sequence identity7and sequence similarity are the BLAST programs, which are described in, e.g., Altschul et al. (1990) “Basic local alignment search tool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification of protein coding regions by database similarity search” Nature Genet. 3:266-272, Madden et al. (1996) “Applications of network BLAST server” Meth. Enzymol. 266: 131-141, Altschul et al. (1997) "’’Gapped BLAST and PSI-BLAST: a new generation of protein database search programs” Nucleic Acids Res. 25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation"Genome Res. 7:649-656, which are each incorporated by reference. Many other optimal alignment algorithms are also known in the art and are optionally utilized to determine percent sequence identity. In the invention, the therapeutic nucleic acid or gene can be at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 99% or 100% identical to the wild-ty pe gene of interest.

[0068] '‘Nucleic acid” or ‘'nucleic acid molecule” refers to a multimeric compound comprising two or more covalently bonded nucleosides or nucleoside analogs having nitrogenous heterocyclic bases, or base analogs, where the nucleosides are linked together by phosphodiester bonds or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and analogs thereof. A nucleic acid backbone can be made up of a variety of linkages, including one or more of sugarphosphodiester linkages, peptide-nucleic acid bonds, phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of the nucleic acid can be ribose, deoxyribose, or similar compounds having known substitutions (e.g. 2'-methoxy substitutions and 2'-halide substitutions). Nitrogenous bases can be conventional bases (A,G, C, T, U) or analogs thereof (e.g., inosine, 5 -methylisocytosine, isoguanine). A nucleic acid can comprise only conventional sugars, bases, and linkages as found in RNA and DNA, or can include conventional components and substitutions (e.g., conventional bases linked by a 2'-methoxy backbone, or a nucleic acid including a mixture of conventional bases and one or more base analogs). Nucleic acids can include “locked nucleic acids” (LNA), in which one or more nucleotide monomers have a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhances hybridization affinity toward complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA). Nucleic acids can include modified bases to alter the function or behavior of the nucleic acid (e.g., addition of a 3'-terminal dideoxynucleotide to block additional nucleotides from being added to the nucleic acid). Synthetic methods for making nucleic acids in vitro are well known in the art although nucleic acids can be purified from natural sources using routine techniques. Nucleic acids can be single-stranded or double-stranded.

[0069] A nucleic acid is typically single-stranded or double-stranded and will generally contain phosphodiester bonds, although in some cases, as outlined, herein, nucleic acid analogs are included that may have alternate backbones, including, for example and without limitation, phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10): 1925 andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; and Pauwels et al. (1986) Chemica Scripta 26: 1419, which are each incorporated by reference), phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19: 1437; and U.S. Pat. No.5,644,048, which are both incorporated by reference), phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111:2321, which is incorporated by reference). O- methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press (1992), which is incorporated by reference), and peptide nucleic acid backbones and linkages (see, Egholm (1992) J. Am. Chem. Soc.114: 1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31 : 1008; Nielsen (1993) Nature 365:566; and Carlsson et al. (1996) Nature 380:207, which are each incorporated by reference). Other analog nucleic acids include those with positively charged backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92:6097, which is incorporated by reference); non-ionic backbones (U.S. Pat. Nos. 5.386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew (1991) Chem. Inti. Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghvi and P. Dan Cook; Mesmaeker et al. (1994) Bioorganic & Medicinal Chem: Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34: 17; and Tetrahedron Lett. 37:743 (1996), which are each incorporated by reference) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. Y. S. Sanghvi and P. Dan Cook, which references are each incorporated by reference. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al.(1995) Chem. Soc. Rev. pp 169-176, which is incorporated by reference). Several nucleic acid analogs are also described in, e.g., Rawls, C & E News Jun. 2, 1997 page 35, which is incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to alter the stability and half-life of such molecules in physiological environments.

[0070] In addition to these naturally occurring heterocyclic bases that are typically found in nucleic acids (e.g., adenine, guanine, thymine, cytosine, and uracil), nucleic acid analogs also include those having non-naturally occurring heterocyclic or modified bases,many of which are described, or otherwise referred to, herein. In particular, many non- naturally occurring bases are described further in, e.g.. Seela et al. (1991) Helv. Chim. Acta 74: 1790, Grein et al. (1994) Bioorg. Med. Chem. Lett. 4:971-976, and Seela et al. (1999) Helv. Chim. Acta 82: 1640, which are each incorporated by reference. To further illustrate, certain bases used in nucleotides that act as melting temperature (TO modifiers are optionally included. For example, some of these include 7-deazapurines (e.g., 7-deazaguanine. 7- deazaadenine, etc.), pyrazolo[3.4-d]pyrimidines, propynyl-dN (e.g., propynyl-dU, propynyl- dC, etc.), and the like. See, e.g., U.S. Pat. No. 5,990,303, entitled “SYNTHESIS OF 7- DEAZA-2'-DEOXYGUANOSINE NUCLEOTIDES,” which issued Nov. 23, 1999 to Seela, which is incorporated by reference. Other representative heterocyclic bases include, e.g., hypoxanthine, inosine, xanthine; 8-aza derivatives of 2-aminopurine, 2,6-diaminopurine, 2- amino-6-chloropurine, hypoxanthine, inosine and xanthine; 7 -deaza-8-aza derivatives of adenine, guanine, 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 6-azacytosine; 5 -fluorocytosine; 5 -chlorocytosine; 5-iodocytosine; 5- bromocytosine; 5-methylcytosine; 5-propynylcytosine; 5-bromovinyluracil; 5 -fluorouracil; 5- chlorouracik 5-iodouracik 5-bromouracik 5-trifluoromethyluracil; 5-methoxymethyluracil; 5- ethynyluracil; 5-propynyluracil, and the like.

[0071] In an aspect, the disclosure provides a pharmaceutical composition comprising the nucleic acid sequences of the disclosure and one or more pharmaceutically acceptable excipients or diluents.

[0072] As used herein the term “pharmaceutical composition” refers to pharmaceutically acceptable compositions, wherein the composition comprises a pharmaceutically active agent, and in some embodiments further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination of pharmaceutically active agents and carriers.

[0073] As used herein the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and / or non-human mammals.

[0074] As used herein the term “pharmaceutically acceptable diluent or excipient” or“pharmaceutically acceptable carrier” refers to an excipient, diluent, preservative, solubilizer,emulsifier, adjuvant, and / or vehicle with which a nucleic acid of the disclosure, is administered. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Antibacterial agents such as benzy l alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. Methods for producing compositions in combination with carriers are known to those of skill in the art. In some embodiments, the language “pharmaceutically acceptable diluent or excipient’' is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

[0075] Formulations of a pharmaceutical composition suitable for administration typically generally comprise the active ingredient combined with a pharmaceutically acceptable diluents or excipients, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. Formulations may also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents or sterile, pyrogen-free, water. Exemplary administration forms may include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.

[0076] The term “combination" refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where one or more active compounds and a combination partner (e.g., another drug as explained below, also referred toas “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals. In some circumstances, the combination partners show a cooperative, e.g., synergistic effect. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g.. the administration of three or more active ingredients.

[0077] In an aspect, the disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering the nucleic acid sequences of the disclosure or the pharmaceutical composition of the disclosure to the subject. In some embodiments, the disease or disorder is a cardiomyopathy.

[0078] The terms “subject.” “patient” and “individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. A “subject,” “patient” or “individual” as used herein, includes any animal that exhibits cardiomyopathy that can be treated with the vectors, compositions, and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0079] In some embodiments, administering comprises administering a therapeutically effective amount to a subject.

[0080] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of a composition to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results. As used herein, “therapeutically effective amount” refers to an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases and medical conditions. When used with reference to a method, the method is sufficiently effective to treat or ameliorate, or in some manner reduce the symptoms associated with diseases or conditions. For example, an effective amount in reference to diseases is that amount which is sufficient to block or prevent onset; or if disease pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. In any case, an effective amount may be given in single or divided doses.

[0081] As used herein, the terms “treat,” “treatment,” or “treating” embraces at least an amelioration of the symptoms associated with diseases in the patient, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. a symptom associated with the disease or condition being treated. As such, “treatment” also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g. prevented from happening) or stopped (e.g. terminated) such that the patient no longer suffers from the condition, or at least the symptoms that characterize the condition.

[0082] As used herein, and unless otherwise specified, the terms "prevent," "preventing" and "prevention" refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In certain embodiments, subjects with familial history of a disease are potential candidates for preventive regimens. In certain embodiments, subjects who have a history of recurring symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be interchangeably used with the term "prophylactic treatment."

[0083] As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with one or more other agent(s), which provides a prophylactic benefit in the prevention of the disease. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. In some embodiments, the pharmaceutical composition of the disclosure is administered in a prophylactically effective amount. EXAMPLES

[0084] Alpha Protein Kinase 3 (ALPK3) is an atypical protein kinase that is a member of the alpha (a)-kinase family. Individuals carrying homozygous or compound heterozygous loss-of-function pathogenic ALPK3 mutations often exhibit a range of cardiomyopathy phenotypes. These frequently present at birth or early childhood as severe early-onset dilated cardiomyopathy (DCM) and subsequently progress towards a hypertrophic cardiomy opathy (HCM) phenotype over time. In contrast, individuals carrying heterozygous truncated variants in ALPK3 typically exhibit a late onset of hypertrophic cardiomyopathy (HCM). This is often accompanied by significant myocardial fibrosis, leading to the progression of heart failure. According to the genoAD database, loss-of- function ALPK3 variants are found in approximately 1 in 2,500 individuals. In two separate cohorts of HCM patients, comprising 770 and 2,047 patients respectively, it was found that 1.56% of HCM patients carried heterozygous loss-of-function ALPK3 variants. Currently, no specific treatments are available for patients diagnosed with ALPK3 cardiomyopathy.

[0085] This invention provides a novel and effective approach for gene replacement to treat ALPK3 cardiomyopathy. In embodiments, the method involves the use of either the full-length wild-type ALPK3 or a functional, smaller version of ALPK3.

[0086] ALPK3 knockout mice exhibited normal cardiac function at birth but rapidly developed dilated cardiomyopathy within the first week of life, resulting in premature death. These mice also demonstrated increased left ventricular cavity dimensions and significantly reduced contractile function. By the sixth week, substantial ventricular hypertrophy was observed in Alpk3 knockout mice. The objective was to utilize adeno-associated viruses(AAV)9 or other modified AAV vectors as a vector to deliver ALPK3 cDNA into cardiomyocytes for the treatment of cardiomyopathy in ALPK3 knockout mice. However, the wild- type ALPK3 cDNA sequence, encoding 1608 amino acids (AAs), exceeds the 4.7 kb AAV packaging limit, rendering AAV infeasible. Consequently, the disclosure provides a functional, smaller version of ALPK3 (miniALPK3), which can be efficiently packaged into AAV to treat ALPK3 cardiomyopathy.

[0087] As a proof of concept for the functionality of this mini ALPK3 gene, a mouse model was developed that carries the miniALPK3 in place of the normal alpk3 allele. This was achieved by deleting a segment of the exon 6 sequence that encodes 373 AAs. Mice homozygous for this miniALPK3 demonstrated normal cardiac function extending into adulthood, suggesting that this miniALPK3 can fully compensate for the function of the full- length ALPK3 in the heart.

[0088] Alpk3 knockout mice exhibited normal cardiac function at birth but rapidly developed dilated cardiomyopathy within the first week of life, resulting in premature death (Fig. 1). These mice also demonstrated increased left ventricular cavity dimensions and significantly reduced contractile function (Fig. 2).

[0089] Fig. 1 shows that loss of ALPK3 in mice induces premature death. (Fig. 1 Panel A) RT-qPCR shows that ALPK3 mRNA is significantly downregulated in ALPK3 global knockout (gKO) mice. (Fig. 1 Panel B) Survival curve showed that ALPK3 gKO mice starts to die after one week of birth, half of gKO mice died before wean age.

[0090] Fig. 2 shows that loss of ALPK3 in mice induces cardiac dysfunction. Fraction shortening (FS) is used for measurement of contractile function. Left ventricle internal dimension at diastole (LVIDd) and left ventricle internal dimension at systole (LVIDs) are used for measurement of chamber size.

[0091] By the eighth week, substantial left ventricular hy pertrophy as indicated by left ventricular posterior wall end diastole (LVPWd) and interv entricular septum thickness end diastole (IVSd) was observed in Alpk3 knockout mice (Fig. 3). Fig. 3 shows that loss of ALPK3 in mice induces left ventricular hypertrophy. The left ventricular posterior wall end diastole (LVPWd) and interventricular septum thickness end diastole (IVSd) are used for measurement of left ventricular hypertrophy.

[0092] The objective in this example was to utilize adeno-associated viruses (AAV) 9 or other modified AAV vectors as a vector to deliver ALPK3 cDNA into cardiomyocytes for the treatment of cardiomyopathy in ALPK3 knockout mice. However, the wild-type ALPK3 cDNA sequence, encoding 1608 amino acids (AAs), exceeds the 4.7 kb AAV packaging limit, rendering AAV infeasible. Consequently, a functional, smaller version of ALPK3 (miniALPK3) was developed, which can be efficiently packaged into AAV to treat ALPK3 cardiomyopathy.

[0093] A mouse model was developed that carries the miniALPK3 in place of the normal alpk3 allele (Figure 4). This was achieved by deleting a segment of the exon 6 sequence, which encodes 373 AAs. Fig. 4 shows generation of miniALPK3 mouse model.(Fig. 4 Panel A) Schematics shows the ALPK3 Exon-6 was cleaved by CRISPR complex. This two-cut strategy generates 1119 bp in-frame deletion (373 aa) of miniALPK3 allele. (Fig. 4 Panel B) PCR of mouse genomic DNA from tails using P1 / P2 primers in (Fig. 4 Panel A) amplifies the miniALPK3 allele DNA fragment. (Fig. 4 Panel C) E Sanger sequencing confirming 1119 bp in-frame deletion (373 aa from position 671 to 1045) in miniALPK3 mice.

[0094] The amino acid sequence of wild-type mouse ALPK3 is provided in SEQ ID No.: 1. The amino acid sequence of mouse miniALPK3 is provided in SEQ ID No.: 3. The amino acid sequence of wild-type human ALPK3 is provided in SEQ ID No.: 5. The amino acid sequence of human miniALPK3 is provided in SEQ ID No.: 7.

[0095] Mice homozygous for this miniALPK3 demonstrated normal cardiac function extending into adulthood (Fig. 5), suggesting that this miniALPK3 can fully compensate for the function of the full-length ALPK3 in the heart. Fig. 5 shows echocardiography assessment the control and homozygous miniALPK3 mice. Fraction shortening (FS) is used for measurement of contractile function. Left ventricle internal dimension at diastole (LVIDd) and left ventricle internal dimension at systole (LVIDs) are used for measurement of chamber size. The left ventricular posterior wall end diastole (LVPWd) was used for measurement of left ventricular hypertrophy.

[0096] To ensure appropriate expression levels of the mini-ALPK3 protein, the optimal viral titer for injection can be determined. Unfortunately, all presently available commercial ALPK3 antibodies failed to yield specific signals in wild-type samples (data notshown). Due to this limitation, a novel ALPK3-3xFLAG tagged knock-in mouse model was generated using CRISPR / Cas9 technology, in which a 3xFLAG tag was incorporated at the C-terminus of ALPK3 (Fig. 6 Panels A-B). Homozygous knock-in (A / ty3FLAG / FLAG) mice exhibited normal cardiac function and displayed no discernible developmental defects (Fig. 6 Panel C), suggesting that the addition of the 3xFLAG tag at the C-terminus of ALPK3 did not impact its functionality. Importantly, this allowed reliable and consistent detection of ALPK3 protein levels using an antibody against the FLAG tag (Fig. 6 Panel B).

[0097] Fig. 6 shows generation of ALPK3-3xFLAG tagged knock-in mouse model. (Fig. 6 Panel A) Schematic representation of the CRISPR / Cas9 strategy used to generate ALPK3-3xFLAG tagged knock-in mice. (Fig. 6 Panel B) Western blot analysis of FLAG- tagged ALPK3 in control (Ctrl) and ALPK3-3xFLAG tagged (FLAG / FLAG) knock-in hearts. Histone H3 was used as a loading control. (Fig. 6 Panel C) Echocardiography measurements of fractional shortening (FS), left ventricular (LV) internal dimensions at enddiastole (LVIDd) and end-systole (LVIDs), as well as LV posterior wall thickness at end- diastole (LVPWd) in ALPK3-3xFLAG tagged knock-in (n=7) and Ctrl mice (n=6) at 3 months of age. NS, not significant.

[0098] Double heterozygous mice carrying both the FLAG-tagged and null Alpk3 alleles (A / / 2^3FLAG‘) were generated by crossing the ALPK3 3xFLAG-tagged mice with Alpk3 heterozygous null mutants (Alpk3+I~). The vector MyoAAV-2A-cTnT-miniALPK3- 3xFLAG was administered to Alpk3 global knockout (GKO) mice at a dose of 1 x 10A14 vg / kg on postnatal day 1, aiming to intervene before the manifestation of cardiac phenotypes. This result contrasts with the observation that 70% of untreated global knockout mice had premature death before reaching weaning age (postnatal day 21). Notably, all three GKO mice receiving the MyoAAV-2A-cTnT-miniALPK3-3xFLAG injection survived beyond this critical period, highlighting the potential protective effect of the treatment. By the sixth week, the heart structure and function of these mice was evaluated using echocardiography (Fig. 7). The analysis revealed that the three gKO mice treated with MyoAAV-2A-cTnT-miniALPK3- 3xFLAG exhibited significant improvements in cardiac function compared to their age- matched gKO counterparts. Furthermore, the cardiac function of the treated gKO mice was found to be comparable to that of control mice, indicating a substantial restoration of normal cardiac performance. This study suggests that early intervention with MyoAAV-2A-cTnT-miniALPK3-3xFLAG can effectively mitigate the adverse cardiac outcomes commonly observed in Alpk3 GKO mice.

[0099] Fig. 7 shows injecting MyoAAV-2A-cTnT-miniALPK3-3xFLAG into ALPK3 gene knockout (GKO) mice restores their heart function. Echocardiography measurements of fractional shortening (FS) in Ctrl (ALPK3-3xFLAG tagged knock-in) (n=3). GKO mice, GKO with MyoAAV-2A-cTnT-miniALPK3-3xFLAG (n=3) at 6 weeks of age.

[0100] It will be understood from the foregoing description that various modifications and changes may be made in the various embodiments of the present disclosure without departing from their true spirit. The description provided herein is intended for purposes of illustration only and is not intended to be construed in a limiting sense. Thus, while the presently disclosed inventive concepts have been described herein in connection with certain embodiments so that aspects thereof may be more fully understood and appreciated, it is not intended that the presently disclosed inventive concepts be limited to these particular embodiments. On the contrary, it is intended that all alternatives, modifications and equivalents are included within the scope of the presently disclosed inventive concepts as defined herein. Thus the examples described above, which include particular embodiments, will serve to illustrate the practice of the presently disclosed inventive concepts, it being understood that the particulars show n are by way of example and for purposes of illustrative discussion of particular embodiments of the presently disclosed inventive concepts only and are presented in the cause of providing what is believed to be a useful and readily understood description of procedures as well as of the principles and conceptual aspects of the inventive concepts. Changes may be made in the construction and formulation of the various components and compositions described herein, the methods described herein or in the steps or the sequence of steps of the methods described herein without departing from the spirit and scope of the presently disclosed inventive concepts.

[0101] SEQUENCE LISTING

[0102] Amino acid sequence of mouse full length ALPK3 SEQ ID No.: 1

[0103] 1680aa

[0104] MGSRRAAGRGWGLGGRAGAGGDSEDDGPVWTPGPASRSYLLSVRPEASLSSNRLSHPSSGRSTFCSIIAQLTEETQPLFETTLKSRAVSEDSDVRFTCIVTGYPEPEVTWYKDDIELDRYCGLPKYEITHQGNRHTLQLYRCQEEDAAIYQASARNTKGIVSCSGVLEVGTMTEYKIHQRWFAKLKRKAAAKMREIEQSWKHGKEASGEADTLLRKISPD RFQRKRRLSGVEEAVLSTPVREMEEGSSAAWQEGETESAQHPGLGLINSFAPGEAPT NGEPAPENGEDEERGLLTYICEVMELGPQNSPPKESGAKKKRKDEESKPGEQKLELE KAEGSQCSSENVVPSTDKPNSSRREKSTDTQPAQTQPRGRVARGPGIESTRKTASVLGIQDKVQDVPAPAPAPVPAPALAPAPVPVPAPTPVPSRSSEQVYFSLKDMFMETTRAGRSQEEEKPPPPSTRVAGESPPGKTPVKSRLEKVPMVSSQPTSSMVPPPIKPLNRKRFAPPKSKVESTTTSLSSQTSESMAQSLGKALPSASTQVPTPPARRRHGTRDSPLQGQTSHKTPGEALESPATVAPTKSANSSSDTVSVDHDSSGNQGATEPMDTETQEDGRTLVDGRT GSRKKTHTDGKLQVDGRTQGDGAQDRAHASPRTQAGEKAPTDVVTQGSERPQSDRSSWKNLVTQRRVDMQVGQMQAGERWQQDPGDARIQEEEKETQSAAGSIPVAFETQSEQLSMASLSSLPGALKGSPSGCPRESQAIECFEKSTEAPCVQERSDLMLRSEEAAFRSHEDGLLGPPSGNRTYPTQLPPEGHSEHLGGQTHQRSEQEDSLSQCPKKEQPQEPLHVGLSGGHSTGLSQEVPAMPSLPGTGLTSSLQEELPGTAASLHTNTDVPLPSRDQDFPSS APTLQLGPGSPTQSHPPEAMATSSEGACAKEPNVDGRSSGTRSCDPGLIDSLKNYLLLLLKLSSPETSEARAESQEVADTGGLTSSSTLVPTMEVAGLSPRTSRRILERVENNHLVQSAQTLLLSPCTSRRLTGLLDREVQAGQQALAAAQCSRGPCPTPLTIPAIVVGEEGSAGEDSEERTSQESDKKGLLGEVEGHTVESRTQEPCQEEAMPGEALTGLPAATPEELALGARRKRFLPKVRAGSDGEANKAEERESPTVSPRGPRKGLTPGSPGTPGRERRSPTQA RKASMLEVPGAEEEPATGDLVSRSKDSGLDSEPAVDEGKQEALAKQRKAKDLLKAPQVIRKTRVEQFPDSSGSLKLWCQFFNIVSDSVLTWAKDQHPVGEVNRSAGDEGPAALAIVQASPTDCGVYRCTIQNEHGSASTDFCLSPEVLSGFISREEGEVGEEIEMTPMVFAKGLADSGCWGDKLFGRLVSEELRGGGHGLQKASRAKVIYGLEPIFESGRTCIIKVSSLLVFGPSSETSLLGRNYDVTIQGCKIQNMSREYCKIFAAEARAASGFGEVPEIIPLYLIYR PANNIPYATLEEDLGKPLQTYCSRQWGCAGAPAAASSSEALQKCQTFQHWLYQWTNGSFLVTDLTGADWKMTDVQIATKLRGYQGLKESCFPALLDQFASSHQCNTYCDM LGLKPLKGPEAAHPQAKAKGSKSPSAGRKGSQLSPQPQKKGLPSPQGSRKSAPSSRA TLQASQAATVQLLGQPPVQDGSSKAQSMR

[0105] Nucleic acid sequence of mouse full length ALPK3 SEQ ID No: 2

[0106] 5043na

[0107] ATGGGGTCGCGGAGGGCCGCGGGCCGGGGCTGGGGCCTGGGTGGTCGAGCAGGAGCTGGTGGAGATAGCGAGGACGACGGGCCGGTGTGGACGCCCGGCCCAGCCAGTCGCAGCTACTTGCTTAGCGTGCGGCCAGAGGCTAGCTTATCAAGCAACCGGTTGTCTCACCCCAGCTCTGGAAGGAGCACCTTCTGCTCCATCATTGCTCAGCTCACAGAGGAGACCCAGCCACTGTTCGAGACCACACTCAAGTCCCGTGCCGTGTCCGAAGACAGTGACGTCAGGTTCACCTGCATTGTCACAGGATACCCAGAGC CAGAGGTGACCTGGTACAAGGATGACATAGAACTGGACCGTTACTGTGGCTTGCCAAAATACGAGATCACTCATCAAGGCAACCGTCACACCCTGCAGCTGTACAGGTGTCAGGAAGAAGATGCTGCCATCTACCAAGCCTCTGCCCGGAACACCAAGGGCATCGTGTCCTGCTCAGGGGTCCTAGAGGTGGGCACTATGACGGAGTACAAGATTCACCAGCGCTGGTTCGCCAAGTTGAAGCGCAAGGCTGCAGCCAAGATGAGGGAGA TTGAGC AGAGCTGGAAGC ATGGAAAAGAGGCTTC AGGGGAGGCTGAC ACGCTTCTTCGCAAGATCAGCCCCGACCGCTTCCAAAGAAAGCGCCGACTGAGTGGAGTCGAAGAGGCTGTCCTCTCCACGCCAGTCAGGGAAATGGAGGAAGGCTCCTCAGCGGCTTGGCAGGAAGGAGAGACTGAGTCTGCTCAGCACCCGGGGTTGGGTTTGATCAACAGTTTTGCTCCTGGAGAGGCGCCCACCAATGGGGAGCCTGCTCCAGAGAACG GGGAAGACGAAGAGCGTGGCTTGCTGACATAC ATCTGCGAGGTCATGGAACTGGGGCCTCAGAACAGCCCTCCAAAGGAGTCTGGGGCTAAGAAGAAAAGGAAGGATGAGGAATCTAAACCAGGAGAGCAGAAGCTGGAGTTAGAAAAGGCAGAAGGGAGCCAGTGCTCTTCAGAAAACGTCGTCCCCAGTACAGACAAACCCAACTCCAGTAGAAGGGAGAAGTCCACGGATACACAGCCAGCTCAGACCCAGCCCAGAGGCCGGG TAGCACGGGGGCCTGGGATAGAAAGCACCAGGAAGACAGCCTCTGTCCTGGGCATTCAAGACAAGGTCCAGGATGTCCCCGCCCCCGCCCCTGCCCCGGTCCCCGCCCCAGCCCTGGCCCCGGCCCCTGTCCCAGTCCCTGCACCCACCCCTGTTCCAAGCCGCAGCTCAGAGCAGGTGTATTTCTCCCTGAAGGACATGTTCATGGAGACCACCCGGGCAGGCAGGTCCCAGGAAGAGGAAAAACCTCCACCTCCAAGTACCAGGGTAGCT GGAGAAAGTCCCCCAGGAAAGACACCAGTCAAGTCTAGACTGGAGAAGGTACCGATGGTCTCCAGCCAGCCCACATCTTCCATGGTTCCCCCGCCCATTAAGCCTTTGAACAGGAAGAGATTTGCCCCTCCTAAATCCAAAGTGGAGTCAACTACTACCTCTCTCTCAAGTCAGACTTCAGAATCTATGGCCCAGAGCTTAGGGAAGGCTCTACCTTCAGCCTCTACCCAGGTCCCAACACCCCCTGCTCGACGGAGACACGGCACCCGAGA TAGCCCCTTGCAAGGACAAACGAGCCACAAGACTCCAGGAGAGGCTCTGGAGTCCCCAGCAACCGTGGCTCCCACCAAGTCTGCCAACAGCAGCTCCGATACCGTCTCTGTTGATCACGACAGCTCTGGAAATCAAGGGGCCACGGAGCCCATGGATACAGAAACTCAGGAAGATGGAAGGACACTTGTGGATGGGAGAACTGGAAGCAGGAAGAAAACACACACAGATGGAAAGCTGCAAGTGGATGGGAGGACTCAGGGAGACGGAGCACAAGACAGAGCACACGCTTCGCCAAGGACACAGGCAGGTGAGAAGGCACCGACGGACGTTGTGACACAAGGAAGTGAGAGGCCACAGTCAGACAGGAGTTCATGGAAGAATTTGGTGACACAGAGAAGAGTGGATATGCAGGTAGGACAGATGCAGGCAGGTGAGAGGTGGCAGCAAGACCCTGGAGACGCAAGGATACAGGAGGAAGAAA AAGAGACACAGTCAGCAGCAGGCAGCATTCCTGTAGCTTTCGAAACCCAATCAGAGCAGTTGTCCATGGCCAGCCTCAGCTCACTTCCTGGAGCTCTCAAAGGCTCACCATCAGGATGCCCTAGAGAGTCCCAGGCTATAGAATGTTTTGAGAAGAGCACAGAGGCACCCTGTGTCCAAGAAAGATCTGACTTGATGCTGCGGTCTGAAGAGGCAGCCTTCAGAAGCCATGAGGATGGGCTGCTAGGCCCCCCATCAGGGAACCGTACCTA CCCAACACAGTTGCCTCCCGAGGGGCACTCAGAGCATTTGGGAGGACAAACACATCAAAGGTCAGAGCAAGAGGACAGCCTGTCTCAGTGCCCCAAGAAGGAGCAGCCCCAGGAACCGCTTCATGTGGGTCTCTCTGGTGGGCATTCAACTGGCTTGAGTCAGGAGGTGCCTGCCATGCCTTCTCTTCCTGGGACGGGCCTAACCAGTAGTCTGCAGGAGGAGCTACCAGGCACCGCAGCTTCTCTGCACACAAACACAGATGTCCCCCTCCC CTCCAGGGACCAGGACTTTCCGAGTTCTGCTCCCACTCTGCAATTGGGGCCAGGGTCCCCCACTCAGAGTCACCCACCAGAAGCCATGGCTACTAGCAGTGAGGGAGCCTGTGCCAAGGAGCCAAATGTGGACGGGAGGTCCTCAGGTACCCGGAGCTGTGACCCTGGCCTTATAGATTCCCTGAAGAACTACTTGCTTCTGCTGCTGAAGCTATCCAGTCCAGAGACAAGTGAAGCCAGGGCCGAGTCCCAGGAAGTGGCAGACACCGGG GGCTTAACCTCCTCCTCTACTCTGGTCCCCACCATGGAGGTGGCTGGGCTGAGTCCCAGGACGTCGAGGCGAATCCTGGAACGCGTGGAGAACAATCACTTAGTGCAGAGTGCACAGACCCTGTTGCTGAGCCCCTGCACCTCCCGCCGCCTTACTGGCCTTCTGGACCGTGAGGTACAGGCTGGCCAGCAGGCTCTGGCTGCTGCCCAGTGCTCTCGGGGCCCGTGCCCCACCCCCCTCACCATCCCTGCCATTGTGGTGGGTGAGGAAGGA TCTGCGGGAGAGGATTCCGAGGAGAGGACTTCGCAGGAAAGTGACAAGAAGGGACTGCTAGGGGAAGTGGAGGGGCACACAGTGGAAAGCAGAACCCAGGAGCCCTGCCAAGAAGAAGCAATGCCAGGGGAGGCTTTGACGGGTCTCCCTGCAGCTACACCCGAGGAACTGGCTCTGGGGGCCCGGAGGAAGAGGTTCCTCCCTAAGGTCAGAGCAGGGTCAGATGGAGAGGCAAACAAGGCTGAAGAAAGGGAGAGCCCCACGGTT TCCCCCCGGGGACCCAGGAAGGGCCTGACACCTGGGTCACCAGGGACTCCAGGGCGGGAGAGACGGTCCCCTACCCAGGCCCGAAAAGCCAGCATGTTAGAGGTGCCTGGCGCAGAAGAAGAGCCTGCAACTGGAGACTTGGTCTCCAGATCCAAAGACAGTGGCCTGGACTCAGAGCCTGCGGTGGATGAAGGCAAGCAGGAAGCTCTGGCCAAGCAAAGGAAAGCTAAGGACCTGCTAAAAGCCCCACAGGTGATCCGGAAAATTCGGGTGGAACAGTTTCCAGATTCTTCTGGTAGTCTGAAGCTTTGGTGCCAGTTTTTCAACATTGTTAGTGACTCAGTCTTGACATGGGCGAAGGATCAGCACCCAGTGGGCGAAGTGAACAGGAGTGCAGGGGACGAGGGGCCAGCGGCTTTGGCCATCGTGCAGGCGTCTCCCACGGACTGTGGTGTGTATCGCTGTACCATCCAAAACGAGCATGGCTCA GCGTCCACTGACTTCTGCCTCAGCCCCGAGGTATTGTCCGGCTTCATCTCCAGAGAGGAAGGTGAAGTTGGAGAAGAGATTGAGATGACCCCCATGGTGTTTGCTAAGGGTCTGGCTGACTCTGGCTGCTGGGGGGACAAGCTCTTTGGGCGATTGGTGAGCGAGGAACTTCGAGGGGGTGGACATGGCCTTCAGAAGGCGTCCCGGGCCAAGGTCATCTATGGGCTGGAACCCATCTTCGAATCTGGCCGCACGTGCATCATCAAAGTATCC AGCCTGCTTGTGTTCGGACCCAGCAGTGAGACCTCTCTTCTGGGCAGAAACTATGACGTCACTATCCAGGGATGCAAGATCCAGAACATGAGTCGAGAGTACTGCAAAATCTTTGCAGCTGAAGCCCGGGCGGCCTCTGGCTTCGGAGAGGTGCCCGAGATCATCCCACTCTACTTGATCTACCGGCCTGCAAACAATATACCATATGCAACCCTGGAGGAAGATCTGGGCAAGCCCCTGCAGACTTACTGTTCCAGGCAGTGGGGCTGTGCT GGGGCCCCCGCAGCAGCCAGCAGCTCCGAGGCCTTGCAGAAATGCCAAACCTTCCAGCACTGGCTGTATCAGTGGACAAACGGCAGCTTTCTTGTCACAGATCTGACAGGAGCTGACTGGAAGATGACTGATGTACAGATTGCTACCAAACTTCGAGGATACCAAGGCCTCAAGGAGAGCTGTTTTCCTGCCCTGCTGGACCAGTTTGCCTCTTCCCACCAGTGTAACACCTACTGTGACATGCTGGGGCTGAAGCCCCTCAAAGGCCCTGA GGCTGCCCACCCTCAAGCCAAGGCCAAAGGCTCCAAAAGTCCATCTGCTGGCAGAAAAGGCTCACAGCTGAGTCCTCAACCCCAGAAGAAAGGCCTTCCCAGTCCCCAGGGCTCCAGGAAGAGCGCTCCAAGCTCCAGGGCTACACTTCAGGCCTCCCAGGCAGCCACTGTTCAGTTACTGGGACAGCCTCCTGTCCAAGATGGGAGCTCTAAGGCCCAGAGCATGCGGTAG

[0108] Amino acid sequence of mouse miniALPK3 SEQ ID No: 3

[0109] 1307aa

[0110] MGSRRAAGRGWGLGGRAGAGGDSEDDGPVWTPGPASRSYLLSVRPEASLSSNRLSHPSSGRSTFCSIIAQLTEETQPLFETTLKSRAVSEDSDVRFTCIVTGYPEPEVTWYKDDIELDRYCGLPKYEITHQGNRHTLQLYRCQEEDAAIYQASARNTKGIVSCS GVLEVGTMTEYKIHQRWFAKLKRKAAAKMREIEQSWKHGKEASGEADTLLRKISPDRFQRKRRLSGVEEAVLSTPVREMEEGSSAAWQEGETESAQHPGLGLINSFAPGEAPTNGEPAPENGEDEERGLLTYICEVMELGPQNSPPKESGAKKKRKDEESKPGEQKLELEKAEGSQCSSENVVPSTDKPNSSRREKSTDTQPAQTQPRGRVARGPGIESTRKTASVLGIQDKVQDVPAPAPAPVPAPALAPAPVPVPAPTPVPSRSSEQVYFSLKDMFMETTRAGRSQEEEKPPPPSTRVAGESPPGKTPVKSRLEKVPMVSSQPTSSMVPPPIKPLNRKRFAPPKSKVESTTTSLSSQTSESMAQSLGKALPSASTQVPTPPARRRHGTRDSPLQGQTSHK TPGEALESPATVAPTKSANSSSDTVSVDHDSSGNQGATEPMDTETQEDGRTLVDGRTGSRKKTHTDGKLQVDGRTQGDGAQDRAHASPRTQAGEKAPTDVVTQGSREVQAGQQALAAAQCSRGPCPTPLTIPAIVVGEEGSAGEDSEERTSQESDKKGLLGEVEGHTVESRTQEPCQEEAMPGEALTGLPAATPEELALGARRKRFLPKVRAGSDGEANKAEERESPTVSPRGPRKGLTPGSPGTPGRERRSPTQARKASMLEVPGAEEEPATGDLVSRSKDSG LDSEPAVDEGKQEALAKQRKAKDLLKAPQVIRKIRVEQFPDSSGSLKLWCQFFNIVSDSVLTWAKDQHPVGEVNRSAGDEGPAALAIVQASPTDCGVYRCTIQNEHGSASTDFCLSPEVLSGFISREEGEVGEEIEMTPMVFAKGLADSGCWGDKLFGRLVSEELRGGGHGLQKASRAKVIYGLEPIFESGRTCIIKVSSLLVFGPSSETSLLGRNYDVTIQGCKIQNMSREYCKIFAAEARAASGFGEVPEIIPLYLIYRPANNIPYATLEEDLGKPLQTYCSRQWGC AGAPAAAS SSEALQKCQTFQHWLYQWTNGSFLVTDLTGADWKMTDVQIATKLRGYQGLKESCFPALLDQFASSHQCNTYCDMLGLKPLKGPEAAHPQAKAKGSKSPSAGRKGSQLSPQPQKKGLPSPQGSRKSAPSSRATLQASQAATVQLLGQPPVQDGSSKAQSMR

[0111] Nucleic acid sequence of mouse mini-ALPK3 SEQ ID No: 4

[0112] 3924na

[0113] ATGGGGTCGCGGAGGGCCGCGGGCCGGGGCTGGGGCCTGGGTGGTCGAGCAGGAGCTGGTGGAGATAGCGAGGACGACGGGCCGGTGTGGACGCCCGGCCCAGCCAGTCGCAGCTACTTGCTTAGCGTGCGGCCAGAGGCTAGCTTATCAAGCAACCGGTTGTCTCACCCCAGCTCTGGAAGGAGCACCTTCTGCTCCATCATTGCTCAGCTCACAGAGGAGACCCAGCCACTGTTCGAGACCACACTCAAGTCCCGTGCCG TGTCCGAAGACAGTGACGTCAGGTTCACCTGCATTGTCACAGGATACCCAGAGCCAGAGGTGACCTGGTACAAGGATGACATAGAACTGGACCGTTACTGTGGCTTGCCAAAATACGAGATCACTCATCAAGGCAACCGTCACACCCTGCAGCTGTACAGGTGTCAGGAAGAAGATGCTGCCATCTACCAAGCCTCTGCCCGGAACACCAAGGGCATCGTGTCCTGCTCAGGGGTCCTAGAGGTGGGCACTATGACGGAGTACAAGATTC ACCAGCGCTGGTTCGCCAAGTTGAAGCGCAAGGCTGCAGCCAAGATGAGGGAGATTGAGCAGAGCTGGAAGCATGGAAAAGAGGCTTCAGGGGAGGCTGACACGCTTCTTCGC AAGATC AGCCC CGAC CGCTTCC AAAGAAAGC GC CGACTGAGTGGAGTC GAAGAGGCTGTCCTCTCCACGCCAGTCAGGGAAATGGAGGAAGGCTCCTCAGCGGCTTGGCAGGAAGGAGAGACTGAGTCTGCTCAGCACCCGGGGTTGGGTTTGATCAACAGTTTTGCTCCTGGAGAGGCGCCCACCAATGGGGAGCCTGCTCCAGAGAACGGGGAAGACGAAGAGCGTGGCTTGCTGACATACATCTGCGAGGTCATGGAACTGG GGCCTCAGAACAGCCCTCCAAAGGAGTCTGGGGCTAAGAAGAAAAGGAAGGATGAGGAATCTAAACCAGGAGAGCAGAAGCTGGAGTTAGAAAAGGCAGAAGGGAGCCAGTGCTCTTCAGAAAACGTCGTCCCCAGTACAGACAAACCCAACTCCAGTAGAAGGGAGAAGTCCACGGATACACAGCCAGCTCAGACCCAGCCCAGAGGCCGGGTAGCACGGGGGCCTGGGATAGAAAGCACCAGGAAGACAGCCTCTGTCCTGGGCA TTCAAGACAAGGTCCAGGATGTCCCCGCCCCCGCCCCTGCCCCGGTCCCCGCCCCAGCCCTGGCCCCGGCCCCTGTCCCAGTCCCTGCACCCACCCCTGTTCCAAGCCGCAGCTCAGAGCAGGTGTATTTCTCCCTGAAGGACATGTTCATGGAGACCACCCGGGCAGGCAGGTCCCAGGAAGAGGAAAAACCTCCACCTCCAAGTACCAGGGTAGCTGGAGAAAGTCCCCCAGGAAAGACACCAGTCAAGTCTAGACTGGAGAAGGTACC GATGGTCTCCAGCCAGCCCACATCTTCCATGGTTCCCCCGCCCATTAAGCCTTTGAACAGGAAGAGATTTGCCCCTCCTAAATCCAAAGTGGAGTCAACTACTACCTCTCTCTCAAGTCAGACTTCAGAATCTATGGCCCAGAGCTTAGGGAAGGCTCTACCTTCAGCCTCTACCCAGGTCCCAACACCCCCTGCTCGACGGAGACACGGCACCCGAGATAGCCCCTTGCAAGGACAAACGAGCCACAAGACTCCAGGAGAGGCTCTGGAGTC CCCAGCAACCGTGGCTCCCACCAAGTCTGCCAACAGCAGCTCCGATACCGTCTCTGTTGATCACGACAGCTCTGGAAATCAAGGGGCCACGGAGCCCATGGATACAGAAACTCAGGAAGATGGAAGGACACTTGTGGATGGGAGAACTGGAAGCAGGAAGAAAACACACACAGATGGAAAGCTGCAAGTGGATGGGAGGACTCAGGGAGACGGAGCACAAGACAGAGCACACGCTTCGCCAAGGACACAGGCAGGTGAGAAGGCACCG ACGGACGTTGTGACACAAGGAAGTCGTGAGGTACAGGCTGGCCAGCAGGCTCTGGCTGCTGCCCAGTGCTCTCGGGGCCCGTGCCCCACCCCCCTCACCATCCCTGCCATTGTGGTGGGTGAGGAAGGATCTGCGGGAGAGGATTCCGAGGAGAGGACTTCGCAGGAAAGTGACAAGAAGGGACTGCTAGGGGAAGTGGAGGGGCACACAGTGGAAAGCAGAACCCAGGAGCCCTGCCAAGAAGAAGCAATGCCAGGGGAGGCTTTGAC GGGTCTCCCTGCAGCTACACCCGAGGAACTGGCTCTGGGGGCCCGGAGGAAGAGGTTCCTCCCTAAGGTCAGAGCAGGGTCAGATGGAGAGGCAAACAAGGCTGAAGAAAGGGAGAGCCCCACGGTTTCCCCCCGGGGACCCAGGAAGGGCCTGACACCTGGGTCACCAGGGACTCCAGGGCGGGAGAGACGGTCCCCTACCCAGGCCCGAAAAGCCAGCATGTTAGAGGTGCCTGGCGCAGAAGAAGAGCCTGCAACTGGAGACTTGGTCTCCAGATCCAAAGACAGTGGCCTGGACTCAGAGCCTGCGGTGGATGAAGGCAA GCAGGAAGCTCTGGCCAAGCAAAGGAAAGCTAAGGACCTGCTAAAAGCCCCAC AGGTGATCCGGAAAATTCGGGTGGAACAGTTTCCAGATTCTTCTGGTAGTCTGAA GCTTTGGTGC C AGTTTTTC AAC ATTGTTAGTGACTC AGTCTTGAC ATGGGCGAAG GATCAGCACCCAGTGGGCGAAGTGAACAGGAGTGCAGGGGACGAGGGGCCAGC GGCTTTGGCCATCGTGCAGGCGTCTCCCACGGACTGTGGTGTGTATCGCTGTACC ATCCAAAACGAGCATGGCTCAGCGTCCACTGACTTCTGCCTCAGCCCCGAGGTAT TGTCCGGCTTCATCTCCAGAGAGGAAGGTGAAGTTGGAGAAGAGATTGAGATGA CCCCCATGGTGTTTGCTAAGGGTCTGGCTGACTCTGGCTGCTGGGGGGACAAGCT CTTTGGGCGATTGGTGAGCGAGGAACTTCGAGGGGGTGGACATGGCCTTCAGAA GGCGTCCCGGGCCAAGGTCATCTATGGGCTGGAACCCATCTTCGAATCTGGCCGC ACGTGCATCATCAAAGTATCCAGCCTGCTTGTGTTCGGACCCAGCAGTGAGACCT CTCTTCTGGGCAGAAACTATGACGTCACTATCCAGGGATGCAAGATCCAGAACA TGAGTCGAGAGTACTGCAAAATCTTTGCAGCTGAAGCCCGGGCGGCCTCTGGCTT CGGAGAGGTGCCCGAGATC ATCCCACTCTACTTGATCTACCGGCCTGCAAAC AAT ATACCATATGCAACCCTGGAGGAAGATCTGGGCAAGCCCCTGCAGACTTACTGTT CCAGGCAGTGGGGCTGTGCTGGGGCCCCCGCAGCAGCCAGCAGCTCCGAGGCCT TGCAGAAATGCCAAACCTTCCAGCACTGGCTGTATCAGTGGACAAACGGCAGCT TTCTTGTCACAGATCTGACAGGAGCTGACTGGAAGATGACTGATGTACAGATTGC TACCAAACTTCGAGGATACCAAGGCCTCAAGGAGAGCTGTTTTCCTGCCCTGCTG GACCAGTTTGCCTCTTCCCACCAGTGTAACACCTACTGTGACATGCTGGGGCTGA AGCCCCTCAAAGGCCCTGAGGCTGCCCACCCTCAAGCCAAGGCCAAAGGCTCCA AAAGTCCATCTGCTGGCAGAAAAGGCTCACAGCTGAGTCCTCAACCCCAGAAGA AAGGCCTTCCCAGTCCCCAGGGCTCCAGGAAGAGCGCTCCAAGCTCCAGGGCTA CACTTCAGGCCTCCCAGGCAGCCACTGTTCAGTTACTGGGACAGCCTCCTGTCCA AGATGGGAGCTCTAAGGCCCAGAGCATGCGGTAG

[0114] Amino acid sequence of human full length ALPK3 SEQ ID No.: 5

[0115] 1705aa

[0116] MGSRRAPSRGWGAGGRSGAGGDGEDDGPVWIPSPASRSYLLSVRPET SLSSNRLSHPSSGRSTFCSIIAQLTEETQPLFETTLKSRSVSEDSDVRFTCIVTGYPEPEV TWYKDDTELDRYCGLPKYEITHQGNRHTLQLYRCREEDAAIYQASAQNSKGIVSCS GVLEVGTMTEYKIHQRWFAKLKRKAAAKLREIEQSWKHEKAVPGEVDTLRKLSPDRFQRKRRLSGAQAPGPSVPTREPEGGTLAAWQEGETETAQHSGLGLINSFASGEVTTNGEAAPENGEDGEHGLLTYICDAMELGPQRALKEESGAKKKKKDEESKQGLRKPEL EKAAQSRRSSENCIPSSDEPDSCGTQGPVGVEQVQTQPRGRAARGPGSSGTDSTRKP ASAVGTPDKAQKAPGPGPGQEVYFSLKDMYLENTQAVRPLGEEGPQTLSVRAPGES PKGKAPLRARSEGVPGAPGQPTHSLTPQPTRPFNRKRFAPPKPKGEATTDSKPISSLSQAPECGAQSLGKAPPQASVQVPTPPARRRHGTRDSTLQGQAGHRTPGEVLECQTTTAP TMSASSSSDVASIGVSTSGSQGIIEPMDMETQEDGRTSANQRTGSKKNVQADGKIQV DGRTRGDGTQTAQRTRADRKTQVDAGTQESKRPQSDRSAQKGMMTQGRAETQLET TQAGEKIQEDRKAQADKGTQEDRRMQGEKGMQGEKGTQSEGSAPTAMEGQSEQEV ATSLGPPSRTPKLPPTAGPRAPLNIECFVQTPEGSCFPKKPGCLPRSEEAVVTASRNHEQTVLGPLSGNLMLPAQPPHEGSVEQVGGERCRGPQSSGPVEAKQEDSPFQCPKEERPGGVPCMDQGGCPLAGLSQEVPTMPSLPGTGLTASPKAGPCSTPTSQHGSTATFLPSE DQVLMSSAPTLHLGLGTPTQSHPPETMATSSEGACAQVPDVEGRTPGPRSCDPGLIDS LKNYLLLLLKLSSTETSGAGGESQVGAATGGLVPSATLTPTVEVAGLSPRTSRRILER VENNHLVQSAQTLLLSPCTSRRLTGLLDREVQAGRQALAAARGSWGPGPSSLTVPAIVVDEEDPGLASEGASEGEGEVSPEGPGLLGASQESSMAGRLGEAGGQAAPGQGPSAESIAQEPSQEEKFPGEALTGLPAATPEELALGARRKRFLPKVRAAGDGEATTPEERESPTVSPRGPRKSLVPGSPGTPGRERRSPTQGRKASMLEVPRAEEELAAGDLGPSPKAGGLDTEVALDEGKQETLAKPRKAKDLLKAPQVIRKIRVEQFPDASGSLKLWCQFFNIL SDSVLTWAKDQRPVGEVGRSAGDEGPAALAIVQASPVDCGVYRCTIHNEHGSASTDFCLSPEVLSGFISREEGEVGEEIEMTPMVFAKGLADSGCWGDKLFGRLVSEELRGGGYGCGLRKASQAKVIYGLEPIFESGRTCIIKVSSLLVFGPSSETSLVGRNYDVTIQGCKI QNMSREYCKIFAAEARAAPGFGEVPEIIPLYLIYRPANNIPYATLEEDLGKPLESYCSR EWGCAEAPTASGSSEAMQKCQTFQHWLYQWTNGSFLVTDLAGVDWKMTDVQIAT KLRGYQGLKESCFPALLDRFASSHQCNAYCELLGLTPLKGPEAAHPQAKAKGSKSPSAGRKGSQLSPQPQKKGLPSPQGTRKSAPSSKATPQASEPVTTQLLGQPPTQEEGSKA QGMR

[0117] Nucleic acid sequence of human full length ALPK3 SEQ ID No: 6

[0118] 5118na

[0119] ATGGGGTCGCGGAGGGCCCCCAGCCGGGGCTGGGGCGCGGGTGGGCGGTCGGGGGCGGGGGGCGACGGTGAGGACGACGGCCCCGTGTGGATCCCCAGCCCAGCCAGCCGGAGCTACCTGCTCAGCGTGCGGCCCGAGACCAGCTTATCAAGCAACCGGTTGTCTCACCCCAGCTCTGGAAGGAGCACCTTCTGCTCCATCATTGCTCAGCTCACAGAGGAGACCCAGCCGCTATTTGAGACCACGCTCAAGTCCCGGTCTGTGTCCGAGGACAGCGACGTCAGGTTCACCTGCATCGTCACAGGATACCCAGAGCCAGAGGTGACCTGGTACAAGGATGATACGGAGCTGGACCGCTACTGTGGCTTGC CAAAATATGAGATCACTCATCAGGGCAACCGCCACACACTGCAGCTGTACAGGTGTCGAGAAGAAGATGCCGCCATCTACCAGGCCTCTGCCCAGAACAGCAAGGGCATTGTGTCCTGCTCAGGGGTCCTGGAGGTGGGCACCATGACTGAGTACAAGATCCACCAGCGCTGGTTCGCCAAGTTGAAGCGCAAGGCTGCGGCAAAGCTGCGCGAGATCGAGCAGAGCTGGAAGCACGAGAAGGCGGTGCCTGGGGAGGTCGACACTCTGC GCAAGCTCAGCCCCGACCGCTTCCAGCGAAAGCGGCGATTGAGCGGGGCTCAAGCGCCGGGCCCCTCGGTCCCTACCAGGGAGCCTGAGGGTGGGACCCTGGCGGCTTGGCAGGAGGGAGAGACTGAGACTGCTCAGCACTCAGGTTTGGGCCTGATCAACAGTTTTGCTTCTGGAGAAGTGACCACCAACGGGGAGGCTGCCCCCGAGAATGGAGAGGACGGAGAGCATGGCTTGCTGACATACATCTGTGACGCCATGGAGCTGGGGC CTCAGAGAGCCCTCAAAGAGGAGAGTGGGGCCAAGAAGAAAAAGAAAGATGAGGAATCCAAGCAAGGCCTGCGGAAGCCAGAGTTAGAGAAGGCAGCCCAAAGCCGCCGTTCTTCAGAAAACTGCATCCCCAGCTCAGACGAGCCTGACTCCTGTGGGACTCAGGGGCCCGTGGGCGTGGAGCAGGTTCAGACCCAGCCCAGAGGCAGGGCTGCACGGGGGCCTGGGTCCTCTGGCACAGATAGTACCAGGAAGCCAGCCTCTGCTGTG GGCACTCCAGACAAGGCCCAGAAGGCCCCTGGCCCAGGCCCAGGCCAGGAAGTGTATTTCTCCTTGAAGGACATGTACCTGGAGAACACCCAGGCAGTCAGGCCTCTTGGGGAAGAGGGACCCCAGACCCTGAGTGTCCGGGCGCCTGGGGAGAGTCCCAAGGGGAAGGCACCCCTCAGGGCTAGAAGCGAGGGGGTGCCTGGCGCTCCTGGCCAGCCCACACACTCCTTGACCCCCCAGCCGACTAGGCCTTTCAACAGAAAGAGATTTG CCCCTCCAAAGCCCAAAGGAGAGGCCACCACTGACAGCAAGCCCATTTCTTCTCTGAGTCAAGCTCCAGAATGCGGGGCCCAGAGCTTAGGAAAGGCCCCACCTCAGGCCTCTGTGCAGGTGCCGACGCCCCCTGCCCGGCGGAGACATGGCACCCGGGACAGCACGTTGCAGGGGCAAGCAGGCCACAGGACTCCAGGAGAGGTCCTGGAATGCCAGACAACCACGGCTCCTACCATGTCGGCCAGCAGCAGCTCTGATGTAGCCTCCATT GGGGTTAGCACTTCCGGAAGTCAAGGTATCATTGAACCCATGGATATGGAAACCCAGGAGGATGGGAGAACATCTGCTAACCAGAGAACTGGAAGCAAGAAGAATGTGCAGGCAGATGGGAAGATACAAGTGGATGGAAGGACCAGGGGAGATGGAACACAGACAGCCCAGAGGACACGTGCAGATAGGAAGACGCAGGTGGATGCTGGGACACAAGAAAGCAAGAGGCCACAGTCAGACAGGAGTGCACAGAAGGGCATGATGACACAGGGAAGGGCAGAGACACAGCTAGAAACAACACAGGCAGGTGAGAAGATACAGGAAGACAGGAAGGCCCAGGCAGATAAGGGCACACAGGAAGACAGAAGGATGCAGGGAGAGAAGGGGATGCAGGGAGAGAAGGGGACGCAGTCAGAGGGGAGCGCGCCCACAGCCATGGAAGGTCAGTCTGAGCAAGAGGTGGCAACCAGCCTCGGCC CACCATCCAGAACCCCCAAACTCCCACCTACAGCGGGTCCTAGAGCTCCTCTGAATATTGAATGTTTTGTACAGACCCCAGAAGGGTCTTGTTTCCCAAAAAAACCTGGTTGCCTGCCCAGATCTGAGGAGGCAGTAGTAACAGCCTCCAGGAACCATGAGCAAACTGTGCTGGGTCCCCTGTCAGGGAACCTCATGCTCCCAGCACAGCCGCCCCATGAGGGGAGTGTGGAGCAGGTGGGAGGAGAGAGATGCCGAGGGCCACAGTCATCA GGCCCAGTCGAGGCCAAGCAGGAGGACAGCCCGTTCCAGTGCCCCAAGGAGGAGCGGCCAGGGGGAGTGCCGTGTATGGATCAGGGTGGCTGTCCTCTAGCTGGCCTGAGCCAGGAGGTACCCACGATGCCTTCTCTTCCTGGAACTGGGCTGACAGCTAGCCCAAAGGCGGGGCCGTGTAGCACCCCGACTTCTCAGCACGGGAGCACAGCCACCTTCCTGCCCTCTGAGGATCAGGTCCTGATGAGTTCTGCCCCAACACTGCACCTGG GGCTGGGGACCCCCACTCAGAGTCACCCACCAGAAACCATGGCCACCAGCAGTGAGGGGGCCTGCGCCCAGGTACCAGATGTGGAGGGGCGGACCCCAGGTCCCCGGAGCTGTGACCCTGGCCTCATAGATTCCCTGAAGAACTACCTGCTTCTGCTGCTGAAGCTGTCCAGCACAGAGACAAGTGGAGCAGGGGGAGAGTCCCAGGTGGGGGCAGCCACCGGAGGTCTGGTGCCCTCAGCCACTCTGACACCCACTGTGGAAGTGGCTGG GCTTAGTCCCCGGACATCGAGGCGCATCCTGGAGCGTGTGGAGAACAACCACCTGGTGCAGAGTGCACAGACCCTGCTGCTGAGCCCCTGTACCTCCCGCCGCCTCACCGGCCTCCTGGACCGTGAGGTGCAGGCTGGCCGCCAGGCCCTTGCTGCTGCCCGAGGCTCCTGGGGTCCTGGTCCCAGCTCCCTCACTGTCCCTGCCATTGTGGTAGACGAGGAGGACCCTGGGCTGGCCTCAGAAGGAGCCAGTGAGGGTGAAGGAGAGGTT TCCCCTGAGGGGCCTGGCCTCCTGGGGGCCTCTCAGGAGAGCAGCATGGCTGGTCGACTGGGGGAGGC GGGTGGGC AGGC AGCC CCTGGAC AGGGGCC CTC AGC AGAGAGCATAGCCCAGGAGCCCTCCCAAGAGGAGAAGTTCCCAGGGGAGGCTCTGACAGGTCTCCCGGCAGCTACACCTGAGGAACTGGCTCTAGGGGCCCGGAGGAAGAGATTTCTCCCTAAGGTCAGAGCAGCAGGAGACGGGGAGGCAACCACACCTGAAGA AAGGGAGAGCCCCACGGTTTCCCCCCGGGGGCCCAGGAAAAGCCTGGTGCCTGGGTCCCCAGGGACTCCAGGGCGGGAGAGACGCTCCCCTACGCAGGGCAGAAAGGCGAGCATGCTGGAGGTGCCTCGGGCAGAGGAGGAGCTGGCGGCAGGAGACCTGGGCCCCAGCCCCAAGGCCGGCGGTCTGGACACAGAGGTGGCCCTGGATGAAGGCAAGCAGGAGACACTGGCCAAGCCCAGGAAAGCCAAAGACCTGCTGAAAGCCCCACAGGTGATCCGGAAGATTCGGGTGGAGCAGTTTCCTGATGCCTCCGGTAGCCTGAAGCTGTGGTGCCAGTTTTTCAACATTCTTAGTGACTCAGTCTTGACATGGGCCAAGGATCAGCGCCCAGTGGGCGAGGTGGGCAGGAGCGCAGGGGATGAGGGGCCGGCGGCCTTGGCCATCGTGCAGGCCTCCCCCGTAGACTGCGGTGTGTATCGGTGCAC CATCCACAATGAGCACGGCTCGGCCTCCACCGACTTCTGCCTCAGCCCTGAGGTGTTGTCAGGATTCATCTCCAGAGAAGAAGGTGAAGTTGGAGAAGAGATTGAGATGACCCCTATGGTGTTTGCTAAGGGTCTGGCTGACTCTGGCTGCTGGGGGGACAAGCTCTTTGGGCGACTGGTAAGCGAGGAGCTCCGAGGGGGTGGATATGGGTGTGGCCTTCGGAAGGCCTCCCAGGCCAAGGTCATCTACGGGCTGGAACCCATCTTCGAGTC GGGCCGCACGTGCATCATCAAGGTGTCCAGCCTGCTTGTGTTTGGGCCCAGCAGTGAGACTTCTCTTGTGGGCAGAAACTACGACGTCACCATCCAGGGGTGCAAGATCCAGAACATGAGTCGGGAGTACTGCAAAATCTTCGCAGCAGAAGCCCGGGCCGCGCCTGGCTTTGGGGAGGTGCCTGAGATCATCCCACTGTATCTGATCTACCGGCCTGCAAACAATATCCCATATGCTACCCTGGAGGAAGACCTGGGCAAGCCCCTGGAGT CTTACTGTTCTCGGGAATGGGGCTGTGCTGAGGCTCCGAC AGC ATCTGGC AGCTCTGAGGCCATGCAGAAATGCCAGACCTTCCAACACTGGCTGTATCAGTGGACAAATGGCAGCTTCCTTGTCACAGACTTGGCAGGGGTTGACTGGAAGATGACTGATGTGCAGATTGCTACCAAACTCCGAGGATACCAGGGCCTCAAGGAAAGCTGCTTCCCTGCCCTGCTGGACCGGTTCGCCTCCTCCCACCAGTGCAATGCCTACTGTGAGCTGC TGGGGCTGACACCTCTCAAGGGCCCGGAGGCGGCCCACCCCCAAGCCAAAGCCAAAGGCTCTAAGAGTCCATCTGCTGGCAGGAAAGGCTCCCAGCTGAGTCCTCAGCCCCAGAAGAAAGGCCTCCCTAGTCCTCAGGGCACCCGGAAGAGTGCTCCAAGTTCCAAGGCCACCCCTCAGGCCTCAGAGCCAGTCACCACTCAGTTGTTGGGACAGCCTCCCACCCAAGAGGAGGGCTCCAAGGCCCAGGGCATGCGGTAG

[0120] Amino acid sequence of human miniALPK3 SEQ ID No: 7

[0121] 1308aa

[0122] MGSRRAPSRGWGAGGRSGAGGDGEDDGPVWIPSPASRSYLLSVRPETSLSSNRLSHPSSGRSTFCSIIAQLTEETQPLFETTLKSRSVSEDSDVRFTCIVTGYPEPEVTWYKDDTELDRYCGLPKYEITHQGNRHTLQLYRCREEDAAIYQASAQNSKGIVSCS GVLEVGTMTEYKIHQRWFAKLKRKAAAKLREIEQSWKHEKAVPGEVDTLRKLSPDRFQRKRRLSGAQAPGPSVPTREPEGGTLAAWQEGETETAQHSGLGLINSFASGEVTTNGEAAPENGEDGEHGLLTYICDAMELGPQRALKEESGAKKKKKDEESKQGLRKPELEKAAQSRRSSENCIPSSDEPDSCGTQGPVGVEQVQTQPRGRAARGPGSSGTDSTRKPASAVGTPDKAQKAPGPGPGQEVYFSLKDMYLENTQAVRPLGEEGPQTLSVRAPGES PKGKAPLRARSEGVPGAPGQPTHSLTPQPTRPFNRKRFAPPKPKGEATTDSKPISSLSQ APECGAQSLGKAPPQASVQVPTPPARRRHGTRDSTLQGQAGHRTPGEVLECQTTTAP TMSASSSSDVASIGVSTSGSQGIIEPMDMETQEDGRTSANQRTGSKKNVQADGKIQVDGRTRGDGTQTAQRTRADRKTQVDAGTQESREVQAGRQALAAARGSWGPGPSSLT VPAIVVDEEDPGLASEGASEGEGEVSPEGPGLLGASQESSMAGRLGEAGGQAAPGQ GPSAESIAQEPSQEEKFPGEALTGLPAATPEELALGARRKRFLPKVRAAGDGEATTPE ERESPTVSPRGPRKSLVPGSPGTPGRERRSPTQGRKASMLEVPRAEEELAAGDLGPSP KAGGLDTEVALDEGKQETLAKPRKAKDLLKAPQVIRKIRVEQFPDASGSLKLWCQFFNILSDSVLTWAKDQRPVGEVGRSAGDEGPAALAIVQASPVDCGVYRCTIHNEHGSASTDFCLSPEVLSGFISREEGEVGEEIEMTPMVFAKGLADSGCWGDKLFGRLVSEELRGGGYGCGLRKASQAKVIYGLEPIFESGRTCIIKVSSLLVFGPSSETSLVGRNYDVTIQGCKIQNMSREYCKIFAAEARAAPGFGEVPEIIPLYLIYRPANNIPYATLEEDLGKPLESYC SREWGCAEAPTASGSSEAMQKCQTFQHWLYQWTNGSFLVTDLAGVDWKMTDVQIATKLRGYQGLKESCFPALLDRFASSHQCNAYCELLGLTPLKGPEAAHPQAKAKGSK SPSAGRKGSQLSPQPQKKGLPSPQGTRKSAPSSKATPQASEPVTTQLLGQPPTQEEGS KAQGMR

[0123] Nucleic acid sequence of human mini-ALPK3 SEQ ID No: 8

[0124] 3927na

[0125] ATGGGGTCGCGGAGGGCCCCCAGCCGGGGCTGGGGCGCGGGTGGGCGGTCGGGGGCGGGGGGCGACGGTGAGGACGACGGCCCCGTGTGGATCCCCAGCCCAGCCAGCCGGAGCTACCTGCTCAGCGTGCGGCCCGAGACCAGCTTATCAAGCAACCGGTTGTCTCACCCCAGCTCTGGAAGGAGCACCTTCTGCTCCATCATTGCTC AGCTCACAGAGGAGACCCAGCCGCTATTTGAGACCACGCTCAAGTCCCGGTCTGTGTCCGAGGACAGCGACGTCAGGTTCACCTGCATCGTCACAGGATACCCAGAGCCAGAGGTGACCTGGTACAAGGATGATACGGAGCTGGACCGCTACTGTGGCTTGCCAAAATATGAGATCACTCATCAGGGCAACCGCCACACACTGCAGCTGTACAGGTGTCGAGAAGAAGATGCCGCCATCTACCAGGCCTCTGCCCAGAACAGCAAGGGCA TTGTGTCCTGCTCAGGGGTCCTGGAGGTGGGCACCATGACTGAGTACAAGATCCACCAGCGCTGGTTCGCCAAGTTGAAGCGCAAGGCTGCGGCAAAGCTGCGCGAGATCGAGCAGAGCTGGAAGCACGAGAAGGCGGTGCCTGGGGAGGTCGACACTCTGCGCAAGCTCAGCCCCGACCGCTTCCAGCGAAAGCGGCGATTGAGCGGGGCTCAAGCGCCGGGCCCCTCGGTCCCTACCAGGGAGCCTGAGGGTGGGACCCTGGCGGCTTGGCAGGAGGGAGAGACTGAGACTGCTCAGCACTCAGGTTTGGGCCTGATCAACAGTTTTGCTTCTGGAGAAGTGACCACCAACGGGGAGGCTGCCCCCGAGAATGGAG AGGACGGAGAGCATGGCTTGCTGACATACATCTGTGACGCCATGGAGCTGGGGCCTCAGAGAGCCCTCAAAGAGGAGAGTGGGGCCAAGAAGAAAAAGAAAGATGAGGAATCCAAGCAAGGCCTGCGGAAGCCAGAGTTAGAGAAGGCAGCCCAAAGCCGCCGTTCTTCAGAAAACTGCATCCCCAGCTCAGACGAGCCTGACTCCTGTGGGACTCAGGGGCCCGTGGGCGTGGAGCAGGTTCAGACCCAGCCCAGAGGCAGGGCTGCA CGGGGGCCTGGGTCCTCTGGCACAGATAGTACCAGGAAGCCAGCCTCTGCTGTGGGCACTCCAGACAAGGCCCAGAAGGCCCCTGGCCCAGGCCCAGGCCAGGAAGTGTATTTCTCCTTGAAGGACATGTACCTGGAGAACACCCAGGCAGTCAGGCCTCTTGGGGAAGAGGGACCCCAGACCCTGAGTGTCCGGGCGCCTGGGGAGAGTCCCAAGGGGAAGGCACCCCTCAGGGCTAGAAGCGAGGGGGTGCCTGGCGCTCCTGGCCAG CCCACACACTCCTTGACCCCCCAGCCGACTAGGCCTTTCAACAGAAAGAGATTTGCCCCTCCAAAGCCCAAAGGAGAGGCCACCACTGACAGCAAGCCCATTTCTTCTCTGAGTCAAGCTCCAGAATGCGGGGCCCAGAGCTTAGGAAAGGCCCCACCTCAGGCCTCTGTGCAGGTGCCGACGCCCCCTGCCCGGCGGAGACATGGCACCCGGGACAGCACGTTGCAGGGGCAAGCAGGCCACAGGACTCCAGGAGAGGTCCTGGAATGCCA GACAACCACGGCTCCTACCATGTCGGCCAGCAGCAGCTCTGATGTAGCCTCCATTGGGGTTAGCACTTCCGGAAGTCAAGGTATCATTGAACCCATGGATATGGAAACCCAGGAGGATGGGAGAACATCTGCTAACCAGAGAACTGGAAGCAAGAAGAATGTGCAGGCAGATGGGAAGATACAAGTGGATGGAAGGACCAGGGGAGATGGAACACAGACAGCCCAGAGGACACGTGCAGATAGGAAGACGCAGGTGGATGCTGGGACA CAAGAAAGCCGTGAGGTGCAGGCTGGCCGCCAGGCCCTTGCTGCTGCCCGAGGCTCCTGGGGTCCTGGTCCCAGCTCCCTCACTGTCCCTGCCATTGTGGTAGACGAGGAGGACCCTGGGCTGGCCTCAGAAGGAGCCAGTGAGGGTGAAGGAGAGGTTTCCCCTGAGGGGCCTGGCCTCCTGGGGGCCTCTCAGGAGAGCAGCATGGCTGGTCGACTGGGGGAGGCGGGTGGGCAGGCAGCCCCTGGACAGGGGCCCTCAGCAGAGAGC ATAGCCCAGGAGCCCTCCCAAGAGGAGAAGTTCCCAGGGGAGGCTCTGACAGGTCTCCCGGCAGCTACACCTGAGGAACTGGCTCTAGGGGCCCGGAGGAAGAGATTTCTCCCTAAGGTCAGAGCAGCAGGAGACGGGGAGGCAACCACACCTGAAGAAAGGGAGAGCCCCACGGTTTCCCCCCGGGGGCCCAGGAAAAGCCTGGTGCCTGGGTCCCCAGGGACTCCAGGGCGGGAGAGACGCTCCCCTACGCAGGGCAGAAAGGCGAGCATGCTGGAGGTGCCTCGGGCAGAGGAGGAGCTGGCGGCAGGAGACCTGGGCCCCAGCCCCAAGGCCGGCGGTCTGGACACAGAGGTGGCCCTGGATGAAGGCAAGCAGGAGACACTGGCCAAGCCCAGGAAAGCCAAAGACCTGCTGAAAGCCCCACAGGTGATCCGGAAGATTCGGGTGGAGCAGTTTCCTGATGCCTCCGGTAGCCTGAA GCTGTGGTGCCAGTTTTTCAACATTCTTAGTGACTCAGTCTTGACATGGGCCAAGGATCAGCGCCCAGTGGGCGAGGTGGGCAGGAGCGCAGGGGATGAGGGGCCGGCGGCCTTGGCCATCGTGCAGGCCTCCCCCGTAGACTGCGGTGTGTATCGGTGCACCATCCACAATGAGCACGGCTCGGCCTCCACCGACTTCTGCCTCAGCCCTGAGGTGTTGTCAGGATTCATCTCCAGAGAAGAAGGTGAAGTTGGAGAAGAGATTGAGATGA CCCCTATGGTGTTTGCTAAGGGTCTGGCTGACTCTGGCTGCTGGGGGGACAAGCTCTTTGGGCGACTGGTAAGCGAGGAGCTCCGAGGGGGTGGATATGGGTGTGGCCTTCGGAAGGCCTCCCAGGCCAAGGTCATCTACGGGCTGGAACCCATCTTCGAGTCGGGCCGCACGTGCATCATCAAGGTGTCCAGCCTGCTTGTGTTTGGGCCCAGCAGTGAGACTTCTCTTGTGGGCAGAAACTACGACGTCACCATCCAGGGGTGCAAGATC CAGAACATGAGTCGGGAGTACTGC AAAATCTTCGC AGCAGAAGCCCGGGCCGCGCCTGGCTTTGGGGAGGTGCCTGAGATCATCCCACTGTATCTGATCTACCGGCCTGCAAACAATATCCCATATGCTACCCTGGAGGAAGACCTGGGCAAGCCCCTGGAGTCTTACTGTTCTCGGGAATGGGGCTGTGCTGAGGCTCCGACAGCATCTGGCAGCTCTGAGGCCATGCAGAAATGCCAGACCTTCCAACACTGGCTGTATCAGTGGACAAA TGGCAGCTTCCTTGTCACAGACTTGGCAGGGGTTGACTGGAAGATGACTGATGTGCAGATTGCTACCAAACTCCGAGGATACCAGGGCCTCAAGGAAAGCTGCTTCCCTGCCCTGCTGGACCGGTTCGCCTCCTCCCACCAGTGCAATGCCTACTGTGAGCTGCTGGGGCTGACACCTCTCAAGGGCCCGGAGGCGGCCCACCCCCAAGCCAAAGCCAAAGGCTCTAAGAGTCCATCTGCTGGCAGGAAAGGCTCCCAGCTGAGTCCTCAGC CCCAGAAGAAAGGCCTCCCTAGTCCTCAGGGCACCCGGAAGAGTGCTCCAAGTTCCAAGGCCACCCCTCAGGCCTCAGAGCCAGTCACCACTCAGTTGTTGGGACAGCCTCCCACCCAAGAGGAGGGCTCCAAGGCCCAGGGCATGCGGTAG

Claims

What is claimed is:

1. A method of treating cardiomyopathy, comprising administering to a subject in need thereof an effective amount of a composition comprising a nucleic acid encoding all or a portion of an ALPK3 protein.

2. The method of claim 1, wherein the nucleic acid is a mini ALPK3 gene comprising a sequence that encodes at least 700, 800. 900, 1000. 1100, 1200, 1300 or 1400 amino acids of a the ALKP3 protein.

3. The method of claim 1, wherein the nucleic acid is a mini ALPK3 gene comprising a sequence that encodes about 1308 amino acids of the ALPK3 protein.

4. The method of claim 1, wherein the nucleic acid contains fewer nucleotides than a naturally occurring ALPK3 gene.

5. The method of claim 1, wherein the nucleic acid is a mini ALPK3 gene having at least a deletion of a portion of exon 6 sequence of the naturally occurring ALKP3 gene.

6. The method of claim 5, wherein the composition further comprises a vector operationally linked to the portion of the mini ALPK3 gene.

7. The method of claim 6, wherein the vector is a muscle adeno-associated virus 2A (MyoAAV 2A).

8. The method of claim 1, wherein the nucleic acid is cDNA or RNA.

9. The method of claim 1, wherein the cardiomyopathy is ALPK3 cardiomyopathy.

10. The method of claim 9, wherein the cardiomyopathy exhibits left ventricular hypertrophy.

11. The method of claim 1, wherein the cardiomyopathy is dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM).

12. The method of claim 1, wherein the subject is a human.

13. A pharmaceutical composition comprising a nucleic acid encoding all or a portion of an ALPK3 protein.

14. The pharmaceutical composition of claim 13, wherein the nucleic acid is a mini ALPK3 gene comprising a sequence that encodes at least 700, 800. 900, 1000. 1100, 1200, 1300 or 1400 amino acids of the ALKP3 protein.

15. The pharmaceutical composition of claim 13, wherein the nucleic acid encodes a mini ALPK3 gene comprising a sequence that encodes about 1308 amino acids of the ALKP3 protein.

16. The pharmaceutical composition of claim 13, wherein the nucleic acid contains fewer nucleotides than a naturally occurring ALPK3 gene.

17. The pharmaceutical composition of claim 13, wherein the nucleic acid encodes a mini ALPK3 gene having at least a deletion of a portion of exon 6 sequence of the naturally occurring ALKP3 gene.

18. The pharmaceutical composition of claim 17, wherein the composition further comprises a vector operationally linked to the portion of ALPK3 gene.

19. The pharmaceutical composition of claim 18. wherein the vector is a muscle adeno- associated virus 2A (MyoAAV 2A).

20. The pharmaceutical composition of claim 13, wherein the nucleic acid is cDNA or RNA.

21. A composition comprising a nucleic acid encoding all or a portion of an ALPK3 protein linked to a capture tag.

22. The composition of claim 21, wherein the tag is a 3xFLAG tag.

23. The composition of claim 22, wherein the tag is incorporated at the C-terminus of the ALPK3 protein.

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