Treatment of alpha-1 antitrypsin deficiency

Abstract

A therapeutic genetic construct for use in the treatment of alpha-1 antitrypsin deficiency (AATD) and related diseases. The genetic construct encodes: (a) a first precursor miRNA; (b) a second precursor miRNA; and (c) a transgene encoding AAT (M). The present invention relates to a precursor miRNA that inhibits the expression of alleles encoding an AAT mutant variant, such as AAT (Z), AAT (S), AAT (I), AAT (Siiyama), and AAT (Malton). The construct can further comprise a liver specific promoter. A vector, such as a chimpanzee adenovirus vector, comprises the genetic construct. A method of treating a disease or disorder, such as chronic obstructive pulmonary disease (COPD), bronchiectasia, cholestasis in neonates, cirrhosis, fibrosis, and hepatocellular carcinoma, comprising administering a therapeutically effective amount of the vector described above. The use of said genetic constructs in the preparation of a medicament for the treatment of a disease or disorder. A composition comprising the genetic construct is also disclosed. The invention also provides a kit comprising the genetic construct.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 512,773, filed July 10, 2023. Background Technology

[0003] Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder that affects a large number of individuals, particularly those of European descent. It is estimated that approximately 1 in 1,500 to 3,500 people in this population are affected, although the disease can also occur less frequently in other ethnic groups.

[0004] This genetic disorder originates from a mutation in the SERPINA1 gene, which encodes the α-1 antitrypsin (AAT) protein, synthesized in the liver. One type of mutation involves a single nucleotide change, resulting in the substitution of lysine for glutamic acid at position 342 of the AAT protein sequence, producing the AAT(Z) variant. Another mutation produces the AAT(S) variant, which involves the substitution of valine for methionine at position 264 of the AAT protein. The AAT(I) variant is produced when methionine at position 50 of the AAT protein is replaced by isoleucine. Another variant, AAT(S... iiyama AAT is produced when the glutamic acid at position 288 of the AAT protein is replaced by a lysine. There is also another variant, AAT(M). malton The mutation, described above, is characterized by the absence of a four-amino acid segment in the AAT protein. In each of these cases, the mutation results in the formation of a misfolded form of AAT compared to normal AAT (encoded by the AAT(M) variant, an example sequence of which can be seen in SEQ ID NO:600), thereby reducing its efficiency of secretion from the liver. This leads to the accumulation of mutant AAT in the liver and a decrease in the amount of normal, functional AAT in the bloodstream.

[0005] AAT acts as a protease inhibitor and plays a crucial role in protecting lung tissue from damage caused by enzymes released by immune cells, particularly neutrophil elastase. When functional AAT levels are insufficient, patients with acute lung disease (AATD) have a significantly increased risk of developing early-onset emphysema, chronic obstructive pulmonary disease (COPD), and other respiratory illnesses. Approximately 75% of AATD patients develop emphysema, and about 10% of children and adults with AATD develop cirrhosis and require liver transplantation. Symptoms of AATD include chronic cough, shortness of breath, and wheezing. Simultaneously, the abnormal accumulation of mutated AAT in the liver can lead to liver diseases, including inflammation, fibrosis, and, in severe cases, cirrhosis and hepatocellular carcinoma.

[0006] Currently, the FDA-approved treatment for AAT (Alephrine Anti-Acid Tract Disease) involves regular infusions of plasma-derived AAT protein throughout the patient's life to maintain adequate levels of this protein in the blood. This therapy aims to restore the protective function of AAT in the lungs and reduce the progression of respiratory symptoms. However, there are currently no approved therapies that address the associated liver disease that may occur in patients with AAT. Therefore, there is an urgent need for effective therapies that can simultaneously address the pulmonary and hepatic manifestations of AAT.

[0007] Gene therapy using microRNAs provides an alternative strategy for AATD treatment by silencing the expression of the mutant AAT allele while providing transgenes that allow the expression of normal, functional AAT.

[0008] MicroRNAs (miRNAs) are small non-coding RNA molecules that can bind to mRNA molecules produced by specific genes, thereby affecting their protein translation process or exerting their effects by destabilizing mRNA transcripts. Through this mechanism, miRNAs can effectively silence the expression of target genes.

[0009] Previous methods for enhancing gene silencing have involved encoding multiple miRNAs in a single polycistronic genetic construct and delivering them into cells via vectors, as illustrated by Mueller et al. (Mueller et al., Molecular Therapy, 20:590–600 (2012), “Mueller”) Figure 1. This approach has shown significant reductions in target gene expression. However, when using constructs containing repetitive precursor miRNA (precursor miRNA) structures, the stem-loop structure of the precursor miRNA may undergo substitution folding during transcription. This substitution folding may produce alternatively processed miRNAs, unintentionally leading to off-target gene silencing and posing potential safety concerns. Furthermore, using constructs with repetitive precursor miRNA structures may allow for intravector recombination, resulting in a non-pure vector population with sequence variations.

[0010] To address these risks, the present invention relates to a polycistronic construct that encodes multiple distinct precursor miRNAs to reduce the expression of alleles expressing mutant AAT, while ensuring that the precursor miRNAs are not complementary to each other. In some embodiments, a minimum spacing of approximately 7 nucleotides is maintained between the precursor miRNA structures to facilitate proper folding of RNA during co-transcription. Furthermore, in certain embodiments, the precursor miRNAs are engineered to retain predicted stem-loop structures and inner loops based on endogenous human sequence preservation, thereby minimizing the risk of RNAi-based toxicity. These constructs may contain precursor miRNAs targeting different genes or different regions of the same gene. Summary of the Invention

[0011] This invention relates to a therapeutic genetic construct encoding dual precursor miRNAs and normal, functional AAT, wherein the dual miRNAs are used to reduce the expression of mutant AAT variants (e.g., AAT(Z)) while introducing an AAT(M) transgene encoding normal, functional AAT.

[0012] In some embodiments, the genetic construct of the present invention comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO:592 (e.g., having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:592), a codon degenerate variant of SEQ ID NO:592, or a nucleic acid capable of hybridizing with a complementary sequence of SEQ ID NO:592 under strict hybridization conditions.

[0013] In some embodiments, the dual miRNA of the present invention comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 589 (e.g., having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 589), or a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 589 under strict hybridization conditions.

[0014] In some embodiments, the transgene expressing the AAT(M) variant is modified to evade the reduction of miRNA expression. In one embodiment, the miRNA-tolerant AAT(M) transgene comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 590 (e.g., having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 590), a codon degenerate variant of SEQ ID NO: 590, or a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 590 under strict hybridization conditions.

[0015] In some embodiments, the miRNA-tolerant AAT(M) transgene is modified to have a reduced level of CG dinucleotide (also known as CpG). In one embodiment, the CpG-reduced miRNA-tolerant transgene comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 596 or a functional variant thereof (e.g., having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 596), a codon degenerate variant of SEQ ID NO: 596, or a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 596 under strict hybridization conditions.

[0016] In some embodiments, the AAT(M) transgene encodes an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 600 (e.g., having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 600), or a conserved substitution variant of SEQ ID NO: 600.

[0017] In some embodiments, the genetic construct comprises a tissue-specific promoter. In one embodiment, the tissue-specific promoter is specific to the lung. In another embodiment, the tissue-specific promoter is specific to the liver. In a further embodiment, the liver-specific promoter comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 587 or a functional variant thereof (e.g., having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 587), or a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 587 under strict hybridization conditions.

[0018] In another embodiment, the tissue-specific promoter is modified to have a reduced level of CpG. In one embodiment, the CpG-reduced tissue-specific promoter is liver-specific. In a further embodiment, the CpG-reduced liver-specific promoter comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 594 or a functional variant thereof (e.g., having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 594), or a nucleic acid capable of hybridizing with a complementary sequence of SEQ ID NO: 594 under strict hybridization conditions.

[0019] In some embodiments, a vector is used to introduce the genetic construct of the present invention into cells. In some embodiments, the vector is an adenovirus or an adenoviral vector. In a preferred embodiment, the vector is an adenoviral vector derived from gorillas, referred to as a gorilla adenovirus vector, such as GC44, GC45, or GC46. In one embodiment, the vector comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 593 or a functional variant thereof (e.g., having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 593), a codon degenerate variant of SEQ ID NO: 593, or a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 593 under strict hybridization conditions.

[0020] This invention also relates in part to a pharmaceutical composition comprising the carriers described herein and pharmaceutically acceptable carriers.

[0021] The present invention also relates in part to a method of treating a disease or condition in a subject with this need, comprising administering to the subject the genetic constructs and / or vectors described herein, or compositions comprising them. In one embodiment, the disease or condition is a liver or lung disease associated with α-1 antitrypsin deficiency.

[0022] This invention also relates in part to the use of the genetic constructs and / or vectors described herein, or compositions comprising them, in the preparation of medicaments for treating diseases or conditions of persons in need. In a preferred embodiment, the genetic constructs described herein, or any vector comprising any genetic construct of this invention, or compositions comprising the nucleic acid or vector, are used to treat liver or lung diseases associated with α-1 antitrypsin deficiency.

[0023] The features of this disclosure are specifically set forth in the appended claims. The features and advantages of this disclosure can be better understood by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments utilizing the principles of this disclosure. Attached Figure Description

[0024] Figure 1A This demonstrates the intravenous injection of the GC44 vector (dose 1 × 10⁻⁶) encoding firefly luciferase and expressed under the control of the CMV promoter. 10 On day 2 after PU, the expression location of firefly luciferase transgene in BALB / c mice.

[0025] Figure 1B This demonstrates the intravenous injection of the GC44 vector (dose 1 × 10⁻⁶) encoding firefly luciferase and expressed under liver-specific promoter (LSP) control. 10 On day 2 after PU, the expression location of firefly luciferase transgene in BALB / c mice.

[0026] Figure 1C This illustrates a vector encoding firefly luciferase, expressed under controlled conditions by a CMV promoter or a liver-specific promoter (LSP), administered via intravenous injection (dose: 1 × 10⁻⁶). 11 On day 1 after PU, the expression location of firefly luciferase transgene in NSG-PiZ mice.

[0027] Figure 2A This demonstrates the effect of administering a GC44 vector (dose 1 × 10⁻⁶) encoding firefly luciferase and controlling its expression by a CMV promoter or a liver-specific promoter (LSP). 9 1×10 10 Or 1×10 11 Total flux (p / s) in mice after PU.

[0028] Figure 2B This illustrates the administration of a vector encoding firefly luciferase and controlled by either a CMV promoter or a liver-specific promoter (LSP) at a dose of 1 × 10⁻⁶. 9 1×10 10 Or 1×10 11 Total flux (p / s) in mice after PU.

[0029] Figure 3A The presence of AAT(Z) in the liver of untreated NSG-PiZ mice, as detected by PAS-D staining, is shown.

[0030] Figure 3B The presence of AAT(Z) in the livers of untreated NSG-PiZ mice was shown 28 days after administration of FFB control buffer.

[0031] Figure 4A The presence of AAT(Z) in the liver of NSG-PiZ mice was shown after administration of the GC44 vector encoding a miRNA targeting AAT(Z) and a transgene of AAT(M).

[0032] Figure 4B The following is an illustration of the administration of the GC44 vector (dose 1 × 10⁻⁶) to a dual miRNA encoding AAT(Z) and an AAT(M) transgene. 11 The presence of AAT(Z) in the liver of untreated NSG-PiZ mice 28 days after PU.

[0033] Figure 5 The adenovirus copy number in each cell in different mouse organs is shown. (Groups and...) Figure 16A The groups in -16D are the same.

[0034] Figure 6 The table shown illustrates the fold change in AAT transgenic RNA expression in other tissues compared to the Group 1 reference sample. (Groups and...) Figure 16A The groups in -16D are the same.

[0035] Figure 7A The levels of human AAT(M)(hAAT(M)) in C57BL / 6 mice were shown after treatment with the GC44 vector encoding a dual miRNA targeting AAT(Z) and an AAT(M) transgene.

[0036] Figure 7B The levels of human AAT(M)(hAAT(M)) in NSG mice were shown after treatment with the GC44 vector encoding a dual miRNA targeting AAT(Z) and an AAT(M) transgene.

[0037] Figure 8AThe levels of human AAT(M) secreted in HepG2 hepatocyte cell culture media transfected with the following plasmid DNAs are shown: (A) Plasmid DNA encoding a liver-specific promoter, 5'UTR, dual miRNA targeting AAT(Z), AAT(M) transgene, and 3'UTR (labeled 5924); (B) Plasmid DNA encoding a liver-specific promoter, 5'UTR, dual miRNA targeting AAT(Z), CpG-decreased form of AAT(M) transgene, and 3'UTR (labeled 6568); (C) Plasmid DNA encoding a CpG-decreased form of a liver-specific promoter, 5'UTR, dual miRNA targeting AAT(Z), CpG-decreased form of AAT(M) transgene, and 3'UTR (labeled 6569); (D) (I) Plasmid DNA encoding a liver-specific promoter, a CpG-reduced 5'UTR, a dual miRNA targeting AAT(Z), a CpG-reduced form of AAT(M) transgene, and a 3'UTR (labeled 6570); and (E) Plasmid DNA encoding a CpG-reduced form of a liver-specific promoter, a CpG-reduced 5'UTR, a dual miRNA targeting AAT(Z), a CpG-reduced form of AAT(M) transgene, and a 3'UTR (labeled 6571).

[0038] Figure 8B The relative body weights of mice given the RA-1276 or RA-1330 vector constructs relative to the control (Final Formulation Buffer (FFB)) are shown.

[0039] Figure 9 The concentration (µM) of human AAT (hAAT) in mouse serum previously administered with the RA-1276 or RA-1330 vector construct is shown as a comparison with the control (FFB).

[0040] Figure 10 The relative percentage of body weight of mice administered different formulations is shown. Body weight is calculated relative to the starting point of the experiment. (Groups and...) Figure 12A-12D The groups in the text are the same.

[0041] Figure 11 The following is an illustration of the administration of the GC44 vector (dose 1 × 10⁻⁶) to a dual miRNA encoding AAT(Z) and an AAT(M) transgene. 11 PU, 5×10 10 PU, 1×10 10 PU, 5×10 9 PU and 1×10 9 Images of hepatocytes obtained by PAS-D staining in NSG-PiZ mice 28 days after PU, as well as control NSG-PiZ mice without the vector and control NSG mice (not expressing AAT(Z)).

[0042] Figure 12A-12D The efficacy of GC44 and GC45 vectors in mice was demonstrated. Group 1 consisted of NSG mice administered the control formulation (FFB). Group 2 consisted of NSG-PiZ mice administered the control formulation (FFB). Group 3 consisted of NSG-PiZ mice administered the RA-1275 vector (RA-1275 is a GC44 vector with a liver-specific promoter driving dual miRNA construct expression and AAT(M) expression). Group 4 consisted of NSG-PiZ mice administered the RA-1330 vector (RA-1330 is a GC44 vector with CpG reduction modification and a liver-specific promoter driving dual miRNA construct expression and AAT(M) expression). Group 5 consisted of NSG-PiZ mice administered the RA-1331 vector (RA-1331 is a GC45 vector with CpG reduction modification and a liver-specific promoter driving dual miRNA construct expression and AAT(M) expression). Figure 12A Representative images of liver tissue stained with PAS-D are shown to illustrate the hepatic spheroids. Figure 12B The relative expression of the AAT(Z) allele transcript of SERPINA1 in NSG-PiZ mice on day 28 post-drug administration is shown. Figure 12C The copy number of the AAT(M) allele in mouse liver is shown. Figure 12D The number of adenovirus copies per cell in the mouse liver is shown.

[0043] Figure 13 This is a schematic diagram of a study that evaluated the expression behavior of vectors containing a dual design of miRNAs targeting AAT(Z), a liver-specific promoter, a miRNA-tolerant AAT(M) transgene, and 5'UTR and 3'UTR (groups 4-6, NSG-PiZ mice).

[0044] Figure 14A Representative images of PAS-D staining in NSG-PiZ mouse liver tissue 28 days after administration of each vector design. Figure 14B For the 28th day Figure 14A The chart shows the percentage of PAS-D staining in liver tissue.

[0045] Figure 15This paper presents liver-specific assays of adenovirus vector DNA in NSG-PiZ mice after administration of a dual-design miRNA targeting AAT(Z), a liver-specific promoter, a miRNA-tolerant AAT(M) transgene, and vectors containing 5'UTR and 3'UTR (groups 4–6), relative to the lowest-tested sample and to results from other tissues. Assays were performed on days 14 and 28 post-sampling. Pancreatic expression values ​​in group 3 at day 14 were crossed out because they appeared artificially elevated. DNA assay results were below the limit of quantitation.

[0046] Figure 16A Dot plots are shown to illustrate liver-specific AAT(M) transgene expression on day 28 after treatment with a vector containing a dual design of miRNAs targeting AAT(Z), a liver-specific promoter, a miRNA-tolerant AAT(M) transgene, and 5'UTR and 3'UTR (groups 4–6, NSG-PiZ mice). Figure 16B The table shown illustrates the fold change in AAT transgenic RNA expression relative to the Group 1 reference sample in different tissues.

[0047] Figure 17A and Figure 17B The following diagram shows the miRNA expression in the livers of NSG-PiZ mice after administration of a vector containing a dual design of miRNA targeting AAT(Z), a liver-specific promoter, a miRNA-tolerant AAT(M) transgene, and 5'UTR and 3'UTR (groups 4-6): Expression on day 28 (dot plot) Figure 17A ) and the average expression over the entire 28-day period (Table - Figure 17B (), compared to miRNA expression in other tissues. Detailed Implementation

[0048] The following description and examples illustrate embodiments of this disclosure in detail. It should be understood that this disclosure is not limited to the specific embodiments described herein, and therefore variations are possible. Those skilled in the art will recognize that various changes and modifications are possible with this disclosure, all of which are included within the scope of this invention.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0050] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0051] While the various features of this disclosure may be described in the context of a single implementation, these features may also be provided individually or in any suitable combination. Conversely, although for clarity this disclosure may be described in the context of various implementations, this disclosure may also be implemented in a single implementation.

[0052] I. Definition

[0053] The following definitions supplement those in the art and are specific to this application, and should not be attributed to any related or unrelated cases, such as any shared patents or applications. The terminology used herein is for describing particular embodiments only and is not intended to be limiting.

[0054] In this application, the use of the singular form includes the plural form unless otherwise specified. As used in the specification, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise.

[0055] In this application, unless otherwise stated, “or” means “and / or”. The terms “and / or” and “any combination thereof” and their grammatical equivalents are used interchangeably. These terms may indicate that any combination is explicitly contemplated. For illustrative purposes only, the phrases “A, B and / or C” or “A, B, C or any combination thereof” may mean “A alone; B alone; C alone; A and B; B and C; A and C; and A, B and C.” The term “or” may be used in combination or separately unless the context clearly indicates separate use.

[0056] Furthermore, the use of the term "including" and other forms such as "contains," "comprises," and "includes" is not restrictive.

[0057] The use of terms such as "some embodiments," "one embodiment," "an embodiment," or "other embodiments" in the specification means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments, but not necessarily in all embodiments of this disclosure.

[0058] The terms “comprising” (and any form of inclusion, such as “comprising” and “containing”), “having” (and any form of having, such as “containing” and “possessing”), “including” (and any form of inclusion, such as “comprising” and “containing”), or “containing” (and any form of containing, such as “including” and “containing”) as used in this specification and claims are closed or open-ended and do not exclude other unreferenced elements or method steps. Any embodiment described in this specification may be implemented in combination with any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure may be used to implement the methods of this disclosure.

[0059] The term "about" or "approximately" indicates an acceptable range of error for a particular value, which is determined by those skilled in the art and depends in part on the method of measurement or determination of the value, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may refer to one or more standard deviations. Alternatively, "about" may refer to a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. As another example, the quantity of "about 10" includes 10 and any quantity between 9 and 11. As another example, the term "about" in relation to a reference value may also include a range of values ​​added to or subtracted from that value by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. Alternatively, particularly in the context of biological systems or processes, the term "about" may mean within an order of magnitude, preferably within five times the value, more preferably within two times. Where a particular value is described in this application and claims, unless otherwise stated, the term "about" should be assumed to indicate that the particular value is within an acceptable range of error.

[0060] "Therapeutic effective amount" or "therapeutic effective dose" refers to the amount or dose that is effective within the time required to achieve the desired therapeutic effect. This amount can vary depending on factors such as disease state, individual age, sex, weight, and the ability of the nucleic acid sequence of this invention to elicit the desired response in an individual.

[0061] "Polynucleotide" or "oligonucleotide" refers to a polymeric form of nucleotides or nucleic acids of any length, which can be ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded deoxyribonucleic acid (DNA), triple-stranded DNA, and double-stranded and single-stranded ribonucleic acid (RNA). It also includes modified forms, such as those modified by methylation and / or capping, as well as unmodified forms of polynucleotides. The term is also intended to include molecules that include non-naturally occurring or synthetic nucleotides and nucleotide analogues.

[0062] Unless otherwise stated, the nucleic acid sequences in this manual are given in the 5′ to 3′ direction when read from left to right.

[0063] The terms “transfection,” “conversion,” “nuclear transfection,” or “transduction” refer to the introduction of one or more exogenous polynucleotides into a host cell or organism by physical, chemical, and / or electrical methods. The nucleic acid sequences and vectors disclosed herein can be introduced into cells or organisms by any of these methods, including, for example, electroporation, calcium phosphate coprecipitation, strontium phosphate DNA coprecipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, multi-cation-mediated transfection, tungsten particle-promoted microparticle bombardment, virus-mediated, and / or non-viral-mediated transfection. In some cases, methods for introducing nucleic acids into cells or organisms involve the use of vectors mediated by viruses, retroviruses, lentiviruses, or transposons or transposition elements (e.g., Sleeping Beauty transposon). Sleeping Beauty )).

[0064] The terms “polypeptide,” “peptide,” and their grammatical equivalents refer to polymers of amino acid residues. A polypeptide may optionally include glycosylation or other modifications typical of a given protein in a particular cellular environment. The polypeptides and proteins disclosed herein (including functional fragments and functional variants thereof) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, dihydroindole- 2-Carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornene)carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, isophenylalanine, and α-tert-butylglycine. This disclosure also contemplates that the expression of the polypeptides or proteins described herein in engineered cells may be related to post-translational modifications of one or more amino acids of the polypeptides or proteins. Non-limiting examples of post-translational modifications include: phosphorylation, acylation, including acetylation and formylation, glycosylation, including N-linking and O-linking, amidation, hydroxylation, alkylation, including methylation and ethylation, ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bonds, sulfation, cardiomylation, palmitoylation, isoprenelation, farnesylation, geranylation, glypiation, lipoylation, and iodination.

[0065] The term "conserved amino acid substitution" or "conserved mutation" refers to the substitution of one amino acid for another with a shared property. A functional approach to defining the shared properties among amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins in homologous organisms (Schulz, GE, and Schirmer, RH, *Principles of Protein Structure*, Springer-Verlag, New York (1979). Based on these analyses, amino acid groups can be defined where amino acids within a group preferentially exchange with each other and are therefore most similar in their effect on the overall protein structure (Schulz, GE, and Schirmer, RH, ibid.). Examples of conserved mutations include amino acid substitutions between amino acids within subgroups, such as lysine replacing arginine and vice versa to maintain a positive charge; glutamic acid replacing aspartic acid and vice versa to maintain a negative charge; serine replacing threonine to maintain free -OH; and glutamine replacing asparagine to maintain free -NH2. Exemplary conserved amino acid substitutions are shown in the table below:

[0066] An amino acid sequence that differs from a reference amino acid sequence solely through conserved amino acid substitutions is referred to herein as a “conserved substitution variant” of the reference sequence.

[0067] In some embodiments, the functional variant may comprise the amino acid sequence of the reference protein and have at least one nonconserved amino acid substitution. The term "nonconserved mutation" refers to amino acid substitutions between different groups, such as lysine replaced with tryptophan, or phenylalanine replaced with serine, etc. In this case, it is preferable that the nonconserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Nonconserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity compared to its homologous parent protein. A more detailed discussion of amino acid substitutability can be found, for example, in LY Yampolsky and A. Stoltzfus, "The Exchangeability of Amino Acids in Proteins." Genetics August 2005, 170(4):1459-1472.

[0068] When used in the context of two nucleic acid sequences or two amino acid sequences, the terms "identity" and "sequence identity" refer to the identical nucleotides or residues in two sequences when compared within a specified comparison window to obtain the maximum correspondence. A "comparison window" refers to a segment of at least about 20 consecutive positions, typically about 50 to about 200 consecutive positions, and more typically about 100 to about 150 consecutive positions, whereby, after optimal alignment of the two sequences, one sequence can be compared with a reference sequence having the same number of consecutive positions. Methods for aligning sequences are well known in the art. Optimal sequence alignment for comparison can be performed by Smith and Waterman, Adv. Appl. Math., Local homology algorithm of 2:482 (1981); Needleman and Wunsch, J Mal. Biol., The alignment algorithm of 48:443 (1970); by Pearson and Lipman, Proc. Nat. Acad Sci US.A., The similarity search method of 85:2444 (1988); computerized implementations of these algorithms (including, but not limited to, CLUSTAL in the Intelligentics (Mountain View, California) PC / Gene program, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group (GCG), 575 Science Avenue, Madison, Wisconsin, USA); the CLUSTAL program fully described in the following literature: Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CABIOS 5:151-153 (1989); Corpet et al. Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology24:307-331 (1994). Alignment is also frequently performed through inspection and manual comparison. In one embodiment, the polypeptide described herein is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% identical to a reference polypeptide (i.e., its full length) or a fragment thereof, for example, determined by using BLASTP (or CLUSTAL or any other available alignment software) with default parameters. Similarly, nucleic acids can also be described relative to the starting nucleic acid, for example, they can have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a reference nucleic acid (i.e., its full length) or a fragment thereof, for example, determined by using BLASTN (or CLUSTAL or any other available alignment software) with default parameters. When a molecule is said to have a certain percentage of sequence identity with a larger molecule, it means that when the two molecules are best aligned, the percentage of residues in the smaller molecule finds matching residues in the larger molecule in the order of the best alignment of the two molecules.

[0069] For the purposes of this specification and claims, the phrase "having at least about 50% sequence identity with a reference sequence," or any reference to any range thereof (e.g., "having at least about 80% sequence identity with a reference sequence"), should be understood to include the reference sequence itself. Therefore, for example, the phrase "nucleic acid having at least about 80% sequence identity with SEQ ID NO: 0" as described in the claims also includes SEQ ID NO: 0 itself.

[0070] When applied to nucleic acid or amino acid sequences, the term "substantially identical" and its grammatically equivalent forms mean that the nucleic acid or amino acid sequence contains a sequence that has at least 95% sequence identity with a reference sequence, which is determined by the procedure described above (e.g., BLAST using standard parameters).

[0071] "Homology" is generally inferred based on the sequence similarity between two or more nucleic acids or proteins (or their sequences). The specific percentage of sequence similarity used to determine homology varies depending on the nucleic acid or protein involved, but as low as 25% sequence similarity is commonly used to determine homology. Higher levels of sequence similarity, such as 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher, can also be used to determine homology. Methods for determining the percentage of sequence similarity (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available. Nucleic acids and / or nucleic acid sequences are said to be "homologous" when they originate from the same ancestral nucleic acid or nucleic acid sequence (whether natural or artificial). Proteins and / or the encoding DNA of a protein sequence are said to be "homologous" when they originate from the same ancestral nucleic acid or nucleic acid sequence (whether natural or artificial). These homologous molecules can be called "homologous molecules". For example, any naturally occurring protein can be modified using any available mutagenesis method. When expressed, the mutagenized nucleic acid encodes a polypeptide homologous to the protein encoded by the original nucleic acid.

[0072] This article also considers and includes nucleic acid molecules that hybridize with the published sequences. Hybridization conditions can be selected as mild, moderate, or stringent as needed.

[0073] Suitable stringent conditions for promoting DNA hybridization include, for example, using a 6× sodium chloride / sodium citrate (SSC) solution at approximately 45°C, followed by washing with 2× SSC at 50°C. Such conditions are known or can be found in *Current Protocols in Molecular Biology*, John Wiley & Sons, New York (1989), 6.3.1–6.3.6. “Stringent hybridization conditions” refer to conditions involving incubation at 37°C for 4 to 12 hours in 50% formamide containing 1.0 M NaCl, followed by washing with 0.1× SSC at 60–65°C.

[0074] As will be understood by those skilled in the art, slight changes in the nucleic acid sequence do not necessarily alter the amino acid sequence of the encoded polypeptide. This disclosure includes codon usage degeneracy as understood by those skilled in the art. For example, as is known in the art, different codons can encode the same amino acid, as shown in the table below.

[0075]

[0076] When used in relation to nucleic acid sequences, the phrase “codon degenerate variant” refers to a nucleic acid sequence of a polypeptide that is different from the reference sequence but encodes a polypeptide with the same amino acid sequence as the reference sequence.

[0077] Furthermore, those skilled in the art will understand that partial sequences are often just as effective as the full-length form. Those skilled in the art are aware of ways in which nucleotide sequences can be varied or shortened, and of ways to test the suitability or effectiveness of the altered gene. In some embodiments, the suitability and / or effectiveness of the altered gene can be easily tested, for example, by conventional gas chromatography. Therefore, all such gene variants are included as part of this disclosure.

[0078] The term "isolated" and its grammatical equivalents refer to the removal of nucleic acids from their natural environment. However, it should be understood that nucleic acids and proteins can be formulated with diluents or adjuvants and can still be considered isolated in a practical sense.

[0079] The term "purified" and its grammatical equivalents refer to a molecule or composition whose purity has been enhanced, whether derived from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and / or amplified under laboratory conditions. "Purity" is a relative term, not "absolute purity." For example, nucleic acids are typically mixed with an acceptable vector or diluent when used for introduction into cells. The term "substantially purified" and its grammatical equivalents refer to a nucleic acid sequence, polypeptide, protein, or other compound that is substantially free of the naturally associated polynucleotides, proteins, polypeptides, and other molecules of that nucleic acid, polypeptide, protein, or other compound—that is, free of approximately 50%, approximately 70%, or approximately 90% of the naturally associated polynucleotides, proteins, polypeptides, and other molecules of that nucleic acid, polypeptide, protein, or other compound.

[0080] "Transposons," "transposable elements," or "TEs" are DNA sequences capable of changing their position within the genome, sometimes producing or reversing mutations and altering the size of the cell's genome. Transposition typically results in the replication of the transposon. Type I transposons replicate in two stages: first, DNA is transcribed into RNA, and then the resulting RNA is reverse transcribed back into DNA. This replicated DNA is then inserted into a new location in the genome. The reverse transcription step is catalyzed by a reverse transcriptase, which can be encoded by the transposon itself. Retrotransposons are characterized similarly to retroviruses, such as HIV. The cut-and-paste transposition mechanism of Type II transposons does not involve an RNA intermediate. Transposition is catalyzed by several transposases. Some transposases can nonspecifically bind to any target site in DNA, while others bind to target sites specific to a DNA sequence. Transposases create staggered cuts at the target site, resulting in single-stranded 5' or 3' DNA overhangs (sticky ends). This step cuts out the DNA transposon and then ligates it to the new target site; this process involves the activity of DNA polymerase filling the gaps and DNA ligase closing the sugar-phosphate backbone. This results in the duplication of the target site. Insertion sites for DNA transposons can be identified by short direct repeat sequences, generated by interleaved cuts in the target DNA and filled in by DNA polymerase, subsequently forming a series of inverted repeat sequences important for transposase cleavage of the transposon. If transposition occurs during the S phase of the cell cycle (when the donor site has replicated but the target site has not), cut-and-paste transposons can be replicated. In class I and II transposons, transposition can be classified as "autonomous" or "non-autonomous." Autonomous transposons can move on their own, while non-autonomous transposons require the presence of another transposon to move. This is usually because non-autonomous transposons lack transposase (for class II) or reverse transcriptase (for class I).

[0081] A transposase is an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome via a cut-and-paste mechanism or a replication-type transposition mechanism. In some implementations, the catalytic activity of transposases can be used to move genes from a vector to the genome.

[0082] "Expression vector" or "vector" refers to any genetic element, such as a plasmid, mini-circle, nanoplasmid, chromosome, virus, or transposon, that functions as an autonomous unit of polynucleotide replication within a cell (i.e., capable of replicating under its own control), or acquires replication capability by inserting into the host cell's chromosome, and is attached to another polynucleotide segment, thereby enabling the linked segment to replicate and / or be expressed. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages, and granules. A vector may contain the polynucleotide sequences necessary for ligation or insertion into a target host cell and for expression of the linked segment. These sequences vary depending on the host organism; they include promoter sequences for promoting transcription, enhancer sequences for enhancing transcription, ribosome binding site sequences, and transcription and translation termination sequences. Alternatively, an expression vector may be able to directly express the nucleic acid sequence product encoded therein without ligating or integrating the vector into the host cell's DNA sequence. In some implementations, the vector is an "attachment expression vector" or "attachment" that is capable of replicating in the host cell and persists within the host cell as an extrachromosomal segment of DNA in the presence of appropriate selective pressure (see, for example, Conese et al.). Gene Therapy, 11 References 1735-1742 (2004)). Representative commercially available augmented expression vectors include, but are not limited to, augmented plasmids utilizing EB nuclear antigen 1 (EBNA1) and EB virus (EBV) origin of replication (oriP). Vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA) and pBK-CMV from Stratagene (La Jolla, CA) represent non-limiting examples of augmented vectors using T antigen and SV40 origin of replication instead of EBNA1 and oriP. Vectors may also contain selection marker genes. In some embodiments using nanoparticles, for example, the R6K strain may be used, which utilizes antisense RNA to select markers (e.g., sucrose tolerance).

[0083] The term "selection marker gene" refers to a nucleic acid sequence that allows for specific selection or anti-selection of cells expressing that nucleic acid sequence in the presence of a suitable selector. Suitable selection marker genes are known in the art and described, for example, in International Patent Application Publications WO 1992 / 08796 and WO 1994 / 28143; Wigler et al. Proc. Natl. Acad. Sci. USA, 77 : 3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78 :1527 (1981); Mulligan and Berg, Proc. Natl. Acad. Sci. USA, 78 : 2072 (1981); Colberre-Garapin wait J. Mol. Biol., 150 :1 (1981); Santerre et al., Gene, 30 : 147 (1984); Kent et al., Science, 237 : 901-903 (1987); Wigler et al., Cell, 11 : 223 (1977); Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA, 48 : 2026 (1962); Lowy et al., Cell, 22 : 817 (1980); and U.S. Patent Nos. 5,122,464 and 5,770,359.

[0084] The term "coding sequence" refers to a multinucleotide region that encodes a protein or polypeptide. This region or sequence is defined by a start codon near the 5' end and a stop codon near the 3' end. A coding sequence can also be called an open reading frame.

[0085] The term "operational linkage" refers to a physical and / or functional connection between two DNA segments that enables them to function as intended. An operational linkage occurs when a DNA sequence encoding a gene product is linked to a regulatory sequence (e.g., a promoter, enhancer, and / or silencer) in a manner that allows direct or indirect regulation of transcription of that DNA sequence. For example, an operational linkage occurs when a DNA sequence is linked downstream of a promoter transcription start site and in the correct reading frame relative to that site, allowing transcriptional elongation to pass through the DNA sequence. An operational linkage occurs when an enhancer or silencer is linked to a DNA sequence encoding a gene product in a manner that increases or decreases transcription of the DNA sequence, respectively. Enhancers and silencers can be located upstream, downstream, or embedded within the coding region of a DNA sequence. An operational linkage occurs when a signal sequence is linked to DNA encoding a polypeptide, enabling the signal sequence to be expressed as a precursor protein and participate in polypeptide secretion. The ligation of DNA sequences to regulatory sequences is typically achieved by ligating at appropriate restriction sites or by inserting adapters or linkers into the sequence using restriction endonucleases known to those skilled in the art.

[0086] The terms “induction”, “inducing effect”, and their grammatical equivalents refer to an increase in nucleic acid sequence transcription, promoter activity, and / or expression relative to a given baseline transcriptional level caused by transcriptional regulatory factors.

[0087] The term "transcriptional regulatory factor" refers to a biochemical element that, under certain environmental conditions, prevents or inhibits the transcription of a promoter-driven DNA sequence (e.g., repressors or nuclear repressor proteins), or under certain environmental conditions, allows or stimulates the transcription of a promoter-driven DNA sequence (e.g., inducers or enhancers).

[0088] The term "enhancer" refers to a DNA sequence capable of increasing transcription of a nucleic acid sequence, for example, that is operatively linked to it. Enhancers can be located thousands of bases from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, DNA methylation patterns, or changes in DNA structure. A large number of enhancers from a variety of different sources are known in the art and can be found as or within clonal polynucleotides (e.g., from depositories such as ATCC and other commercial or personal sources). Many polynucleotides containing promoters (e.g., the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream or downstream of the coding sequence, or even within the coding sequence. The term “Ig enhancer” refers to an enhancer element derived from an enhancer region mapped within an immunoglobulin (Ig) gene locus (such enhancers include, for example, heavy chain (μ) 5' enhancers, light chain (κ) 5' enhancers, κ and μ intrinsic enhancers and 3′ enhancers (see, generally, Paul WE, ed., Fundamental Immunology, 3rd ed., Raven Press, New York (1993), pp. 353-363; and U.S. Patent No. 5,885,827).

[0089] The term "promoter" refers to the multinucleotide region that initiates transcription of a sequence that encodes a gene. Promoters are located near the transcription start site of a gene, on the same strand of DNA and upstream (towards the 5' region of the sense strand). Some promoters are constitutive because they are active in all cellular conditions, while others become active in response to specific stimuli, such as inducible promoters. The term "promoter activity" and its grammatical equivalents refer to the degree of expression of the nucleotide sequence operationally linked to the promoter whose activity is being measured. Promoter activity can be measured directly by determining the amount of RNA transcript produced, for example by Northern blotting analysis, or indirectly by the amount of product encoded by the linked nucleic acid sequence, such as the reporter nucleic acid sequence linked to the promoter.

[0090] An "inducible promoter" is a promoter that is induced to become active in the presence or absence of transcriptional regulatory factors, such as biotic or abiotic factors. Inducible promoters are useful because the expression of the gene operationally linked to them can be turned on or off at certain developmental stages or in specific tissues of an organism. Non-restrictive examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenicity-regulated promoters, temperature-regulated promoters, and light-regulated promoters. Inducible promoters can be part of a gene switch or a genetic switch.

[0091] When used in relation to peptides, the phrase "functional fragment" refers to a fragment of the peptide that has the principal function of the reference peptide. For example, a functional fragment of a peptide that is a transmembrane domain is a fragment of the peptide that also has transmembrane domain function. In some embodiments, the functional fragment of the peptide is at its N-terminus and / or C-terminus that is at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue shorter than the reference peptide. When used in relation to nucleic acids, the phrase "functional fragment" refers to a fragment of the reference nucleic acid that encodes a peptide having the same principal function as the peptide encoded by the reference nucleic acid.

[0092] When used in connection with peptides, the phrase "functional variant" refers to a peptide that differs from the reference peptide but has the principal function of the reference peptide. For example, a functional variant of a peptide that is a transmembrane domain is a fragment of that peptide that also has transmembrane domain function. In some embodiments, the functional variant has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the reference amino acid sequence and / or is a conserved substitution variant of the reference sequence. In some embodiments, the functional variant is a conserved substitution variant of the reference sequence that differs from the reference sequence by only 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 conserved amino acid substitution.

[0093] When used in relation to nucleic acids, the phrase "functional variant" refers to a nucleic acid that differs from the reference nucleic acid but encodes a polypeptide that has the same principal function as the reference nucleic acid. In some embodiments, the functional variant has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the reference nucleic acid sequence, is capable of hybridizing with a complementary sequence of the reference nucleic acid sequence under stringent hybridization conditions, or is a codon-degenerate variant of the nucleic acid sequence.

[0094] "Patient" or "object" refers to a mammalian object diagnosed with or suspected of having or likely to develop a disease or condition associated with α-1 antitrypsin deficiency (e.g., liver disease or lung disease). In some embodiments, the term "patient" refers to a mammalian object with a higher-than-average probability of developing a disease (e.g., lung disease or liver disease) caused by α-1 antitrypsin deficiency. Exemplary patients may be humans, apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents, and other mammals who may benefit from the therapies disclosed herein. Exemplary human patients may be male and / or female. "Patient in need" or "object in need" refers to a patient diagnosed with or suspected of having a disease or condition, such as, but not limited to, lung disease or liver disease.

[0095] "Administration" refers to providing a patient or subject with one or more of the compositions described herein. For example, but not limited to, the composition may be administered (e.g., by injection) via intravenous (iv) (e.g., portal vein delivery), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im). One or more such routes may be used. For example, parenteral administration may be performed by bolus injection or gradual perfusion over time. Alternatively, administration may be performed simultaneously via oral route. Furthermore, administration may be performed by surgical deposition of cell clusters or cell particles, or by using a targeted medical device.

[0096] The terms "treatment," "treatment," and their grammatical equivalents refer to achieving a desired pharmacological and / or physiological effect. In some embodiments, this effect is a therapeutic effect, meaning that the effect partially or completely cures the disease and / or adverse symptoms caused by the disease. In some embodiments, the term "treatment" may also include "prevention" of the disease or symptom.

[0097] "Treatment interval" refers to a treatment cycle, such as a course of treatment with a therapeutic agent, which can be repeated, for example, according to a fixed schedule. In some implementations, the dosing regimen may include one or more time periods between treatment intervals during which no therapeutic agent is administered.

[0098] The terms "co-administration," "co-dosing," "co-administered," "co-delivered," and "co-provided" refer to the delivery of two or more different treatments to a subject during the course of the disease, such as after the subject is diagnosed with the disease and before the disease is cured or eliminated or treatment is discontinued for other reasons. In some embodiments, the first treatment is still in progress when the second treatment begins, thus there is an overlap in the timing of administration. This is sometimes referred to herein as "simultaneous" or "parallel delivery." In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins. In some embodiments of any of the above situations, the treatment is more effective due to co-administration. For example, the second treatment is more effective, for example, the same effect can be observed with a smaller amount of the second treatment, or the second treatment reduces symptoms to a greater extent than when given without the first treatment, or a similar effect can be observed with the first treatment. In some embodiments, the delivery method results in a reduction in symptoms or other parameters related to the condition that is greater than the reduction observed when a single treatment is given without the other treatment. The effects of the two treatments can be partially additive, completely additive, or greater than additive. The delivery method allows the effect of the first treatment to still be detectable when the second treatment is delivered.

[0099] In some embodiments, the first and second treatments may be administered simultaneously (e.g., at the same time), in the same or different compositions, or sequentially. Sequential administration means administering one treatment before administering another treatment (e.g., the second treatment), for example, immediately before, less than 5, 10, 15, 30, 45, or 60 minutes before; 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, 96, or more hours before; 4, 5, 6, 7, 8, or 9 days before; or 1, 2, 3, 4, 5, 6, 7, 8, or more weeks before. The order of administration of the first and second treatments may also be reversed.

[0100] The terms "therapeutic effective dose," "therapeutic amount," "immunologic effective dose," "antitumor effective dose," "tumor-suppressive effective dose," and their grammatical equivalents refer to the amount that is effective at the necessary dose and time to achieve the desired therapeutic effect. Therapeutic effective doses can vary depending on factors such as disease state, individual age, sex, weight, and the ability of the compositions described herein to induce a desired response in one or more subjects. The specific dosage of the compositions disclosed herein can be determined by a physician based on differences in the patient's (subject's) age, weight, disease or symptom severity, and individual condition.

[0101] Alternatively, the pharmacological and / or physiological effects of administering one or more of the compositions described herein to a patient or subject may be “preventative,” meaning that the effect completely or partially prevents the disease or its symptoms. “Preventatively effective amount” refers to the amount that is effective at the necessary dose and time to achieve the desired preventative effect (e.g., prevention of disease onset).

[0102] As used herein, terms used in identifying biological entities may include or not include a hyphen “-”. The presence or absence of a hyphen does not alter the intended meaning or identification of the biological entity. For illustrative purposes only and not limited to these biological entities, each of the following pairs of terms (shown with or without a hyphen) represents and identifies the same biological entity: CCR-4 / CCR4, CD-3 / CD3, CD-4 / CD4, CD-33 / CD33, EGFR-2 / EGFR2, FLT-1 / FLT1, HER-1 / HER1, HER-1t / HER1t, IL-12 / IL12, IL-15 / IL15, IL-15Rα / IL15Rα, MUC-1 / MUC1, MUC-16 / MUC16, ROR-1 / ROR1, ROR-1R / ROR1R, TGF-Beta / TGFBeta, VEGF-1 / VEGF1, VEGF-R2 / VEGFR2.

[0103] II. miRNA

[0104] The terms “miR,” “mir,” and “miRNA” are used to refer to microRNAs, which are a class of small non-coding RNA molecules that can affect gene (“target gene”) expression by regulating the translation of messenger RNA produced by their transcription (increasing or decreasing gene expression) and / or by destabilizing the messenger RNA.

[0105] A primary miRNA (or simply primary miRNA) is a miRNA containing at least one RNA hairpin structure. In the cell nucleus, the RNA hairpin structure is cleaved by the primary miRNA to form one or more precursor miRNAs ("precursor miRNAs"). This precursor miRNA is then exported to the cytoplasm, where its stem-loop structure is cleaved to produce a double-stranded miRNA containing a miRNA-5p strand from the original 5' arm of the hairpin loop and a miRNA-3p strand from the original 3' arm of the hairpin loop. The Argonaute protein then binds to this double-stranded miRNA, releasing one strand (either the miRNA-5p sequence or the miRNA-3p sequence). The remaining bound strand becomes the "guide strand" (also called the guide miRNA), while the released strand is called the "passenger strand" (also called the passenger miRNA) and is preferably degraded. The guide strand then interacts with messenger RNA derived from the target gene, thereby influencing its translation or stability. Once the miRNA is guided to interact with the target mRNA, the RNA-induced silencing complex (RISC, also known as the microRNA-ribonucleoprotein complex (miRNP)) is recruited into the complex, for example, to cleave the target mRNA into at least two fragments, destabilize the target mRNA by shortening its poly(A) tail, or reduce the translation of the target mRNA.

[0106] Both miRNA-5p and miRNA-3p sequences are referred to as "mature miRNA" sequences in this paper. The remaining portions of the primary miRNA or precursor miRNA (i.e., the portion located at the 5' end of the miRNA-5p sequence, the portion located at the 3' end of the miRNA-3p sequence, and the stem-loop sequence located between the miRNA-5p and miRNA-3p sequences) are collectively referred to as miRNA backbone sequences in this paper. The term "5' backbone sequence" is used in this paper to refer to the backbone sequence located at the 5' end of the miRNA-5p sequence in the primary or precursor miRNA. The term "3' backbone sequence" is used in this paper to refer to the backbone sequence located at the 3' end of the miRNA-3p sequence in the primary or precursor miRNA. The term "loop sequence" refers to the backbone sequence located between the miRNA-5p and miRNA-3p sequences in the primary or precursor miRNA.

[0107] Unless otherwise stated, the term “miRNA” generally refers to the mature, primary, and precursor forms of a particular microRNA, as well as its functional fragments and functional variants.

[0108] The miRNA may be non-natural. The terms “non-natural,” “synthetic,” and “artificial” used herein to describe miRNA are used interchangeably and refer to miRNAs having sequences that do not exist in nature.

[0109] This invention relates in part to a ribonucleic acid comprising one or more non-natural precursor miRNA sequences, wherein at least one precursor miRNA sequence comprises a guide miRNA that inhibits the expression of the SERPINA1 allele encoding an α-1 antitrypsin (AAT) mutant variant, such as AAT(Z), AAT(S), AAT(I), AAT(S) iiyama ) or AAT(M malton In some embodiments, the RNA comprises more than two such non-natural precursor miRNA sequences, such as three, four, five, six, seven, eight, nine, ten, or more such sequences. It should be understood that each guide miRNA may target the same or different alleles. In embodiments where two or more guide miRNAs target the same allele, such guide miRNAs may target the same or different regions of the gene. In some embodiments, the RNA comprises two or more non-natural precursor miRNA sequences, wherein at least one precursor miRNA sequence contains a guide miRNA that inhibits AAT(Z) expression, and a second precursor miRNA sequence contains a guide miRNA that inhibits the expression of any AAT allele (e.g., AAT(M), AAT(Z), or AAT(S)).

[0110] In some embodiments, the non-natural precursor miRNA sequences in the ribonucleic acid form a stem-loop secondary structure that is different from and not complementary to the structure formed by another different non-natural precursor miRNA sequence in the ribonucleic acid. In some embodiments, the sequence identity between the non-natural precursor miRNA sequences is less than about 99%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 55%, or less than about 50%. For example, the non-natural precursor miRNA sequences have about 25% to about 99% sequence identity with each other.

[0111] In some embodiments, the secondary structure of each non-natural precursor miRNA is sufficiently similar to the secondary structure of a naturally occurring precursor miRNA sequence to reduce or prevent cytotoxicity based on cellular RNAi. In some such embodiments, the nucleotide sequence of the non-natural precursor miRNA has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the naturally occurring precursor miRNA and / or is capable of hybridizing with the naturally occurring precursor miRNA under stringent hybridization conditions. For example, the nucleotide sequence of the non-natural precursor miRNA has about 50% to about 100% sequence identity with the naturally occurring precursor miRNA and / or is capable of hybridizing with the naturally occurring precursor miRNA under stringent hybridization conditions.

[0112] In some embodiments, the secondary structure of each primary miRNA containing a non-natural precursor miRNA (hereinafter referred to as "non-natural primary miRNA") is sufficiently similar to the secondary structure of a naturally occurring primary miRNA sequence to reduce or prevent cytotoxicity based on cellular RNAi. In some such embodiments, the nucleotide sequence of the non-natural primary miRNA has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the naturally occurring primary miRNA and / or is capable of hybridizing with the naturally occurring primary miRNA under stringent hybridization conditions. For example, the nucleotide sequence of the non-natural primary miRNA has about 50% to about 100% sequence identity with the naturally occurring primary miRNA and / or is capable of hybridizing with the naturally occurring primary miRNA under stringent hybridization conditions.

[0113] The non-natural precursor miRNA of the present invention can be obtained by removing the natural mature miRNA sequence from a naturally occurring precursor miRNA and replacing it with a non-natural mature miRNA sequence, one of which can serve as a guide miRNA for targeting genes of interest.

[0114] In some embodiments, each non-natural precursor miRNA comprises a backbone sequence derived from a naturally occurring precursor miRNA, such as a backbone sequence from mice, rats, or humans. In some embodiments, the backbone sequences (3' backbone sequence, 5' backbone sequence, and loop sequence) of the non-natural precursor miRNA have at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the corresponding backbone segment under stringent hybridization conditions. In some embodiments, the backbone segment of the non-natural precursor miRNA sequence is identical to the corresponding backbone segment of the naturally occurring precursor miRNA. In some embodiments, the natural precursor miRNA is miR16, miR17, miR19, miR21, miR22, miR26a1, miR29b1, miR30a, miR122, miR126, miR133a1, miR142, miR150, miR155, miR181a, miR204, miR206, miR214, miR412, miR486, miR494, or miR1915. In some embodiments, the natural precursor miRNA is miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206. In some embodiments, the natural precursor miRNA is miR16, miR17, miR21, miR22, miR204, or miR206. In some embodiments, the natural precursor miRNA is miR204 or miR206. In some embodiments, the natural precursor miRNA is miR16, miR17, miR22, miR29b, miR133a1, miR150, miR181a1, miR204, miR206, or miR486.

[0115] In some embodiments, each non-natural primary miRNA comprises a backbone sequence derived from a naturally occurring primary miRNA, such as a backbone sequence derived from a mouse, rat, or human. In some embodiments, the backbone sequence (3' backbone sequence, 5' backbone sequence, and loop sequence) of the non-natural primary miRNA has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the corresponding backbone sequence of the naturally occurring primary miRNA and / or is capable of hybridizing with the corresponding backbone segment under stringent hybridization conditions. For example, the backbone sequence of the non-natural primary miRNA has about 50% to 100% sequence identity with the corresponding backbone sequence of the naturally occurring primary miRNA and / or is capable of hybridizing with the corresponding backbone segment under stringent hybridization conditions. In some embodiments, the backbone region of the non-natural primary miRNA sequence is identical to the corresponding backbone region of a naturally occurring primary miRNA. In some embodiments, the natural primary miRNA is miR16, miR17, miR19, miR21, miR22, miR26a1, miR29b1, miR30a, miR122, miR126, miR133a1, miR142, miR150, miR155, miR204, miR206, miR214, miR412, miR486, miR494, or miR1915. In some embodiments, the natural precursor miRNA is miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206. In some embodiments, the natural precursor miRNA is miR16, miR17, miR21, miR22, miR204, or miR206. In some embodiments, the natural precursor miRNA is miR204 or miR206.

[0116] Although miRNA-5p and miRNA-3p sequences hybridize with each other, they are not necessarily perfectly complementary. When designing non-natural miRNAs, compensatory mutations can be made in the miRNA-5p and / or miRNA-3p sequences to maintain the RNA folding structure and free energy of the natural miRNA. In some implementations, the sequence encoding the miRNA-3p sequence has at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the complementary sequence encoding the miRNA-5p sequence, or is capable of hybridizing with the sequence encoding the miRNA-5p sequence under strict hybridization conditions.

[0117] In some embodiments, the two non-natural precursor miRNA sequences are separated from each other by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, or at least about 250 nucleotides. In some embodiments, the two non-natural precursor miRNA sequences are separated from each other by approximately 5 to 250 nucleotides, approximately 10 to 250 nucleotides, approximately 10 to 200 nucleotides, approximately 10 to 150 nucleotides, approximately 10 to 100 nucleotides, approximately 10 to 50 nucleotides, approximately 10 to 40 nucleotides, approximately 10 to 30 nucleotides, approximately 10 to 20 nucleotides, approximately 16 to 250 nucleotides, approximately 16 to 200 nucleotides, approximately 16 to 150 nucleotides, approximately 16 to 100 nucleotides, approximately 16 to 50 nucleotides, and approximately 1... Separated by 6 to 40 nucleotides, approximately 16 to 30 nucleotides, approximately 16 to 20 nucleotides, approximately 20 to 200 nucleotides, approximately 20 to 150 nucleotides, approximately 20 to 100 nucleotides, approximately 20 to 50 nucleotides, approximately 20 to 45 nucleotides, approximately 20 to 40 nucleotides, approximately 20 to 35 nucleotides, approximately 20 to 30 nucleotides, approximately 20 to 25 nucleotides, approximately 30 to 200 nucleotides, approximately 30 to 150 nucleotides, approximately 30 to 100 nucleotides, approximately 30 to 50 nucleotides, or approximately 30 to 40 nucleotides.In some embodiments, the two non-natural precursor miRNA sequences are separated from each other by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72. 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 1 37, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194 Separated by 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 nucleotides. In some embodiments, the two non-natural precursor miRNA sequences are separated from each other by at least about 7 nucleotides. In some embodiments, the two non-natural precursor miRNA sequences are separated from each other by at least about 10 nucleotides.

[0118] In some implementations, two non-natural primary miRNA sequences are adjacent to each other, with the 3' nucleotide of one primary miRNA directly linked to the 5' nucleotide of the other. In such implementations, the nucleotides separating the non-natural precursor miRNAs contained in each primary miRNA constitute part of the primary miRNA sequence.

[0119] In some embodiments, the non-natural precursor miRNA comprises a mature miRNA sequence capable of binding to mRNA, thereby interfering with its translation and / or promoting its degradation. The mRNA may be derived from the expression of the target gene.

[0120] In some embodiments, the target gene encodes a mutant variant of AAT. Therefore, the precursor miRNA sequence inhibits the expression of the gene that expresses the mutant AAT by targeting it. In some such embodiments, the mutant variant of AAT is AAT(Z), AAT(S), AAT(I), or AAT(S... iiyama ) or AAT(M malton ).

[0121] In some implementations, each non-natural precursor miRNA targets a different allele. In some implementations, each non-natural precursor miRNA targets a different region of the same allele. In some implementations, each non-natural precursor miRNA targets a different region of a different allele.

[0122] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting an AAT(Z) allele; and (b) a precursor miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting an AAT(Z) allele.

[0123] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting a gene expressing AAT(Z); and (b) a primary miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting a gene expressing AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting a gene expressing AAT(Z); and (b) a primary miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting an AAT(Z) allele.

[0124] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR16 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the RNA comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR16 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR16 and a guide miRNA targeting any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) allele.

[0125] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence derived from miR16 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR16 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR16 and a guide miRNA targeting any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) allele.

[0126] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting an AAT(Z) allele.

[0127] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting an AAT(Z) allele.

[0128] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR142 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR142 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence derived from miR142 and a guide miRNA targeting an AAT(Z) allele.

[0129] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR142 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR142 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence derived from miR142 and a guide miRNA targeting an AAT(Z) allele.

[0130] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting an AAT(Z) allele.

[0131] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting an AAT(Z) allele.

[0132] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR21 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the RNA comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR21 and a guide miRNA targeting any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence derived from miR21 and a guide miRNA targeting an AAT(Z) allele.

[0133] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR21 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR21 and a guide miRNA targeting any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR22 and a guide miRNA targeting any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence derived from miR206 and a guide miRNA targeting an AAT(Z) allele.

[0134] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR21 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a precursor miRNA comprising a backbone sequence derived from miR21 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence derived from miR21 and a guide miRNA targeting an AAT(Z) allele.

[0135] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR21 and a guide miRNA targeting an AAT(Z) mutant gene. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting an AAT(Z) mutant gene; and (b) a primary miRNA comprising a backbone sequence derived from miR21 and a guide miRNA targeting any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence derived from miR204 and a guide miRNA targeting any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence derived from miR21 and a guide miRNA targeting an AAT(Z) allele.

[0136] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting an allele encoding AAT(Z).

[0137] In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting an allele encoding AAT(Z).

[0138] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting an allele encoding AAT(Z).

[0139] In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting an allele encoding AAT(Z).

[0140] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting an allele encoding AAT(Z).

[0141] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR142 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence from miR21 and a guide miRNA targeting an allele encoding AAT(Z).

[0142] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting an allele encoding AAT(Z).

[0143] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting an allele encoding AAT(Z).

[0144] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting an allele encoding AAT(Z).

[0145] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting an allele encoding AAT(Z).

[0146] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting an allele encoding AAT(Z).

[0147] In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting an allele encoding AAT(Z).

[0148] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting an allele encoding AAT(Z).

[0149] In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting an allele encoding AAT(Z).

[0150] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting an allele encoding AAT(Z).

[0151] In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting an allele encoding AAT(Z).

[0152] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting an allele encoding AAT(Z).

[0153] In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence from miR26a1 and a guide miRNA targeting an allele encoding AAT(Z).

[0154] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting an allele encoding AAT(Z).

[0155] In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting an allele encoding AAT(Z).

[0156] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting an allele encoding AAT(Z).

[0157] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting an allele encoding AAT(Z).

[0158] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0159] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0160] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0161] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR150 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0162] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0163] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0164] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid (RNA) comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid (RNA) comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0165] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0166] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid (RNA) comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid (RNA) comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0167] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR16 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR22 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0168] In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid (RNA) comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0169] In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z). In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a mutant gene encoding AAT(Z).In some embodiments, the ribonucleic acid (RNA) comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; and (c) a primary miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the RNA comprises: (a) a primary miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a primary miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a primary miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein. In some embodiments, the ribonucleic acid comprises: (a) a precursor miRNA comprising a backbone sequence from miR204 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein; (b) a precursor miRNA comprising a backbone sequence from miR206 and a guide miRNA targeting a mutant gene encoding AAT(Z); and (c) a precursor miRNA comprising a backbone sequence from miR17 and a guide miRNA targeting a gene encoding any of the AAT alleles described herein.

[0170] The present invention also relates in part to deoxyribonucleic acid encoding any of the above-mentioned ribonucleic acids.

[0171] Examples of deoxyribonucleic acid (DNA) sequences encoding backbone sequences that can be used in the practice of this invention include, but are not limited to, the sequences listed in Table 1 below. In Table 1, the symbols “X” and “Y” represent nucleic acid sequences encoding a guide miRNA (which may be miRNA-5p or miRNA-3p) and a passenger miRNA (which may be miRNA-5p or miRNA-3p), respectively, while the symbol “n” represents the number of nucleotides in such sequences, for example, 16-30, preferably 18-25. In one embodiment, n may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides. In some embodiments, the DNA encoding the backbone sequence is a DNA sequence that hybridizes under strict hybridization conditions to a complementary sequence of any of the sequences listed in Table 1.

[0172] Table 1: Deoxyribonucleic acid sequences encoding miRNA backbone sequences

[0173] In any of the above embodiments, the sequence encoding the precursor miRNA may include: Specifically, SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; Specifically, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; Specifically, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9; Specifically, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12; Specifically, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15; Specifically, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18; Specifically, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21; Specifically, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24; Specifically, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27; Specifically, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30; Specifically, SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33; Specifically, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36; Specifically, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39; Specifically, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42; Specifically, SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45; Specifically, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48; Specifically, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51; Specifically, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54; Specifically, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57; Specifically, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60; Specifically, SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63; Specifically, SEQ ID NO: 338, SEQ ID NO: 339 and SEQ ID NO: 340; Specifically, SEQ ID NO: 341, SEQ ID NO: 342 and SEQ ID NO: 343; Specifically, SEQ ID NO: 344, SEQ ID NO: 345 and SEQ ID NO: 346; Specifically, SEQ ID NO: 597, SEQ ID NO: 598 and SEQ ID NO: 599; SEQ ID NO: 589; Or a sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the above sequences, or a sequence that can hybridize with complementary sequences of such sequences under strict hybridization conditions.

[0174] In some embodiments, the dual miRNA of the present invention comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 589 (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 589, or a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 589 under strict hybridization conditions).

[0175] Non-restrictive examples of nucleic acid sequences encoding guide miRNAs targeting mutant AAT variants are listed in Table 2. Table 2 also lists sequences encoding passenger chains. As previously mentioned, the guide and passenger chains are not necessarily complementary. It is also envisioned that passenger chains could be used to target messenger RNAs associated with target genes. It is also envisioned that sequences that hybridize to complementary sequences listed in Table 2 under strict hybridization conditions could be used. The mature miRNA sequences used can be combined with specific primary miRNA backbones. Table 2 also lists backbones that can be combined with the mature guide and passenger miRNAs listed therein.

[0176] Table 2: Deoxyribonucleic acid sequences encoding mature miRNA sequences

[0177] In some embodiments, the present invention relates to a deoxyribonucleic acid (DNA) comprising a nucleic acid sequence having at least about 80% sequence identity with any of the sequences in SEQ ID NO: 64-67, or a nucleic acid sequence capable of hybridizing with the complementary sequence of any of the sequences in SEQ ID NO: 64-67 under stringent hybridization conditions. In some such embodiments, the present invention relates to a DNA comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the sequences in SEQ ID NO: 64-67, or a nucleic acid sequence capable of hybridizing with the complementary sequence of any of the sequences in SEQ ID NO: 64-67 under stringent hybridization conditions.

[0178] In some embodiments, the sequence encoding the guide miRNA sequence has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 64 or 66, or is capable of hybridizing with the complementary sequence of SEQ ID NO: 64 or 66 under strict hybridization conditions.

[0179] In some embodiments, the sequence encoding the passenger miRNA sequence has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 65 or 67, or is capable of hybridizing with the complementary sequence of SEQ ID NO: 65 or 67 under strict hybridization conditions.

[0180] In some embodiments, the present invention relates to a deoxyribonucleic acid, wherein the sequences encoding precursor miRNAs comprise: a) The sequence encoding the 5' miRNA backbone sequence; b) The sequence encoding the guide miRNA sequence; c) The sequence encoding the stem-loop sequence; d) The sequence encoding the passenger miRNA sequence; and e) The sequence that encodes the 3' skeleton sequence.

[0181] In some implementations, the sequence encoding the precursor miRNA includes: a) A guide miRNA sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 64 or 66, or a guide miRNA sequence capable of hybridizing with the complementary sequence of SEQ ID NO: 64 or 66 under strict hybridization conditions; and b) A passenger sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 65 or 67, or a passenger sequence capable of hybridizing with the complementary sequence of SEQ ID NO: 65 or 67 under strict hybridization conditions.

[0182] Deoxyribonucleic acid (DNA) encoding exemplary non-natural precursor miRNA sequences targeting AAT(Z) is described in Table 3. In some embodiments, the DNA may comprise a sequence capable of hybridizing with a complementary sequence to any of the sequences listed in Table 3 under stringent hybridization conditions.

[0183] Table 3: Deoxyribonucleic acid sequences encoding non-natural miRNA sequences

[0184] In some embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 178 or 179, or a sequence capable of hybridizing with the complementary sequence of SEQ ID NO: 178 or 179 under strict hybridization conditions.

[0185] In some embodiments, the present invention relates to a deoxyribonucleic acid comprising a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 347 or 348, or a sequence capable of hybridizing with the complementary sequence of SEQ ID NO: 347 or 348 under strict hybridization conditions.

[0186] In embodiments of the present invention, each of the two or more precursor miRNAs encoded by the deoxyribonucleic acid may contain a guide miRNA sequence targeting the same target allele, or different guide miRNAs may target different alleles. Furthermore, the design of each precursor miRNA, or the design of the primary miRNA containing them, may be based on different naturally occurring miRNA backbones to reduce the likelihood of one miRNA misfolding with another. Table 4 provides examples of deoxyribonucleic acid sequences encoding two or more primary miRNAs.

[0187] Table 4: Deoxyribonucleic acid sequences containing two or more primary miRNAs

[0188] In some embodiments, the present invention relates to a deoxyribonucleic acid comprising a nucleic acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 267, or a nucleic acid sequence capable of hybridizing with a complementary sequence of SEQ ID NO: 267 under strict hybridization conditions.

[0189] III. Proteins of Interest

[0190] In some embodiments, the deoxyribonucleic acid encoding the precursor miRNA is contained in the same genetic construct as one or more genes encoding proteins of interest (e.g., normal, functional α-1 antitrypsin encoded by AAT(M)).

[0191] A. AAT(M)

[0192] In any of the above embodiments, the protein of interest may be a normal, functional α-1 antitrypsin (“AAT”). The genetic constructs of the present invention encode a precursor miRNA and also encode a normal, functional AAT, enabling treatment by using the miRNA to reduce the expression of a mutant AAT variant (e.g., AAT(Z)) while simultaneously introducing a transgene expressing AAT(M). By combining the reduction of mutant AAT expression with the introduction of functional AAT, this type of gene therapy provides a comprehensive approach to addressing the root cause of AATTD. In some embodiments, the transgene encoding AAT(M) differs from the wild-type gene in that it is modified to include a silent mutation, thereby avoiding reduction of expression by certain miRNA constructs contemplated herein, such as miRNA constructs designed to reduce the expression of mutant AAT alleles (e.g., AAT(Z)). In some such embodiments, the miRNA-tolerant AAT(M) gene variant comprises the nucleic acid sequence of SEQ ID NO: 590 or a functional variant thereof. In some such embodiments, the functional variant has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 590 and / or a codon degenerate variant of SEQ ID NO: 590.

[0193] In some embodiments, the AAT(M) variant is modified to reduce the presence of CpG sites and avoid the reduction of expression by certain miRNA constructs as described above. In some such embodiments, the CpG-reduced miRNA-tolerant AAT(M) gene variant comprises the nucleic acid sequence of SEQ ID NO: 596 or a functional variant thereof. In some such embodiments, the functional variant has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 596 and / or a codon degenerate variant of SEQ ID NO: 596. In some embodiments, the functional variant is a codon degenerate variant of SEQ ID NO: 596.

[0194] In some embodiments, the AAT(M) transgene encodes an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 600 (e.g., having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 600), or a conserved substitution variant of SEQ ID NO: 600.

[0195] IV. Genetic constructs

[0196] As previously described, in some embodiments, the deoxyribonucleic acid encoding the precursor miRNA is contained in the same genetic construct as one or more genes encoding proteins of interest (e.g., AAT(M) variants encoding wild-type AAT proteins).

[0197] In a preferred embodiment, the genetic construct comprises at least the following components: (i) a tissue-specific promoter (e.g., a liver-specific promoter); (ii) a 5'UTR; (iii) any miRNA design for mutant AAT disclosed herein (e.g., miR204 + miR206); (iv) a silencer / miRNA-tolerant AAT(M) transgene; and (v) a 3'UTR.

[0198] In another preferred embodiment, the genetic construct comprises the following components: (i) a liver-specific promoter; (ii) a 5'UTR; (iii) two miRNA designs targeting mutant AAT disclosed herein (e.g., miR204 + miR206); (iv) a silent / miRNA-tolerant AAT(M) transgene; and (v) a 3'UTR. Notably, a key inventive aspect of this preferred embodiment is that only one of the two miRNAs specifically targets across mutant AAT variants (e.g., AAT(Z)). The second miRNA targets another site common to other alleles (including AAT(M)). In some embodiments, the genetic construct of the present invention comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 592 (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 592), a codon degenerate variant of SEQ ID NO: 592, or a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 592 under strict hybridization conditions.

[0199] In another embodiment, a vector is used to deliver the genetic construct to a subject or patient. In a further preferred embodiment, a goat adenovirus vector is used to deliver the genetic construct. Any genetic construct described herein can be inserted into a vector in either the forward or reverse direction. In some embodiments, the genetic construct is inserted into a goat adenovirus vector in the reverse direction. In one such embodiment, the goat adenovirus vector is a GC44 vector comprising a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 593 or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 593), a codon degenerate variant of SEQ ID NO: 593, or a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 593 under strict hybridization conditions.

[0200] In one embodiment, the deoxyribonucleic acid encoding the precursor miRNA is contained in the same genetic construct as the one containing the gene variant (i.e., AAT(M)), which is resistant to miRNA downexpression and encodes a normal, functional AAT protein. In one embodiment, the gene variant contains at least one silencing mutation or codon variant that enables the gene variant to avoid miRNA downexpression and encode a normal, functional AAT protein. In a further embodiment, the AAT(M) gene variant is a miRNA-resistant variant and contains the nucleic acid sequence of SEQ ID NO: 590 or a functional variant thereof. In some such embodiments, the functional variant has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 590 and / or a conserved substitution variant of SEQ ID NO: 590. In some embodiments, the functional variant is a conservative substitution variant of SEQ ID NO: 590 and is distinguished from it only by substitution of 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 conservative nucleic acid base.

[0201] In some such embodiments, the genetic construct comprises a nucleic acid sequence encoding a 5' untranslated region (5'UTR) located directly upstream of a gene encoding a protein of interest, and a precursor miRNA sequence is contained within the 5'UTR. In one embodiment, the 5'UTR is derived from human GADPH. In a further embodiment, the 5'UTR comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 588 or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 588).

[0202] In some embodiments, the genetic construct comprises a nucleic acid sequence encoding a 3' untranslated region (3'UTR) located directly downstream of a gene encoding a protein of interest, and a precursor miRNA sequence is contained within the 3'UTR. In one embodiment, the 3'UTR comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 591 or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 591).

[0203] In some embodiments, the genetic construct comprises nucleic acid sequences encoding 5'UTR and 3'UTR, and each region contains at least one precursor miRNA sequence (e.g., each UTR may contain one precursor miRNA, the 5'UTR may contain one precursor miRNA and the 3'UTR may contain two precursor miRNAs, the 5'UTR may contain two precursor miRNAs and the 3'UTR may contain one precursor miRNA, both UTRs may contain two precursor miRNAs, etc.).

[0204] In embodiments where the primary miRNA coding sequence is contained within the corresponding 5'UTR sequence, the transcribed RNA may contain additional sequences, such as splice donor, branch point, and / or acceptor site sequences. The inclusion of splice donor, branch point, and acceptor sites is important for splicing miRNA from the transcribed RNA. Without splicing, highly structured miRNA sequences may prevent ribosome scanning to translation initiation sequences associated with the gene of interest. Examples of sequences encoding such splice donor / acceptor sites include SEQ ID NO: 291 and 292, sequences having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with such sequences, and sequences capable of hybridizing with complementary sequences of such sequences under strict hybridization conditions.

[0205] Therefore, in some embodiments, the deoxyribonucleic acid of the present invention further comprises: a) a nucleic acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 291, or a nucleic acid sequence capable of hybridizing with the complementary sequence of SEQ ID NO: 291 under stringent hybridization conditions; and b) a nucleic acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 292, or a nucleic acid sequence capable of hybridizing with the complementary sequence of SEQ ID NO: 292 under stringent hybridization conditions.

[0206] In some implementations, the deoxyribonucleic acid portion encoding the precursor miRNA is contained within the intron-corresponding region of the gene encoding the protein of interest.

[0207] In other embodiments, at least one precursor miRNA is contained within the 5' UTR. In other embodiments, at least one precursor miRNA is contained within an intron located in the 5' UTR (hereinafter referred to as a "5' UTR intron"). In other embodiments, at least one precursor miRNA is contained within an intron in an open reading frame. In other embodiments, at least one precursor miRNA is contained within the 3' UTR.

[0208] The polynucleotide of the present invention may contain an integration signal for integration into the attP / attB phage genome. The polynucleotide may also contain a 5' homologous arm or a 5' terminal repeat sequence and a 3' homologous arm or a 3' terminal repeat sequence. The polynucleotide may also contain insulators, boundary elements, and S / MARs located adjacent to the 3' side of the 5' homologous arm or 5' terminal repeat sequence and adjacent to the 5' side of the 3' homologous arm or 3' terminal repeat sequence. Between insulators, boundary elements, or S / MARs, polynucleotides from 5' to 3' may include: promoters, which may include silencers, enhancers, transcription factor binding modules, and core promoters; 5' untranslated regions, which may include stability modules, translation control elements, and intron embedding elements, such as miRNA coding sequences; one or more genes, which may include signal peptides, extracellular domains, transmembrane domains, signal transduction domains, antibody domains, peptide linkers, introns, and epitope tags; and 3' untranslated regions, which may include stability modules, translation control sequences, 3' terminal processing signals, and transcription terminators.

[0209] As previously mentioned, miRNAs can encode additional proteins of interest (e.g., normal, functional AAT) in the same genetic construct. The advantages of using a single genetic construct to express two or more such components include stoichiometric expression of these components, reduced product complexity, and lower manufacturing and dosing costs.

[0210] As those skilled in the art will understand, genes encoding polypeptides of interest can be linked via adapters. In carrying out this invention, any adapter known to be suitable for linking genes can be used. Examples of such adapters include those encoding internal ribosome entry sites (IRES), cleavable peptides, and ribosome jumping peptides. Examples of cleavable peptides encoded by such adapters include Furinlink, fmdv, and 2A adapters (e.g., P2A, GSG-P2A, FP2A, T2A, and furin-T2A), or functional fragments or variants thereof.

[0211] The polynucleotides of this invention can be operatively linked to a promoter in the construct. A suitable promoter can be selected based on the host cell and desired effect. Suitable promoters include constitutive promoters and inducible promoters. The promoter can be a tissue-specific promoter, which is well known in the art. See, for example, Zheng C, Baum BJ. Methods Mol Biol 2008;434:205-219. Furthermore, tissue-specific promoters can also be synthetic tissue-specific promoters, such as synthetic liver tissue-specific promoters.

[0212] Examples of constitutive promoters that can be used in this invention include, but are not limited to: immediate early cytomegalovirus (CMV) promoters; human extended growth factor 1α1 (hEF1A1); simian virus 40 (SV40) early promoters; mouse mammary tumor virus (MMTV); human immunodeficiency virus (HIV) long terminal repeat (LTR) promoters; MoMuLV promoters; avian leukosis virus promoters; Epstein-Barr virus immediate early promoters; Rous sarcoma virus promoters; and human gene promoters, such as, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters; and their functional fragments and variants. Compared to constitutive promoters, the use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to it when expression is needed, and turn off its expression when expression is not needed. Examples of inducible promoters include, but are not limited to: metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In one aspect, the inducible promoter can be a gene switch ligand inducible promoter. In some cases, inducible promoters can be gene switches based on small molecule ligand-induced dual-peptide ecdysone receptors, such as the RHEOSWITCH® gene switch.

[0213] Tissue-specific promoters

[0214] A novel aspect of the genetic constructs described in this paper is the presence of tissue-specific promoters. Tissue-specific DNA promoter sequences vary depending on the tissue or cell type they regulate. For example, muscle-specific DNA promoter sequences are responsible for the specific activation of genes in muscle cells. They contain specific DNA sequences that interact with muscle-specific transcription factors such as MyoD, Myogenin, and MEF2 (myotrophic factor 2). These transcription factors bind to the promoter sequence and recruit RNA polymerases, thereby initiating the transcription of muscle-specific genes. Similarly, other tissue or cell types (e.g., brain, heart, kidney, lung, liver, or skin) have their own unique DNA promoter sequences that control gene expression in a tissue-specific manner. These promoter sequences typically contain enhancer and silencer elements for finely modulating gene expression levels and responding to specific signals and factors present in the tissue microenvironment.

[0215] In one embodiment, the tissue-specific promoter is a lung-specific promoter. In embodiments of the genetic construct of the present invention that include a lung-specific promoter, transgene expression is restricted to lung tissue, and the promoter provides high levels and sustained transgene expression therein.

[0216] The genetic construct of the present invention may also include any of the following lung-specific promoters: surfactant protein B (SP-B) promoter, Clara cell secreted protein (CCSP) promoter, mucin promoter and / or prosuractive protein C (proSP-C) promoter.

[0217] In one embodiment, the genetic construct of the present invention comprises a surfactant protein B (SP-B) promoter. Surfactant protein B is a protein involved in reducing lung surface tension, thereby enabling the lungs to function properly and perform gas exchange. The SP-B promoter is a lung-specific DNA promoter sequence that specifically regulates the transcription of the SP-B gene in lung cells and contains binding sites for lung-specific transcription factors such as Nkx2.1 (also known as TTF-1 or thyroid transcription factor-1) and GATA6.

[0218] In another embodiment, the genetic construct of the present invention includes a Clara cell secretory protein (CCSP) promoter. Clara cells are non-ciliated secretory cells present in the epithelium of the bronchioles of the lungs. The CCSP promoter is a lung-specific promoter that drives the specific expression of the CCSP gene in these Clara cells and contains binding sites for lung-specific transcription factors, including Nkx2.1 and Foxa2. CCSP proteins are involved in airway protection and regulate lung inflammation and repair.

[0219] In one embodiment, the genetic construct of the present invention includes a mucin promoter. Mucins are glycoproteins that play a key role in forming a mucus layer in the airways, which helps protect and lubricate the respiratory system. Several mucin genes exhibit lung-specific expression. For example, MUC5AC and MUC5B are mucin genes primarily expressed in the respiratory tract, including the lungs. The promoters of these genes contain lung-specific regulatory elements and are regulated by transcription factors such as Nkx2.1 and Spdef (ETS transcription factors containing the SAM tip domain).

[0220] In another embodiment, the genetic construct of the present invention includes a prosuractive protein C (proSP-C) promoter. Prosuractive protein C is a protein essential for the maintenance and stability of pulmonary surfactant. The proSP-C promoter is a lung-specific promoter that primarily drives the expression of the proSP-C gene in alveolar type II cells and includes binding sites for lung-specific transcription factors, including Nkx2.1 and Foxa2.

[0221] In another embodiment, the tissue-specific promoter is a liver-specific promoter. In embodiments of the genetic construct of the present invention that include a liver-specific promoter, transgene expression is restricted to the liver, and the promoter provides high levels and sustained transgene expression therein.

[0222] The genetic construct of the present invention may also include any of the following liver-specific promoters: albumin promoter, cytochrome P450 promoter, α-1-antitrypsin (AAT) promoter and / or transthyretin (TTR) promoter.

[0223] The genetic constructs of the present invention may also include one or more other types of regulatory sequences involved in the activation or regulation of liver-specific genes, such as: enhancers (e.g., liver-specific enhancer elements of the albumin gene (Alb), alpha-fetoprotein enhancers, transthyretin (TTR) enhancers, cytochrome P450 family 3A (CYP3A) enhancers, and α-1 microglobulin / bikunin (A1-MB) enhancers); transcription factors (e.g., hepatocyte nuclear factors (HNF), particularly HNF1, HNF3, and HNF4); response elements (e.g., CCAAT / enhancer-binding protein (C / EBP) response elements); and silencers (e.g., DNA or RNA sequences as negative regulators of gene expression). In one embodiment, the liver-specific promoter comprises two A1-MB enhancers.

[0224] In one embodiment, the genetic construct of the present invention comprises an alpha-fetoprotein (AFP) promoter. AFP is a protein that is primarily produced in the liver during fetal development, and its expression decreases significantly after birth. The AFP promoter region contains a specific DNA sequence that interacts with liver-specific transcription factors such as HNF and C / EBP. These transcription factors bind to the promoter sequence and promote the recruitment of RNA polymerase, thereby initiating the transcription of the AFP gene.

[0225] In another embodiment, the genetic construct of the present invention comprises an albumin promoter. The albumin gene encodes the protein albumin, which is primarily synthesized in the liver. The albumin promoter enhances transcription of the albumin gene and contains binding sites for liver-specific transcription factors such as HNF1 (hepatocyte nuclear factor 1) and HNF4. These transcription factors bind to the albumin promoter, thereby ensuring specific expression of the gene in hepatocytes.

[0226] In another embodiment, the genetic construct of the present invention comprises a cytochrome P450 promoter. Cytochrome P450 (CYP) enzymes are a superfamily of enzymes involved in drug metabolism, hormone synthesis, and detoxification. Several members of the CYP family exhibit liver-specific expression. For example, the CYP3A4 promoter is primarily active in hepatocytes and is regulated by a combination of liver-specific transcription factors, such as HNF4 and CAR (constitutive androstenedione receptor) . The CYP2E1 promoter is another liver-specific promoter that primarily drives the expression of the CYP2E1 enzyme in hepatocytes.

[0227] In another embodiment, the genetic construct of the present invention includes a transthyretin (TTR) promoter. Transthyretin is a protein primarily synthesized in the liver and involved in the transport of thyroxine and retinol-binding proteins. The TTR promoter specifically drives the expression of the TTR gene in hepatocytes and contains binding sites for liver-specific transcription factors such as HNF1, HNF3, and HNF4. Mutations in the TTR promoter are associated with hereditary transthyretin amyloidosis, a disease characterized by the deposition of abnormal proteins in multiple organs.

[0228] In a preferred embodiment of the invention, the genetic construct of the invention comprises an α-1-antitrypsin (AAT) promoter. As previously described, α-1-antitrypsin is a protein produced in the liver and plays a role in protecting the lungs from damage caused by neutrophil elastase. In one embodiment, the AAT promoter is a liver-specific promoter that regulates the transcription of the AAT gene. In another embodiment, the AAT promoter comprises a binding site for liver-specific transcription factors such as HNF1 and C / EBP.

[0229] AAT promoters can be partial or complete. A complete promoter (also called a full promoter) is a promoter region that contains all the elements necessary for efficient initiation of transcription. On the other hand, a partial promoter is a promoter region that is incomplete or partially functional. It may lack certain essential elements or contain mutations that impair its function. Therefore, compared to a complete promoter, a partial promoter may have reduced or impaired activity in driving gene expression.

[0230] In some embodiments described herein, the liver-specific promoter is an AAT promoter that restricts AAT(M) expression to the liver region or reduces AAT(M) expression in organs other than the liver. In further embodiments, the liver-specific promoter provides high and sustained levels of AAT(M) expression, particularly relative to AAT(M) expression levels controlled by a CMV promoter. In one embodiment, the genetic construct comprises a liver-specific promoter or a functional variant thereof having at least 80% sequence identity with SEQ ID NO: 587 (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 587, or a nucleic acid capable of hybridizing to the complementary sequence of SEQ ID NO: 587 under strict hybridization conditions).

[0231] CpG site

[0232] It is also known in the art that promoters can be side-attached to other specific DNA sequences to enhance or degrade transgene expression. For example, short, scattered DNA sequences that deviate significantly from the average genomic pattern (e.g., CpG sites) can be used to control promoter function. (Deaton AM, Bird A) Genes Dev. 2011;25(10):1010-1022.

[0233] "CpG" refers to a specific DNA nucleotide sequence consisting of cytosine (C) followed by guanine (G) and linked by a phosphate group (p). CpG sites usually exist in clusters, called CpG islands, which are DNA regions containing high-frequency CpG dinucleotides.

[0234] In genetic engineering, CpG sites hold particular significance in gene expression and regulation. CpG sites (including CpG islands) are typically located near gene promoters and can influence gene expression. Normally, DNA methylation occurs at CpG sites, specifically by adding a methyl group (CH3) to a cytosine residue. Methylation of CpG sites in gene promoters is often associated with gene silencing or reduced gene expression. The presence of methyl groups can interfere with the binding of transcription factors and other proteins required for gene transcription, leading to decreased gene activity. Therefore, gene expression can be regulated by manipulating CpG sites. For example, removing or deleting CpG sites can be used to promote the expression of genes of interest.

[0235] In some embodiments, the genetic construct of the present invention is modified to reduce the presence of CpG sites. For example, any region of any vector disclosed herein may be a CpG-reduced form (e.g., backbone portion, promoter, untranslated region, miRNA, and nucleic acid encoding AAT(M)). In some embodiments, the GC44 vector containing the genetic construct of the present invention, the GC45 vector containing the genetic construct of the present invention, or the GC46 vector containing the genetic construct of the present invention are CpG-reduced forms. In one embodiment, the promoter in the genetic construct is modified to reduce the presence of CpG sites. In a further embodiment, the promoter is a liver-specific promoter with reduced CpG content. In one embodiment, the CpG-reducing liver-specific promoter of the genetic construct comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 594 or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 594).

[0236] In addition to promoting gene expression by manipulating CpG sites (including CpG islands) near gene promoters, reducing CpG sites also helps reduce potential immune activation. Unlike mammalian CpGs, which are typically methylated, fewer in number, and more concentrated in / near promoters, CpGs in plasmids or viral vectors are typically more numerous and unmethylated. Unmethylated CpGs in viral vectors can be recognized by TLR9 in cells, thereby activating an immune response against the vector DNA. Therefore, in the context of this invention, CpGs in genetic constructs can be reduced to decrease potential undesirable immune activation.

[0237] In some embodiments, the previously disclosed miRNA tolerance gene encoding AAT(M) is further modified to reduce its CpG content. In one embodiment, the CpG-reduced form of the miRNA tolerance AAT(M) gene variant comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 596 or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 596), or a codon degenerate variant of SEQ ID NO: 596.

[0238] In some embodiments, the 5'UTR of the genetic construct disclosed herein is modified to reduce its CpG content. In one embodiment, the CpG-reduced form of the 5'UTR comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 595 or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 595), or a codon degenerate variant of SEQ ID NO: 595.

[0239] This invention relates in part to a deoxyribonucleic acid comprising the above-described genetic construct.

[0240] V. Carriers and Delivery Systems

[0241] The ribonucleic acid and / or deoxyribonucleic acid of the present invention can be delivered to cells via long oligonucleotides and then inserted into specific genomic locations. In some embodiments, the ribonucleic acid and / or deoxyribonucleic acid of the present invention can be integrated into the cellular genome using gene editing systems utilizing CRISPR, TALEN, or zinc finger nucleases.

[0242] The polynucleotides of the present invention can be delivered to target cells via any suitable delivery system, including non-viral and viral delivery systems. Therefore, the present invention also relates in part to a vector comprising the ribonucleic acid or deoxyribonucleic acid of the present invention.

[0243] Any vector known in the art for delivering ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) can be used to carry out the present invention. In some embodiments, the vector is a plasmid, a small circular DNA, a nanoplasmid, a viral vector, an episome vector, or a non-viral vector. Examples of viral vectors that can be used in the present invention include lentiviral vectors, retroviral vectors, adenovirus (Ad) vectors, adeno-associated virus (AAV) vectors (e.g., AAV1, AAV2, AAV7, AAV8, or AAV9), herpes simplex virus (HSV) vectors, and baculovirus vectors. More specifically, examples of adenovirus vectors that can be used in the present invention include at least GC44, GC45, GC46, Ad14, Ad35, Ad41, Ad28, Ad26, and Ad5. Examples of non-viral vectors that can be used in the present invention include plasmids, lipid-based vectors, polymer-based vectors, peptide-based vectors, nanoparticles, and Sleeping Beauty transposons. In some embodiments, the vector may contain a sequence for serine recombinase-mediated integration (e.g., aatP or attB sites). When the vector is a plasmid, a small circular DNA, or a nanoparticle, the plasmid, small circular DNA, or nanoparticle may also contain a bacterial origin of replication, such as an origin of replication from the ColE1 plasmid.

[0244] One example of a non-viral vector for delivering the deoxyribonucleic acid or ribonucleic acid of the present invention is a lipid formulation. Any lipid formulation known in the art for delivering such nucleic acids can be used to carry out the present invention. In some embodiments, the nucleic acid may be associated with lipids. For example, the nucleic acid may be encapsulated within the aqueous phase of liposomes, embedded in the lipid bilayer of liposomes, attached to liposomes by linkers associated with both liposomes and oligonucleotides, embedded in liposomes, forming a complex with liposomes, dispersed in a lipid-containing solution, mixed with lipids, bound to lipids, present as a suspension in lipids, contained in micelles or forming a complex with micelles, or otherwise associated with lipids.

[0245] Another example of a non-viral vector is a transposon. Any transposon known in the art for delivering deoxyribonucleic acid or ribonucleic acid can be used to implement this invention. When using a transposon to deliver nucleic acid, a transposase or nucleic acid encoding the enzyme is typically also delivered to the cell. A transposon is an enzyme that binds to a transposon and catalyzes its integration into the cellular genome. In some embodiments, the vector is a Sleeping Beauty transposon. When used, a Sleeping Beauty transposon or a functional fragment or variant thereof, or a nucleic acid encoding the enzyme, is also delivered to the cell. Examples of such transposases include, but are not limited to, SB10, SB11, SB100x, and SB110 transposases. The Sleeping Beauty transposon system is known in the art, for example, as described in U.S. Patent Nos. 6,489,458 and 8,227,432.

[0246] Any viral vector known in the art for delivering deoxyribonucleic acid or ribonucleic acid can be used to carry out the present invention. Examples of such vectors include, but are not limited to, adenovirus vectors (e.g., the adenovirus-based Per.C6 system provided by Crucell (Leyden, Netherlands), adeno-associated virus-based vectors, lentivirus-based vectors (e.g., the lentivirus-based pLPI provided by Life Technologies (Carlsbad, California), retroviral vectors (e.g., pFB-ERV plus pCFB-EGSH), and herpesvirus-based vectors.

[0247] In one embodiment, the viral vector is an adenovirus vector.

[0248] Chimpanzee adenovirus vector

[0249] In a preferred embodiment, the adenovirus vector is derived from chimpanzee adenovirus, such as GC44, GC45, or GC46 adenovirus. In some such embodiments, the adenovirus vector is derived from GC44. In other embodiments, the adenovirus vector is derived from GC45.

[0250] In some embodiments, the adenovirus vector is derived from GC44 and comprises a nucleic acid sequence having at least about 80% sequence identity with the sequence disclosed in U.S. Patent No. 9,233,153, or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence disclosed in U.S. Patent No. 9,233,153, or a conserved substitution variant of the sequence disclosed in U.S. Patent No. 9,233,153).

[0251] In some embodiments, the adenovirus vector is derived from GC45 and comprises a nucleic acid sequence having at least about 80% sequence identity with the sequence disclosed in U.S. Patent No. 9,629,906, or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence disclosed in U.S. Patent No. 9,629,906, or a conserved substitution variant of the sequence disclosed in U.S. Patent No. 9,629,906).

[0252] In some embodiments, the adenovirus vector is derived from GC46 and comprises a nucleic acid sequence having at least about 80% sequence identity with the sequence disclosed in U.S. Patent No. 9,617,560, or a functional variant thereof (e.g., a nucleic acid having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence disclosed in U.S. Patent No. 9,617,560, or a conserved substitution variant of the sequence disclosed in U.S. Patent No. 9,617,560).

[0253] Gorilla adenoviruses are closely related to human adenoviruses, but existing immunity against gorilla adenoviruses (such as GC44, GC45, or GC46) is rare and weak in the population. For example, the seropositivity rate for newly isolated GC44, GC45, or GC46 is 6% in the US population, compared to 57% for human adenovirus type 5 (Ad5). Therefore, gorilla adenoviruses retain the advantages of human adenoviruses, including the ability to delete gene regions to ensure replication defects and allow insertion of transgenes of interest, but without being recognized by human serum from healthy donors. Hollingdale MR, Sedegah M, Limbach K: Development of replication-deficient adenovirus malaria vaccines. Expert Rev Vaccines 2017, 16(3):261-271.

[0254] GC44, GC45, and GC46 are newly isolated and unique chimpanzee adenovirus strains, obtained from fecal samples of healthy African gorillas. Based on hexon, DNA polymerase, and exon 4 ORF6 protein sequence comparisons, these three chimpanzee adenoviruses are phylogenetically closely related to and cluster with human type C adenoviruses. (Duncan et al.) Virology , 444:119–123 (2013). In the US population, the seroprevalence of all three chimpanzee adenovirus types is less than approximately 6%. In contrast, the seroprevalence of Ad5 is approximately 57%, with most seropositive individuals having high titers (IC90 greater than 200). Johnson et al., Molecular Therapy , 22:196–205 (2014). Therefore, existing neutralizing activity against GC44, GC45, or GC46 is rare and weak in the US population compared to conventional adenovirus treatments based on Ad5 serotypes. Furthermore, comparative studies of human serum samples from sub-Saharan Africa have confirmed rare and weak neutralizing activity in the population. These data suggest that existing neutralizing activity against GC44, GC45, or GC46 does not significantly interfere with molecular vaccines and therapeutics constructed based on one of these chimpanzee adenovirus platforms, making chimpanzee adenoviruses GC44, GC45, and GC46 highly suitable as backbone viral vectors.

[0255] In particular, adenovirus vectors with deletions in the E1 and / or E4 regions may have advantages in terms of safety and efficacy. (Gao et al., Journal of Virology, 70:8934-8943 (1996)). Therefore, in some embodiments, the adenovirus vectors described herein are engineered to delete part or all of the E1 and / or E4 regions.

[0256] In some embodiments, the adenovirus vector is an engineered chimpanzee adenovirus vector that has partially or completely deleted the E1 and / or E4 regions. For example, deletions in the E1 region can render the adenovirus vector replication-deficient and include bases 459 to 3411, thereby deleting the E1A and E1B promoters and open reading frames. For example, deletions in the E4 region may include bases 34144 to 36824 and remove all E4 open reading frames (ORFs), thereby eliminating key elements required for chimpanzee adenovirus replication. (The chimpanzee adenovirus coordinates provided herein are based on a wild-type adenovirus genome size of 37,213 base pairs.)

[0257] Modified adenovirus vector backbones that delete part or all of the E1 and / or E4 regions offer several advantages. One advantage is that the extended deletion of the adenovirus genome provides a higher payload capacity for the adenovirus vector. A second advantage is the reduced risk of generating replicating adenovirus (RCA) during adenovirus vector production. A third advantage is that eliminating E1 and E4 expression products facilitates further silencing of other regions of the viral genome.

[0258] Therefore, in one aspect described herein, the chimpanzee adenovirus vector described herein deletes the E1 region or a portion thereof. In another aspect, the chimpanzee adenovirus vector described herein deletes the E4 region or a portion thereof. In yet another aspect, the chimpanzee adenovirus vector described herein deletes both the E1 and E4 regions or portions thereof. In one aspect, the deletion length in the E1 and / or E4 regions is approximately 1,500 to approximately 3,500 base pairs (bp) compared to the wild type. In yet another aspect, the deletion length in the E1 and / or E4 regions is approximately 3,000 bp compared to the wild type.

[0259] In some implementations, the deletion of the E4 region removes all predicted open reading frames (ORFs) therein. To avoid potentially low yields when producing adenoviral vectors with an E4 deletion, a spacer sequence may be inserted within the E4 deletion region to prevent any transcription that might be initiated by the retained E4 promoter. In one aspect, the chimpanzee adenoviral vector described herein includes a spacer sequence inserted at the location of the deleted portion of the E4 region. In one aspect, the spacer sequence includes a bovine growth hormone polyadenylation (BGH polyA) signal sequence inserted to replace the deleted E4 ORF, but any suitable spacer sequence may also be used. In some aspects, the spacer sequence is about 10 to about 500 base pairs (bp) in length. For example, the length of this interval subsequence can be approximately 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150, approximately 160, approximately 170, approximately 180, approximately 190, approximately 200, approximately 210, approximately 220, approximately 230, approximately 240, and so on. 250, approximately 260, approximately 270, approximately 280, approximately 290, approximately 300, approximately 310, approximately 320, approximately 330, approximately 340, approximately 350, approximately 360, approximately 370, approximately 380, approximately 390, approximately 400, approximately 410, approximately 420, approximately 430, approximately 440, approximately 450, approximately 460, approximately 470, approximately 480, approximately 490, or approximately 500 bp. Alternatively, compared to the wild type, the length of the spacer sequence can be approximately 50 bp to approximately 100 bp, approximately 100 bp to approximately 150 bp, approximately 150 bp to approximately 200 bp, approximately 200 bp to approximately 250 bp, approximately 250 bp to approximately 300 bp, approximately 300 bp to approximately 350 bp, approximately 350 bp to approximately 400 bp, approximately 400 bp to approximately 450 bp, or approximately 450 bp to approximately 500 bp. The spacer can also be of any length within these ranges. For example, the length of the spacer can be approximately 250 bp to approximately 350 bp, approximately 260 bp to approximately 340 bp, approximately 270 bp to approximately 330 bp, approximately 280 bp to approximately 320 bp, or approximately 290 bp to approximately 310 bp. In one aspect, the length of the spacer sequence is approximately 300 base pairs. In another aspect, the spacer sequence is 278 bp.

[0260] In another aspect, compared to the wild type, the spacer sequence is located at approximately 34,700 to 35,000 base pairs in the vector genome. In yet another aspect, compared to the wild type, the spacer sequence is located at approximately 34,692 to 34,969 base pairs in the vector genome.

[0261] In some embodiments, the vector deletes the entire E1 and E4 regions. In some such embodiments, either the E1 or E4 region is replaced by a genetic construct containing a transgene or a spacer. In some such embodiments, the E1 region is replaced by the genetic construct. In some embodiments, the E4 region is replaced by the genetic construct. In some embodiments, both the E1 and E4 regions are replaced by the genetic construct. In some embodiments, the E1 region is replaced by the genetic construct, while the E4 region is replaced by a spacer.

[0262] On the other hand, the vector has at least a portion of the E2 and / or E3 regions deleted. In some embodiments, the deletion of at least a portion of the E2 and / or E3 regions increases the vector's cloning space and production efficiency.

[0263] In one embodiment, the vector has at least a portion of E1, E3, and / or E4 deleted. In some embodiments, at least a portion of the E2 and / or E3 regions is missing, increasing the vector's cloning space and / or production efficiency. In some embodiments, the E2 and / or E3 regions are replaced by the genetic construct of the present invention.

[0264] In one embodiment, the viral vector is a lentiviral vector. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they enable the long-term stable integration of transgenes and their amplification in daughter cells. Lentiviral vectors have additional advantages over vectors derived from tumor retroviruses (e.g., murine leukemia virus) because they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0265] To assess the expression of one or more miRNAs or portions thereof described herein, the expression vector introduced into cells may also contain a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected by a viral or non-viral vector. In other aspects, the selection marker may be carried on another DNA segment and used during co-transfection. Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences to ensure their expression in host cells. Useful selection markers include, for example, antibiotic resistance genes, such as the neomycin resistance gene (neo) and the ampicillin resistance gene. In some embodiments, a truncated epidermal growth factor receptor (HER1t or HER1t-1) tag may be used as the selection marker gene.

[0266] Reporter genes can be used to identify cells that may have been transfected and to assess the function of regulatory sequences. Typically, a reporter gene is a gene that is absent or not expressed in the recipient organism or tissue, and the expression of its encoded polypeptide is indicated by some easily detectable property, such as enzyme activity. Reporter gene expression is detected at an appropriate time after DNA introduction into the recipient cell. Suitable reporter genes include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., FEBS Letters 479: 79-82 (2000)). Suitable expression systems are known in the art and can be prepared using known techniques or are commercially available. Typically, a construct with the smallest 5' flanking region and exhibiting the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to the reporter gene and used to assess the ability of regulators to control the promoter-driven transcription.

[0267] VI. Methods for introducing miRNA into cells

[0268] This invention relates in part to a method for modifying gene expression in cells, wherein the method includes introducing the ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) of this invention into the cells. This invention also relates in part to the use of the RNA or DNA of this invention in the preparation of medicaments for modifying gene expression.

[0269] The present invention also relates in part to a method for producing genetically engineered cells, wherein the method includes introducing the ribonucleic acid or deoxyribonucleic acid of the present invention into the cells.

[0270] In some embodiments of the above method, the method includes transfecting cells with the ribonucleic acid or deoxyribonucleic acid of the present invention. In some embodiments, transfection involves electroporation.

[0271] In some embodiments, the deoxyribonucleic acid (DNA) may contain transposons, such as the Sleeping Beauty transposon. In embodiments using the Sleeping Beauty transposon, the Sleeping Beauty transposase or a functional fragment or variant thereof, or the nucleic acid encoding it, may be introduced into the cells. In some embodiments of cell transfection with transposons, the method further includes transfecting the cells with a vector encoding the transposon.

[0272] In some embodiments of the above methods, cells are transduced using the ribonucleic acid or deoxyribonucleic acid of the present invention. Cells can also be transduced using viral vectors containing this ribonucleic acid or deoxyribonucleic acid.

[0273] Methods for introducing and expressing genes in cells are known in the art. In the case of expression vectors, the vectors can be readily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method known in the art. For example, the vectors can be transferred into cells by physical, chemical, or biological means.

[0274] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, and electroporation. Methods for generating cells containing vectors and / or exogenous nucleic acids are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001). In some embodiments, the method for introducing polynucleotides into host cells is calcium phosphate transfection or polyethyleneimine (PEI) transfection. In some embodiments, the method for introducing polynucleotides into host cells is electroporation.

[0275] Chemical methods for delivering polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a carrier for in vitro and in vivo delivery is a liposome (e.g., an artificial membrane vesicle).

[0276] As a biological method, the ribonucleic acid or deoxyribonucleic acid of the present invention can be introduced into cells, for example, using a virus-based delivery system. Representative viral expression vectors include, but are not limited to, adenovirus vectors (e.g., the Per.C6 adenovirus vector system provided by Crucell Ltd. (Leiden, Netherlands), adeno-associated virus vectors, lentiviral vectors (e.g., the pLPI lentiviral vector provided by Life Technologies (Carlsbad, California, USA), retroviral vectors (e.g., pFB-ERV plus pCFB-EGSH), and herpesvirus vectors. In one embodiment, the viral vector is a lentiviral vector. Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer because they allow for the long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from tumor retroviruses (e.g., mouse leukemia virus) because they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. Typically, in implementations, a suitable vector includes a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent 6,326,193).

[0277] Regardless of the method used to introduce exogenous nucleic acids into host cells, various tests can be performed to confirm the presence of recombinant DNA sequences in the host cells. These tests include, for example, "molecular biology" tests known to those skilled in the art, such as Southern blotting, Northern blotting, RT-PCR, and PCR, as well as "biochemical" tests, such as those using immunological methods (ELISA and Western blotting) to detect the presence or absence of specific peptides.

[0278] VIII. Kits and Compositions

[0279] This invention relates in part to a kit or composition comprising the ribonucleic acid or deoxyribonucleic acid of the present invention. In some embodiments, the kit or composition comprises the genetic constructs and / or vectors of the present invention. In some embodiments, the kit comprises the compositions of the present invention.

[0280] This invention also relates in part to kits or compositions as described above for modifying gene expression. This invention also relates in part to kits or compositions as described above for treating a disease or condition of a subject, or for producing a medicament for treating a disease or condition of a subject.

[0281] In some embodiments, the kit or composition contains a transposase.

[0282] In some embodiments, the kit or composition contains a gene switch component, such as the RHEOSWITCH® gene switch component.

[0283] In some embodiments, the composition further comprises a carrier, a diluent, and / or an excipient. Any carrier, diluent, or excipient known in the art for use with nucleic acids, vectors, or cells may be used to implement this invention. For example, the compositions of this invention may comprise: a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, dextran, or mannitol; proteins; peptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0284] In some implementations, the kit includes a carrier, packaging, label, container, or instructions for use. Suitable containers include, for example, bottles, vials, syringes, and test tubes.

[0285] IX. Treatment Methods

[0286] Any genetic construct disclosed herein can be used to treat subjects (e.g., humans or other mammals) suffering from diseases or conditions from which they can benefit from receiving the construct. In a preferred embodiment, as detailed in the foregoing disclosure, the genetic construct of the present invention comprises at least the following: (i) a tissue-specific promoter (e.g., a liver-specific promoter); (ii) a 5'UTR; (iii) any AAT miRNA design disclosed herein; (iv) a silencer / miRNA-tolerant AAT(M) transgene; and (v) a 3'UTR. The genetic construct can be delivered to the subject via a vector, preferably a chimpanzee adenovirus vector.

[0287] Alpha-1 antitrypsin deficiency can lead to a variety of diseases, including lung or liver diseases. Furthermore, diabetes (type 1 and type 2) and cystic fibrosis are also associated with AAT deficiency. See Kim M, Cai Q, Oh Y. Therapeutic potential of alpha-1 antitrypsin in human disease. Ann Pediatr Endocrinol Metab. Sep 2018;23(3):131-135. The genetic construct described herein can be given to subjects to help treat any of the aforementioned diseases associated with AAT deficiency attributable to the pathogenic SERPINA1 allele.

[0288] Lung diseases

[0289] Chronic obstructive pulmonary disease (COPD), particularly emphysema and / or chronic bronchitis, is the most common clinical manifestation of acute arterial disease-asthmatic bronchitis (AATD). Bronchiectasis is also associated with AATD. Individuals with severe AATD may exhibit common signs and symptoms of obstructive pulmonary disease, asthma, and chronic bronchitis (e.g., dyspnea, cough, wheezing, and sputum production). Evidence of bronchiectasis is visible on chest CT scans in most patients with severe AATD.

[0290] Liver disease

[0291] The most common manifestation of AATD-related liver disease in childhood is neonatal cholestasis: jaundice, accompanied by hyperbilirubinemia and elevated serum aminotransferase levels, appears in the first few days to months of life. Adult liver disease (manifesting as cirrhosis and fibrosis) can occur without a history of neonatal or childhood liver disease. Individuals with AATD also have a risk of developing hepatocellular carcinoma (HCC). Those with AATD and PI... Individuals with the ZZ genotype have a risk of developing HCC that is several times higher than the risk typically associated with cirrhosis. This increased risk is thought to be attributed to the failure of apoptosis caused by the retained mutant AAT protein in damaged cells, thereby sending a continuous regenerative signal to hepatocytes containing a lower load of retained mutant AAT protein.

[0292] Genotype-phenotype correlation

[0293] The risk of lung and / or liver disease associated with the following SERPINA1 genotypes is summarized in the table below. (Stoller JK et al., Alpha-1 Antitrypsin Deficiency.) Gene Review (2006). The SERPINA1 alleles in the table below are prefixed with PI. (protease inhibitors) The gene is named as an alias. For example, PI. The MM genotype indicates that the individual possesses two copies of the AAT(M) allele and is considered homozygous for the AAT(M) allele. PI The MZ genotype indicates that the individual has one copy of the AAT(M) allele and one copy of the AAT(Z) allele, and is considered a heterozygote of the AAT(M) and AAT(Z) alleles.

[0294]

[0295] Diabetes (Type 1 and Type 2)

[0296] Type 2 diabetes is a metabolic, chronic inflammatory disease. Diabetic inflammation leads to local and systemic insulin resistance, resulting in elevated circulating glucose levels. To compensate for the increased insulin resistance, functional β-cell populations initially proliferate to induce hyperinsulinemia. Subsequently, local and systemic inflammatory responses disrupt the intrinsic β-cell populations within the islets of Langerhans. Blocking the inflammatory pathway can restore insulin response. The mechanism of action of astatin A in type 2 diabetes is not yet fully understood, but it is known that AAT protects pancreatic β-cells from apoptosis by inhibiting caspase-3. Recent studies have shown that the proportion of adults with diabetes who have lower AAT levels (1.0 mg / mL or lower) is 50% higher than in non-diabetic individuals. Administration of AAT to patients with type 2 diabetes may reduce disease severity.

[0297] Type 1 diabetes, also known as juvenile diabetes or insulin-dependent diabetes mellitus, is a progressive disease caused by the production of little or no insulin by pancreatic beta cells. It can be caused by a variety of factors, including genetics, infection, or the destruction of pancreatic beta cells by autoreactive T cells. Control of blood glucose levels and reduction of diabetic complications are closely related to the protection of pancreatic beta cells. Despite decades of in-depth research, a definitive cure for this disease remains undetermined. However, recent studies on AAT suggest that controlling inflammation and immune responses by downregulating interleukin (IL)-1β and other pro-inflammatory cytokines helps maintain pancreatic beta cell function. IL-1β is known to have a damaging effect on insulin-producing cells. Although circulating AAT levels in patients with type 1 diabetes may be within the normal range, AAT function is impaired due to extensive non-enzymatic glycation, suggesting that functional AAT levels may play a role in disease progression. In non-obese diabetic (NOD) mouse models (i.e., autoimmune animal models of type 1 diabetes), serum AAT levels are only half that of most wild-type mice. NOD mice returned to normal blood glucose levels after 14 days of AAT treatment, while NOD mice overexpressing AAT showed reduced islet inflammation and no hyperglycemia. Furthermore, administration of clinical-grade human AAT to chemically induced diabetic mice promoted islet allogeneic transplant survival and produced cytoprotective effects. AAT treatment (80 mg / kg / dose) has been reported to have beneficial effects on β-cell function in adult patients with type 1 diabetes. Another study reported that AAT treatment was feasible in children recently diagnosed with autoimmune diabetes during a 37-week study, without serious adverse complications, and improved glycemic control and peak serum C-peptide levels.

[0298] Cystic fibrosis

[0299] Cystic fibrosis (CF) is caused by mutations in CF transmembrane transport regulators and progresses from childhood. CF patients experience a variety of pulmonary symptoms, such as thick mucus production, chronic airway infections, and inflammation, which lead to decreased lung function and early death. In CF lungs, the number of neutrophils is higher than in healthy lungs, and the secreted NE disrupts the lung's defense mechanisms against infection and inflammation. Therefore, in CF, treatment focuses on reducing neutrophil overactivation and counteracting the effects of NE on the lungs. To inhibit NE in the lungs during CF progression, early studies primarily focused on increasing systemic AAT levels through intravenous injection. Recently, a randomized, double-blind, placebo-controlled phase 2a study was conducted in CF patients to evaluate the safety of once-daily inhalation of 100 mg or 200 mg of AAT for 3 weeks in 30 adult subjects, and the inhaled administration was reported to be safe and well-tolerated. Further studies have shown that inhaled AAT can control neutrophil function, NE levels, and lung inflammation in a dose-dependent manner. However, there are still many obstacles to the application of AAT in CF because the observed results are inconsistent depending on the device used and the patient's lung condition and NE concentration.

[0300] In some embodiments, the genetic construct (including a vector containing the genetic construct) can be used to treat subjects suffering from conditions, diseases, or disorders that can be benefited by reducing or inhibiting AATD, such as liver disease, lung disease, cystic fibrosis, or diabetes. In some embodiments, the disease is a lung disease or liver disease. In a further embodiment, the lung disease or liver disease is attributed to α-1 antitrypsin deficiency. In some embodiments, a therapeutically effective amount of the genetic construct is administered to the subject. The subject can be an adult, adolescent, child, or infant. Pharmaceutical compositions containing the genetic construct can be used to provide methods of treating diseases such as AATD. Such methods include administering the pharmaceutical compositions described herein to humans or animals. Furthermore, such methods can be carried out by any means known in the art, including aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The nucleic acids, vectors, cells, or compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intralymphaticly, intramedullaryly, intramuscularly, intravenously (iv), or intraperitoneally. In some embodiments, intravenous injection can be performed via a peripheral vein, portal vein, or hepatic artery. In some embodiments, the genetic construct (including any pharmaceutical composition comprising the genetic construct) is administered once monthly via intravenous injection. In another embodiment, the genetic construct (including any pharmaceutical composition comprising the genetic construct) is administered once daily via intravenous injection.

[0301] The dosage of the above treatments administered to patients will vary depending on the specific nature of the disease being treated and the individual receiving the treatment. Dosage conversions for human administration can be performed according to accepted practices in the field. Appropriate dosages may be adjusted accordingly for adult or pediatric patients.

[0302] Dosage values ​​may vary depending on the type and severity of the disease requiring relief. It should be understood that, for any particular subject, the specific dosing regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges described herein are merely exemplary and not intended to limit the scope or implementation of the claimed compositions.

[0303] The effectiveness of treatment or prevention can be monitored by regularly assessing patients receiving treatment. For repeated administration over several days or longer, treatment may be repeated until the desired symptom suppression is achieved, depending on the disease condition. However, other dosing regimens may also be useful and fall within the scope of this invention. The required dose can be delivered by a single bolus injection, multiple bolus injections, or continuous infusion of the composition.

[0304] In some embodiments, the genetic construct described herein is used to treat subjects suffering from AATD, including symptoms, diseases, or disorders associated with AATD. AATD-related liver diseases or disorders include, but are not limited to, chronic hepatitis, cirrhosis, hepatocellular carcinoma, tonsillitis, cholestasis, fibrosis, and fulminant hepatic failure. According to some embodiments, the genetic construct is used to treat at least one symptom in subjects with AATD. A therapeutically effective amount of the genetic construct is administered to the subject.

[0305] In some embodiments, the present invention provides a method for treating a patient’s disease or disorder (e.g., AATD), wherein the method includes administering to the patient any of the genetic constructs, vectors, or compositions described herein.

[0306] In some embodiments, the genetic constructs described herein are used to treat or manage the clinical symptoms of a subject suffering from AATTD liver or lung disease or disorder. A therapeutically effective amount of the composition comprising the genetic constructs described herein is administered to the subject. According to some embodiments, the method includes administering the composition comprising the genetic constructs described herein to the subject to be treated.

[0307] In some embodiments, the object has an AAT(Z) variant. In some embodiments, the object has multiple (e.g., 2) copies of the AAT(Z) variant. For example, in some embodiments, the object is homozygous for the AAT(Z) variant. In some embodiments, the object has an AAT(S) variant. In some embodiments, the object has multiple (e.g., 2) copies of the AAT(S) variant. For example, in some embodiments, the object is homozygous for the AAT(S) variant. In some embodiments, the object has an AAT(F) variant. In some embodiments, the object has multiple (e.g., 2) copies of the AAT(F) variant. For example, in some embodiments, the object is homozygous for the AAT(F) variant. In some embodiments, the object has an AAT(I) variant. In some embodiments, the object has multiple (e.g., 2) copies of the AAT(I) variant. For example, in some embodiments, the object is homozygous for the AAT(I) variant. In some embodiments, the object has an allele associated with intrahepatic inclusion bodies (e.g., M... malton S iiyama In some embodiments, the subject has multiple (e.g., two) copies of the AAT allele containing intrahepatic inclusion bodies. For example, in some embodiments, the subject is homozygous for the AAT allele containing intrahepatic inclusion bodies. In some embodiments, the subject has multiple AAT allele variants. For example, in some embodiments, the subject has both an AAT(M) variant and an AAT(Z) variant. In some embodiments, the subject is heterozygous for both the AAT(M) variant and the AAT(Z) variant. In some embodiments, the subject has a pathogenic allele that results in the absence of mRNA product or protein production. In some embodiments, the subject's serum AAT level is below about 57 mg / dL.

[0308] In some embodiments, the genetic construct is used to treat chronic obstructive pulmonary disease, including emphysema and / or chronic bronchitis. In some embodiments, the genetic construct is used to treat bronchiectasis. In some embodiments, the genetic construct is used to treat neonatal cholestasis. In some embodiments, the genetic construct is used to treat cirrhosis. In some embodiments, the genetic construct is used to treat panniculitis. In some embodiments, the genetic construct is used to treat granulomatous polyangiitis.

[0309] Dosage

[0310] Dosing regimens will vary depending on the subject's age, sex, and the type of active agent to be administered. Dosage can be administered hourly, daily, weekly, monthly, or yearly. Dosage can also be given as a single dose.

[0311] In some embodiments, the agent is delivered at intervals of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, the dosing intervals are approximately twice daily, approximately once daily, approximately twice weekly, approximately once weekly, approximately once every two weeks, approximately once every three weeks, approximately once every four weeks, or approximately once every five weeks. In some embodiments, the second dose is given approximately one week, two weeks, three weeks, four weeks, or five weeks after the first dose; the third dose is given approximately two weeks, three weeks, four weeks, five weeks, or six weeks after the second dose; and the fourth dose is given approximately three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or twelve weeks after the third dose. In one embodiment, the second dose is given approximately two weeks after the first dose, the third dose is given approximately six weeks after the second dose, and the fourth dose is given approximately twelve weeks after the third dose.

[0312] In some embodiments, the dosage of the genetic construct may be: about 200 PU / kg or at least about 300 PU / kg or at least about 400 PU / kg or at least about 500 PU / kg or at least about 600 PU / kg, at least about 700 PU / kg, at least about 800 PU / kg, at least about 900 PU / kg or at least about 1000 PU / kg, or at least about 1500 PU / kg, or at least about 2000 PU / kg or at least about 2500 PU / kg, or at least about 3000 PU / kg, or at least about 3500 PU / kg, or at least about 4000 PU / kg, or at least about 4500 PU / kg, or at least about 5000 PU / kg.

[0313] In other embodiments, the dosage of the genetic construct may be: about 60,000 PU / kg, or about 70,000 PU / kg, or about 80,000 PU / kg, or about 90,000 PU / kg, or about 100,000 PU / kg, or about 200,000 PU / kg, or about 300,000 PU / kg, or about 400,000 PU / kg, or about 500,000 PU / kg.

[0314] In other embodiments, the dose of the genetic construct may be approximately 1 × 10⁻⁶. 8 PU / kg to approximately 1×10 13 PU / kg (e.g., approximately 1×10) 9PU / kg to approximately 1×10 12 PU / kg, approximately 1×10 8 PU / kg to approximately 1×10 11 PU / kg or approximately 1×10 10 PU / kg to approximately 1×10 13 (PU / kg). The range may include, but is not limited to, any combination of the lower and upper limits discussed above.

[0315] It should also be understood that in some cases, the initial dose of the genetic construct may be increased beyond the above-mentioned upper limit to rapidly achieve the desired gene expression, protein and / or mRNA transcript levels, or in some cases, the initial dose may be lower than the optimal value.

[0316] In some embodiments, the dose is contained in a composition having a volume of about 0.1 to about 20 ml, about 0.1 to about 15 ml, about 0.1 to about 10 ml, about 0.1 to about 5 ml, about 0.1 to about 4 ml, about 0.1 to about 3 ml, about 0.1 to about 2 ml, about 0.25 to about 1.75 ml, about 0.5 to about 1.5 ml, about 0.75 to about 1.25 ml, or about 1.0 ml. In some embodiments, the dosage comprises volumes of about 0.1 ml, about 0.2 ml, about 0.3 ml, about 0.4 ml, about 0.5 ml, about 0.6 ml, about 0.7 ml, about 0.8 ml, about 0.9 ml, about 1.0 ml, about 1.2 ml, about 1.3 ml, about 1.4 ml, about 1.5 ml, about 1.6 ml, about 1.7 ml, about 1.8 ml, about 1.9 ml, about 2.0 ml, about 2.1 ml, about 2.2 ml, about 2.3 ml, about 2.4 ml, about 2.5 ml, about 2.6 ml, about 2.7 ml, about 2.8 ml, about 2.9 ml, about 3.0 ml, about 3.1 ml, about 3.2 ml, about 3.3 ml, about 3.4 ml, about 3.5 ml, about 3.6 ml, about 3.7 ml, about 3.8 ml, about 3.9 ml, and about 4.0 ml. In a composition of about 4.1 ml, about 4.2 ml, about 4.3 ml, about 4.4 ml, about 4.5 ml, about 4.6 ml, about 4.7 ml, about 4.8 ml, about 4.9 ml or about 5.0 ml.

[0317] In some embodiments, the dose is contained in a composition having a volume of less than about 1 ml, about 1 ml, about 2 ml, about 3 ml, about 4 ml, 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 11 ml, about 12 ml, about 13 ml, about 14 ml, about 15 ml, about 16 ml, about 17 ml, about 18 ml, about 19 ml, about 20 ml, or greater than about 20 ml.

[0318] In some embodiments, the dose is contained in a composition having a volume of less than about 10 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 95 ml, about 100 ml, about 105 ml, about 110 ml, about 120 ml, about 130 ml, about 140 ml, about 150 ml, or greater than about 150 ml.

[0319] In some embodiments, the dose in one dose may contain about 1 × 10 5 Approximately 4×10 14 One virus particle, approximately 1×10 7 To approximately 1×10 12 One virus particle, approximately 1×10 8 To approximately 1×10 11 One virus particle, approximately 3 × 10 8 Approximately 3×10 11 One virus particle, approximately 1×10 9 To approximately 1×10 12 One virus particle, approximately 1×10 9 To approximately 1×10 11 One virus particle, approximately 1×10 9 To approximately 1×10 10 One virus particle, approximately 1×10 10 To approximately 1×10 12 One virus particle, approximately 1×10 12 To approximately 1×10 14 One virus particle, approximately 2 × 10 12 Approximately 2×10 14 One virus particle, approximately 1×10 13 Approximately 3×10 13 One virus particle, approximately 3 × 10 12 Approximately 3×10 14 One virus particle or approximately 4 × 10 12Approximately 4×10 14 A virus particle.

[0320] In some implementations, for example, one dose of the genetic construct may be about 0.1 × 10⁻⁶. 9 Approximately 10×10 12 One virus particle, approximately 0.5 × 10⁻⁶ 9 Approximately 9×10 12 One virus particle, approximately 0.5 × 10⁻⁶ 9 Approximately 8×10 12 One virus particle, approximately 0.5 × 10⁻⁶ 9 Approximately 7×10 12 One virus particle, approximately 0.5 × 10⁻⁶ 9 Approximately 6×10 12 One virus particle, approximately 0.5 × 10⁻⁶ 9 Each virus particle is approximately 5 × 10⁶ 10 One virus particle, approximately 0.1 × 10⁻⁶ 10 One virus particle is approximately 10 × 10 11 One virus particle, approximately 0.5 × 10⁻⁶ 10 Approximately 9×10 11 One virus particle, approximately 0.5 × 10⁻⁶ 10 Approximately 8×10 11 One virus particle, approximately 0.5 × 10⁻⁶ 10 Approximately 7×10 11 One virus particle, approximately 0.5 × 10⁻⁶ 10 Approximately 6×10 11 One virus particle, approximately 0.5 × 10⁻⁶ 10 Each virus particle is approximately 5 × 10⁶ 11 One virus particle, approximately 0.1 × 10⁻⁶ 11 Approximately 10×10 11 One virus particle, approximately 0.5 × 10⁻⁶ 11 To approximately 9.0 × 10 11 One virus particle, approximately 0.5 × 10⁻⁶ 11 To approximately 8.0 × 10 11 One virus particle, approximately 0.5 × 10⁻⁶ 11 From approximately 7.0 × 10 11 One virus particle or approximately 0.5 × 10⁻⁶ 11 From approximately 6.0 × 10 11 One viral particle. For example, one dose of the vector can be about 0.1 × 10⁻⁶. 11 One virus particle, approximately 0.2 × 10⁻⁶ 11 One virus particle, approximately 0.3 × 10⁻⁶ 11 One virus particle, approximately 0.4 × 10⁻⁶ 11 One virus particle, approximately 0.5 × 10⁻⁶11 One virus particle, approximately 0.6 × 10⁻⁶ 11 One virus particle, approximately 0.7 × 10⁻⁶ 11 One virus particle, approximately 0.8 × 10⁻⁶ 11 One virus particle, approximately 0.9 × 10⁻⁶ 11 One virus particle, approximately 1.0 × 10⁻⁶ 11 One virus particle, approximately 0.1 × 10⁻⁶ 10 One virus particle, approximately 0.2 × 10⁻⁶ 10 One virus particle, approximately 0.3 × 10⁻⁶ 10 One virus particle, approximately 0.4 × 10⁻⁶ 10 One virus particle, approximately 0.5 × 10⁻⁶ 10 One virus particle, approximately 0.6 × 10⁻⁶ 10 One virus particle, approximately 0.7 × 10⁻⁶ 10 One virus particle, approximately 0.8 × 10⁻⁶ 10 One virus particle, approximately 0.9 × 10⁻⁶ 10 One virus particle, approximately 1.0 × 10⁻⁶ 10 One virus particle, approximately 0.1 × 10⁻⁶ 9 One virus particle, approximately 0.2 × 10⁻⁶ 9 One virus particle, approximately 0.3 × 10⁻⁶ 9 One virus particle, approximately 0.4 × 10⁻⁶ 9 One virus particle, approximately 0.5 × 10⁻⁶ 9 One virus particle, approximately 0.6 × 10⁻⁶ 9 One virus particle, approximately 0.7 × 10⁻⁶ 9 One virus particle, approximately 0.8 × 10⁻⁶ 9 One virus particle, approximately 0.9 × 10⁻⁶ 9 One virus particle or approximately 1.0 × 10⁻⁶ 9 A virus particle.

[0321] In some embodiments, the dosage in one dose may contain about 0.1 × 10⁻⁶. 9 Approximately 10×10 11 One virus particle, approximately 0.1 × 10⁻⁶ 9 From approximately 1.0 × 10 11 One virus particle, approximately 0.5 × 10⁻⁶ 9 To approximately 0.5 × 10 11 One virus particle, approximately 0.5 × 10⁻⁶ 9 To approximately 0.1 × 10 11 One virus particle, approximately 1.0 × 10⁻⁶ 10 Approximately 10×10 11 One virus particle, approximately 1.0 × 10⁻⁶ 10 To approximately 0.1 × 10 11 One virus particle, approximately 0.1 × 10⁻⁶11 Approximately 10×10 11 One virus particle, approximately 0.5 × 10⁻⁶ 11 Approximately 9×10 11 One virus particle, approximately 0.5 × 10⁻⁶ 11 Approximately 8×10 11 One virus particle, approximately 0.5 × 10⁻⁶ 11 Approximately 7×10 11 One virus particle, approximately 0.5 × 10⁻⁶ 11 Approximately 6×10 11 One virus particle, approximately 1×10 10 One virus particle or approximately 5 × 10 10 A virus particle.

[0322] In some implementations, for example, the dose may contain about 1 × 10⁻⁶. 6 PU, 2×10 6 PU, 4×10 6 PU, 1×10 7 PU, 2×10 7 PU, 4×10 7 PU, 1×10 8 PU, 2×10 8 PU, 3×10 8 PU, 4×10 8 PU, 1×10 9 PU, 2×10 9 PU, 3×10 9 PU, 4×10 9 PU, 1×10 10 PU, 2×10 10 PU, 3×10 10 PU, 4×10 10 PU, 1×10 11 PU, 2×10 11 PU, 3×10 11 PU, 4×10 11 PU, 1×10 12 PU, 2×10 12 PU, 3×10 12 PU, 4×10 12 PU, 1×10 13 PU, 2×10 13 PU, 3×10 13 PU, 4×10 13 PU, 1×10 14 PU, 2×10 14 PU, 3×10 14PU or 4×10 14 PU.

[0323] In some embodiments, the dose may contain about 1.0 × 10⁻⁶. 5 From approximately 1.0 × 10 10 Plaque forming units (PFU), for example, about 0.5 × 10⁻⁶. 5 To approximately 0.5 × 10 10 PFU, approximately 0.1 × 10 5 To approximately 0.1 × 10 10 PFU, approximately 1×10 6 To approximately 1×10 9 PFU, approximately 0.5 × 10 6 To approximately 0.5 × 10 9 PFU, approximately 0.1 × 10 6 To approximately 0.1 × 10 9 PFU, approximately 1×10 7 To approximately 1×10 8 PFU, approximately 0.5 × 10 7 To approximately 0.5 × 10 8 PFU, approximately 0.1 × 10 7 To approximately 0.1 × 10 8 PFU, approximately 1.0 × 10 6 From approximately 1.0 × 10 9 PFU, approximately 0.5 × 10 6 To approximately 0.5 × 10 9 PFU, approximately 1.0 × 10 7 To approximately 1×10 8 PFU, approximately 1.0 × 10 6 From approximately 1.0 × 10 8 PFU, approximately 0.5 × 10 6 To approximately 0.5 × 10 8 PFU or approximately 0.1 × 10 6 To approximately 0.1 × 10 8 PFU.

[0324] In some embodiments, the viral vector can be quantified by quantitative PCR (Q-PCR) or analytical HPLC.

[0325] Inhibit AAT(Z) expression

[0326] In some embodiments, after administration of the genetic construct described herein to a subject, the gene expression level of AAT(Z) and / or the AAT(Z) mRNA transcript level in the subject is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% compared to subjects before administration of the genetic construct or subjects that did not receive the genetic construct. The gene expression level of AAT(Z) and / or the AAT(Z) transcript level in the subject may be reduced in the subject's cells, cell populations, and / or tissues.

[0327] In some embodiments, after administration of the genetic construct described herein to a subject, the level of mutant AAT protein in the subject is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% compared to subjects before administration of the genetic construct or those who did not receive the genetic construct. The level of mutant AAT protein in the subject can be reduced in the subject's cells, cell populations, tissues, blood, and / or other body fluids. The reduction in AAT(Z) gene expression, AAT(Z) mRNA transcripts, or mutant AAT protein levels can be assessed by any method known in the art.

[0328] Enhance AAT(M) expression

[0329] In some embodiments, after administration of the genetic construct described herein to a subject, the gene expression level of AAT(M) and / or the AAT(M) mRNA transcript level in the subject is higher than in subjects who have not received the genetic construct. In some embodiments, after administration of the genetic construct described herein to a subject, the gene expression level of AAT(M) and / or the AAT(M) mRNA transcript level in the subject increases by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% relative to subjects who were not given the genetic construct or who did not receive the genetic construct. The gene expression level of AAT(M) and / or the AAT(M) transcript level in the subject may be increased in the subject's cells, cell populations, and / or tissues.

[0330] In some implementations, after administering the genetic construct described herein to a subject, the level of wild-type functional AAT protein in the subject increases by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% relative to the subject before or without the genetic construct. In other embodiments, after administration of the genetic construct described herein to a subject, the level of wild-type functional AAT protein in the subject increases by at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 times or greater than 10.0 times. The level of wild-type AAT protein in the subject can be increased in the subject's cells, cell populations, tissues, blood, and / or other body fluids. Increased levels of AAT(M) gene expression, AAT(M) mRNA transcripts, or wild-type AAT protein can be assessed by any method known in the art.

[0331] In some embodiments, after administration of the genetic construct described herein to a subject, the serum protein level of wild-type functional AAT in the subject is at least 11 µM (e.g., about 11 µM to about 65 µM, about 11 µM to about 53 µM, or about 20 µM to about 53 µM). In some embodiments, after administration of the genetic construct described herein to a subject, the serum protein level of wild-type functional AAT in the subject is about 11 µM to about 65 µM. In some embodiments, after administration of the genetic construct described herein to a subject, the serum protein level of wild-type functional AAT in the subject is about 11 µM to about 53 µM. In some embodiments, after administration of the genetic construct described herein to a subject, the serum protein level of wild-type functional AAT in the subject is about 20 µM to about 53 µM.

[0332] This invention also relates in part to the use of the genetic constructs and / or vectors described herein, or compositions comprising them, in the preparation of medicaments for treating diseases or conditions of persons in need. In a preferred embodiment, the genetic constructs described herein, or any vector comprising any genetic construct of this invention, or compositions comprising the nucleic acid or vector, are used to treat liver or lung diseases associated with α-1 antitrypsin deficiency.

[0333] Combination therapy

[0334] In some embodiments, compositions comprising the genetic constructs described herein may be administered in combination with another therapeutic agent as a combination therapy. Examples of such therapeutic agents include biologics and small molecules, such as immunosuppressants or autophagy enhancers.

[0335] In some embodiments, the additional therapeutic agent is an immunosuppressant. Immunosuppressants prevent or limit the overactivation of the immune system by reducing or inhibiting its activity. Examples of immunosuppressants include, but are not limited to, corticosteroids (e.g., prednisone, prednisolone, and budesonide), calcineurin inhibitors (e.g., cyclosporine and tacrolimus), mTOR inhibitors (e.g., sirolimus and everolimus), IMDH inhibitors (e.g., azathioprine, leflunomide, and mycophenolate mofetil), and some biologics (e.g., abatacept, adalimumab, and infliximab).

[0336] In some embodiments, the additional therapeutic agent is an autophagy enhancer. Autophagy is an endogenous pathway that maintains cellular function by targeting intracellular components such as proteins or organelles for degradation. Typically, autophagy functions through a mechanism in which cytoplasmic proteins or organelles are surrounded by a double-membrane vesicle called an autophagosome, which then fuses with a lysosome to degrade the intracellular component.

[0337] The autophagy enhancers disclosed herein are compounds and compositions capable of enhancing the autophagy pathway in hepatocytes, thereby promoting the clearance of Z-AAT protein globules and reducing AAT(Z) protein aggregates retained in the endoplasmic reticulum. Various known classes of autophagy enhancers have been observed to enhance the autophagy of aggregate-prone proteins (e.g., AAT(Z)) in cells or experimental animal models, examples of which are shown in the table below:

[0338] Compared with treatment using autophagy enhancers alone, treatment with autophagy enhancers in combination with the genetic construct has a greater effect on hepatocytes containing microspheres.

[0339] Autophagy enhancers are generally classified into two categories: (1) drugs that induce autophagy by acting directly or indirectly on the mammalian target of rapamycin (mTOR) signaling pathway complex, or (2) drugs that act through non-mTOR-dependent mechanisms (Chu et al., BioMed Research Intl (Article ID 459823) (2014)). mTOR is a protein kinase responsible for regulating a variety of functions, including cell proliferation and growth, cell survival, protein synthesis, transcription, and autophagy. Examples of drugs believed to act directly or indirectly through the mTOR signaling complex include rapamycin and its analogues, PI3K inhibitors, and ezetimibe. Examples of drugs that enhance autophagy through non-mTOR-dependent mechanisms include those that reduce intracellular Ca²⁺. + Drugs that prevent calpain-1-mediated autophagy gene cleavage, such as fluspiking, or compounds that act on the phosphatidylinositol signaling pathway by directly or indirectly affecting inositol, such as carbamazepine, valproic acid, and lithium (Chu et al., BioMed Research Int'l (article number 459823) (2014)).

[0340] Non-limiting examples of autophagy enhancers are as follows: Ezetimibe Ezetimibe is considered a potent inhibitor of small intestinal cholesterol absorption (Garcia-Calvo, PNAS 102(23):8132-8137(2005)). Ezetimibe is the active ingredient in the FDA-approved brand-name drug Zetia®, indicated for lowering plasma cholesterol levels. Zetia® is available in 10 mg tablets, administered once daily.

[0341] Regarding autophagy, ezetimibe and related compounds are thought to activate the autophagy mechanism by inhibiting the cholesterol efflux protein Niemann-Pick-type C1-like 1 (NPC1L1), which in turn reduces the recruitment of mTOR to lysosomes and inhibits mTORC1 activity (Yamamura, 2014, Hepatology 59: 1591-1599).

[0342] Carbamazepine

[0343] Carbamazepine is the active ingredient in several FDA-approved brand-name medicines used to treat conditions associated with epilepsy and neuropathic pain. These medicines include Carbatrol® (100, 200, and 300 mg oral extended-release capsules, used as an anticonvulsant); Equetro® (100, 200, and 300 mg oral extended-release capsules, used as a mood stabilizer for acute manic or mixed episodes associated with type I bipolar disorder); Tegretol® (100 and 200 mg oral tablets and 100 mg / 5 mL oral suspension, used as an anticonvulsant and for the treatment of true trigeminal neuralgia-related pain); and Tegretol XR® (100, 200, and 400 mg oral extended-release tablets, used as an anticonvulsant and for the treatment of true trigeminal neuralgia-related pain). The recommended daily dose range for FDA-approved carbamazepine is 10 to 20 mg / kg twice daily for children, and in rare cases in adults, doses up to 1600 mg daily.

[0344] Hidvegi et al. reported that carbamazepine reduced Z-AAT globules in hepatocytes and improved liver fibrosis in an AATD-induced PiZ mouse model (Hidvegi et al., 2010, Science 329: 229-232). Although the exact mechanism by which carbamazepine and related compounds reduce Z-AAT protein load in hepatocytes is not fully understood, its mood-stabilizing effects are similar to those of drugs such as lithium and valproic acid, and therefore it may act through a similar mechanism. This mechanism is thought to involve inhibition of inositol monophosphatase (IMPase), thereby reducing intracellular inositol levels and thus negatively regulating autophagy (Chu et al., BioMed Research Intl (Article No. 459823) (2014) p. 4). Furthermore, related compounds and derivatives of carbamazepine (e.g., oxcarbazepine and imipramine) may also have autophagy-enhancing effects in hepatocytes.

[0345] Fluphenazine

[0346] Fluphenazine is a trifluoromethylphenothiazine derivative used as an antipsychotic and to treat schizophrenia. Fluphenazine can be administered in several salt forms, including fluphenazine hydrochloride, fluphenazine decanoate, and fluphenazine heptaate. Fluphenazine hydrochloride was first marketed over 50 years ago under the brand names Permitil® and Prolixin®. Prolixin® decanoate (fluphenazine decanoate) and Prolixin® heptaate (fluphenazine heptaate) were also first marketed in the United States decades ago. Currently, at least fluphenazine hydrochloride (2.5 mg / 5 mL syrup, 5 mg / mL concentrate, 2.5 mg / mL injection, and 1 mg, 2.5 mg, 5 mg, and 10 mg oral tablets) and fluphenazine decanoate (25 mg / mL injection) are approved in the United States.

[0347] The FDA-recommended dose of fluphenazine hydrochloride is typically 2.5 to 10 mg daily, divided into 6 or 8-hour intervals. For severe cases, the daily dose can be up to 40 mg. Once symptoms are controlled, maintenance therapy can be initiated at a dose as low as 1 mg daily. The FDA-recommended starting dose of long-acting fluphenazine decanoate is typically 12.5 to 25 mg / mL, which can be increased to 100 mg / mL in severe cases. Fluphenazine decanoate usually begins to take effect 24 to 72 hours after administration, and a single 25 mg dose can effectively control schizophrenic behaviors over several weeks.

[0348] Li et al. reported that fluphenazine reduced Z-AAT globules in hepatocytes and improved liver fibrosis in an AATD-induced PiZ mouse model (Li et al., 2014, PLOS ONE 9:e87260). Although the exact mechanism by which fluphenazine and related compounds reduce Z-AAT protein load in hepatocytes is not fully understood, fluphenazine and other phenothiazine compounds may activate autophagy by regulating cellular calcium.

[0349] Pimozide

[0350] Pimozide is a diphenylbutylpiperidine oral antipsychotic that blocks dopaminergic receptors and is used to suppress motor and vocal tics in patients with Tourette syndrome. Pimozide can be administered in several salt forms, including fluphenazine hydrochloride, fluphenazine decanoate, and fluphenazine heptaate. Pimozide is the active ingredient in the brand-name drug Orap® (1 mg and 2 mg oral tablets). The FDA recommends a minimum dose of 0.05 mg / kg / day for children and a maximum dose of 10 mg / kg / day for adults. Doses exceeding 0.2 mg / kg / day or 10 mg / day are not recommended. Pimozide and related compounds may activate autophagy by modulating cellular calcium.

[0351] Fluspirilene

[0352] Fluspidil is a diphenylbutylpiperidine antipsychotic drug used to treat schizophrenia. It was developed and marketed by Janssen Pharmaceutics. Fluspidil was previously marketed globally under the brand names Imap® and Redeptin®, and was available in 2 mg / mL and 10 mg / mL formulations. Fluspidil and related compounds may activate autophagy by regulating cellular calcium.

[0353] Glyburide

[0354] Glibenclamide is a sulfonylurea oral hypoglycemic agent used as adjunctive therapy to improve glycemic control in adults with type 2 diabetes. Micronized glibenclamide is the active ingredient in the brand-name drugs Glynase® PresTab® (1.5, 3, and 6 mg oral tablets) and Diaβeta® (1.25, 2.5, and 5 mg oral tablets). Although there is no fixed dosing regimen for Glynase® PresTab®, the recommended starting dose is 1.5 to 3 mg daily. Doses exceeding 12 mg daily are not recommended. Similarly, there is no fixed dosing regimen for Diaβeta®, but the recommended starting dose is 2.5 to 5 mg daily, with a typical maintenance dose of 1.25 to 20 mg daily. Doses exceeding 20 mg daily are not recommended. Glibenclamide and related compounds may activate autophagy by modulating cellular calcium.

[0355] Clonidine

[0356] Clonidine is a centrally acting alpha-adrenergic receptor agonist antihypertensive drug derived from an imidazoline. It lowers heart rate and blood pressure by stimulating alpha-adrenergic receptors in the brainstem. Clonidine's alpha-2-adrenergic receptor agonist effect has also been shown to be useful in the treatment of attention deficit hyperactivity disorder (ADHD). Clonidine is also considered a centrally acting analgesic and can be used to treat severe pain. Clonidine hydrochloride is the active ingredient in several FDA-approved antihypertensive medications, including the brand-name drugs Catapres® (0.1, 0.2, and 0.3 mg oral tablets for hypertension), Catapres-TTS®-1, Catapres-TTS®-2, and Catapres-TTS®-3 (a 7-day transdermal patch system releasing 0.1, 0.2, and 0.3 mg every 24 hours). Clonidine hydrochloride is also the active ingredient in the FDA-approved drug Kapvay® (0.1 mg and 0.2 mg extended-release tablets) for the treatment of ADHD. For severe pain, clonidine hydrochloride is the active ingredient in the FDA-approved drug Duraclon® (1 mg / 10 mL and 5 mg / 10 mL for continuous epidural infusion).

[0357] The initial dose of Catapres® is 0.1 mg twice daily, with common therapeutic doses ranging from 0.2 mg to 0.6 mg daily, administered in divided doses. Doses of Catapres® up to 2.4 mg daily have been shown to be effective but are rarely used. The recommended starting dose of Kapvay® is 0.1 mg daily, which can be increased to 0.4 mg daily. The recommended starting dose of Duraclon® is 30 μg / hour, which can be titrated as needed and diluted with 0.9% sodium chloride injection to a final concentration not exceeding 100 μg / mL. Clonidine and related compounds may activate autophagy by modulating cAMP levels. Furthermore, related compounds and derivatives of clonidine (e.g., dexmedetomidine, guanifaxine, xylazine, and oxymetazoline) may also have autophagy-enhancing effects in hepatocytes.

[0358] Verapamil

[0359] Verapamil is a calcium channel blocker or slow channel antagonist that inhibits the influx of calcium ions. Verapamil hydrochloride has been approved for a variety of indications, including angina pectoris, arrhythmias, and hypertension.

[0360] Verapamil hydrochloride is the active ingredient in several FDA-approved medications, including the brand-name Calan® (40 mg, 80 mg, and 120 mg oral tablets; and for intravenous administration, 5 mg (2 mL) ampoules, 5 mg (2 mL) and 10 mg (4 mL) syringes, and 5 mg (2 mL) and 10 mg (4 mL) vials), Calan® SR (120 mg, 180 mg, and 240 mg extended-release tablets), and Covera-HS® (180 mg and 240 mg extended-release tablets). The initial dose of Calan® oral tablets can be 40 mg three times daily, but the dose can be increased to 480 mg daily (divided doses). The initial dose of Calan® SR oral tablets can be 180 mg daily, but the dose can be gradually increased to 240 mg every 12 hours (total 480 mg). For Calan® intravenous administration, the recommended initial dose for adults is 5-10 mg via intravenous bolus, which can be repeated with 10 mg after 30 minutes if necessary, until a sufficient response is achieved. In children, doses as low as 0.1 mg / kg may also be effective. The initial dose of Covera-HS® extended-release oral tablets can be 180 mg daily, but the dose can be gradually increased to 480 mg per night. The highest dose tested in clinical trials was 540 mg at bedtime.

[0361] Verapamil hydrochloride is also the active ingredient in the brand-name drug Isoptin® (40 mg, 80 mg, and 120 mg oral tablets and 2.5 mg / mL intravenous formulation). Verapamil hydrochloride is also the active ingredient in Verelan® (120, 180, 240, and 360 mg extended-release granule-filled capsules) and Verelan® PM (100, 200, and 300 mg extended-release capsules). Verelan® can be administered up to 480 mg daily, and Verelan® PM up to 400 mg at bedtime. Verapamil and related compounds may activate autophagy by modulating cellular calcium.

[0362] Loperamide

[0363] Loperamide hydrochloride is a synthetic oral antidiarrheal medication. It is the active ingredient in the brand-name drugs Imodium® (2 mg capsules), Imodium® AD (2 mg tablets and 1 mg / 7.5 mL liquid), Imodium® AD EZ Chews (2 mg chewable tablets), and Imodium® AD pediatric formulation (1 mg / 7.5 mL liquid). The recommended starting dose for adults of Imodium® is 4 mg, followed by 2 mg after each loose stool, with a daily dose not exceeding 16 mg. For Imodium® AD, adults may take up to 4 tablets (8 mg) or 60 mL (8 mg) every 24 hours. Adults may take up to 4 tablets (8 mg) of Imodium® AD EZ Chews every 24 hours. Loperamide and related compounds may activate autophagy by regulating cellular calcium.

[0364] Nimodipine

[0365] Nimodipine is a calcium channel blocker approved for improving neurological outcomes by reducing the incidence and severity of ischemic neurological deficits in patients with subarachnoid hemorrhage caused by rupture of intracranial berry-like aneurysms.

[0366] Nimodipine is the active ingredient in the brand-name drug Nimotop® (30 mg oral capsules). The recommended dose of Nimotop® oral capsules is 60 mg every 4 hours for 21 consecutive days (or to be started within 96 hours after bleeding stops). Nimodipine is also the active ingredient in the brand-name drug Nymalize® (60 mg / 20 mL oral solution), with a recommended dose of 20 mL (60 mg) every 4 hours for 21 consecutive days (or to be started within 96 hours after bleeding stops). Nimodipine and related compounds may activate autophagy by regulating cellular calcium.

[0367] Nitrendipine

[0368] Nifedipine is a calcium channel blocker with significant vasodilatory effects and is considered an effective antihypertensive drug. Nifedipine has been approved for the treatment of hypertension in several regions worldwide and has been found to reduce the cardiotoxicity of cocaine. It is marketed in 10 mg and 20 mg tablets, with a recommended daily dose not exceeding 40 mg. Nifedipine and related compounds may activate autophagy by regulating cellular calcium.

[0369] Amiodarone

[0370] Amiodarone is an antiarrhythmic drug. Amiodarone hydrochloride is the active ingredient in the FDA-approved drugs Cordarone® (200 mg tablets) and Nextarone® (150 mg / 100 mL and 360 mg / 200 mL premixed intravenous solutions) for the treatment of life-threatening recurrent ventricular arrhythmias. Cordarone® is recommended as a loading dose of 800 to 1600 mg daily for up to three weeks; after approximately one month, the dose is 600 to 800 mg daily; the usual maintenance dose is 400 mg daily. Nextarone® is recommended to be administered at approximately 1000 mg within the first 24 hours; clinical studies have shown that average daily doses exceeding 2100 mg are associated with an increased risk of hypertension. Amiodarone and related compounds may activate autophagy by inhibiting mTORC1 signaling and / or by regulating the mTOR-independent pathway of cellular calcium.

[0371] Lithium

[0372] Lithium is known to alter sodium transport in nerve and muscle cells and promote the conversion of catecholamines into neuronal metabolism. Lithium has been approved for the treatment of manic episodes in bipolar disorder and manic-depressive illness. Lithium carbonate is the active ingredient in several pharmaceutical products, including Eskalith® (lithium carbonate 300 mg oral capsules), Eskalith CR® (lithium carbonate 450 mg controlled-release tablets), Lithobid® (300 mg extended-release tablets), and Lithonate® (300 mg oral capsules and 300 mg / 5 mL oral syrup). For Eskalith® and Eskalith CR®, most patients have been reported to remain stable at a daily dose of 900 mg lithium, but optimal patient response has been reported at a daily dose of 1800 mg. The recommended dose of Lithobid® for the treatment of acute mania is 1800 mg daily, 900 mg twice a day; the maintenance dose is 1200 mg daily, 600 mg twice a day. For lithium carbonate in a 300 mg / 5 mL syrup formulation, the optimal patient response to acute mania is typically established by administration of 10 mL three times daily. Lithium is thought to exert its effects by inhibiting inositol monophosphatase, thereby reducing inositol-1,4,5-triphosphate (OP3) levels.

[0373] Rapamycin

[0374] Rapamycin (also known as sirolimus) is a macrolide produced by bacteria that has immunosuppressive effects in humans. Rapamycin is the active ingredient in the FDA-approved drug Rapamune® (0.5 mg, 1 mg, and 2 mg oral tablets and 60 mg / 60 mL solution) used to prevent organ rejection in kidney transplant patients. The recommended initial dose may be as low as 2 mg daily, depending on the patient's condition, and the maximum total daily dose should not exceed 40 mg. Regarding autophagy, rapamycin is thought to exert its effects by inhibiting the mammalian target of rapamycin (mTOR), a negative regulator of autophagy. Furthermore, related compounds and derivatives of rapamycin (such as tesilimus, everolimus, deformolimus, and ATP-competitive mTOR kinase inhibitors) may also have autophagy-enhancing effects in hepatocytes.

[0375] Minoxidil

[0376] Minoxidil is an antihypertensive peripheral vasodilator. It is the active ingredient in Loniten® (2.5 mg and 10 mg tablets), used to treat hypertension. The recommended starting dose of Loniten® is 5 mg daily, which can be increased to 40 mg daily (single or divided doses), with a maximum recommended dose of 100 mg daily. Minoxidil and related compounds may activate autophagy by regulating cellular calcium.

[0377] Beclin1 peptide

[0378] In terms of autophagy, the Beclin1 peptide is thought to function by interacting with GAPR-1 (also known as GLIPR2), which is a negative regulator of autophagy.

[0379] Bile acid derivatives, ursodeoxycholic acid / norursodeoxycholic acid

[0380] Other compounds that can mimic the activation or upregulation of autophagy can also be used in combination with expression-inhibiting oligomers to treat AADTD and related diseases, manifestations, and symptoms caused by AADTD. For example, ursodeoxycholic acid (UDCA) and other bile acid derivatives, including bile salts such as taurocholate and glycocholate, are increasingly used to treat cholestatic liver disease. Studies have shown that UDCA can improve the clinical condition and liver function test results of some children with AADTD-related liver disease (Lykavieris et al., 2008, Journal of Pediatric Gastroenterology and Nutrition 47:623-629). Experimental studies have shown that bile acids mainly exert their effects through three mechanisms: (1) by regulating the mixed micelle composition rich in phospholipids to protect bile duct cells from the cytotoxic effects of hydrophobic bile acids, thereby reducing the cytotoxicity of bile acids in bile and possibly reducing the concentration of hydrophobic bile acids in bile duct cells; (2) by Ca 2+ The protein kinase C-α-dependent mechanism and / or activation of p38 (MAPK) and extracellular signal-regulated kinase (Erk) stimulate hepatobiliary secretion, thereby allowing transport protein molecules (such as bile salt efflux pump BSEP and conjugate efflux pump MRP2) to insert into the hepatocyte tubular membrane and potentially activate these transport proteins; (3) protect hepatocytes from bile acid-induced apoptosis by inhibiting mitochondrial membrane permeability transition (MMPT) and may stimulate cell survival pathways (Paumgartner and Beuers, 2002, Hepatology 36:525-531). Recent studies in the AATD PiZ mouse model have shown that modified bile acid norursodeoxycholic acid (nor-UDCA) can reduce apoptotic signaling and reduce the accumulation of mutant Z-AAT in hepatocytes, and these effects are associated with increased hepatic autophagy (Tang et al., 2016, American Journal of Physiology—Gastrointestinal and Liver Physiology 311:G156-G165). Any drug that can improve hepatocyte health (e.g., as assessed by ALT, AST, and GGT indicators), such as the bile acid derivatives disclosed herein, may make hepatocytes more likely to clear Z-AAT polymers and achieve better long-term efficacy in AAT-inhibiting oligonucleotide compounds in AAT-inhibiting therapy than using AAT expression-inhibiting oligonucleotide compounds alone. While not wishing to be limited by any theory, it is believed that bile acid derivatives function at least partially through autophagy.

[0381] The autophagy enhancers explicitly listed herein and identified in the table above are merely examples and are not intended to limit the scope of this application. Any suitable autophagy enhancer capable of reducing or eliminating Z-AAT spheres beyond the effects achievable with monotherapy using only AAT expression-inhibiting oligomers may be used. Furthermore, various derivative compounds and analogues related to the compounds explicitly described herein have similar or related properties and may therefore act on autophagy or other pathways through the same or similar mechanisms, thereby providing benefit in AATD treatment. These compounds are considered to be within the scope of this invention.

[0382] The autophagy enhancers disclosed herein can be provided as compositions comprising pharmaceutically acceptable carriers or pharmaceutically acceptable excipients (including, for example, loads, transporters, and / or diluents). Excipients may include, but are not limited to: absorption enhancers, anti-adhesives, defoamers, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, dextran, glucose, diluents, disintegrants, emulsifiers, spreaders, fillers, flavorings, flow aids, humectants, lubricants, oils, polymers, preservatives, physiological saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, osmotic pressure regulators, loads, water-repellent agents, and wetting agents. Pharmaceutically acceptable excipients may or may not be inert substances.

[0383] The pharmaceutical composition may also contain other additional components commonly found in pharmaceutical compositions. The pharmaceutically active substance may include, but is not limited to, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). The autophagy enhancer may be any suitable dosage form known in the art for a particular autophagy enhancer. For example, the autophagy enhancers disclosed herein may be administered orally in capsule or tablet form, or in any other suitable unit dosage form, in combination with the administration of an AAT expression-inhibiting oligomeric compound. Of course, it is also contemplated that, depending on the specific autophagy enhancer selected, the two drugs may be administered in other suitable ways, such as nasal spray, buccal or sublingual dosage forms, transdermal administration, parenteral administration, suppository administration, sustained-release dosage forms, etc. Any route of administration may be used as long as an appropriate dose can be delivered.

[0384] In some embodiments, the pharmaceutical composition containing the autophagy enhancer may further comprise the genetic construct. In some embodiments, the pharmaceutical composition containing the autophagy enhancer is separate from the pharmaceutical composition containing the AAT expression-inhibiting oligomeric compound. The pharmaceutical composition can be used to treat subjects suffering from diseases or disorders that can benefit from reduced or inhibited AAT expression. The pharmaceutical composition can also be used to treat subjects at risk of developing diseases or disorders that can benefit from reduced or inhibited AAT expression and Z-AAT sphere formation. Diseases and / or disorders that can benefit from such reduction or inhibition may be selected from a list including: AATD, chronic hepatitis, cirrhosis, hepatocellular carcinoma, and fulminant hepatic failure. Preferably, the subject is a mammal, and most preferably a human patient.

[0385] Example

[0386] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention as described in the claims.

[0387] The table below contains abbreviations and special terms applicable only to these embodiments. These abbreviations and special terms are not restrictive and do not replace or narrow the broader definitions described above, which continue to apply to the claims.

[0388] Table 6: Abbreviations and special terms used in the examples.

[0389]

[0390] Example 1: miRNA design for reducing AAT(Z) expression

[0391] This embodiment describes the design of artificial primary miRNAs and artificial mature miRNAs, which are the subject of this invention. The artificial primary miRNA structure is designed to reduce the expression of a mutant gene encoding a mutant AAT(Z) variant using a transgene expressing a wild-type AAT(M) variant. The artificial primary miRNA structure is designed such that the polynucleotide sequence encoding at least one miRNA structure is sufficiently different from the polynucleotide structure encoding at least another primary miRNA structure to reduce or prevent vector recombination and / or reduce or prevent pairing or binding between these primary miRNAs. The primary miRNAs are also designed such that the stem-loop secondary structure of each primary miRNA is sufficiently similar to the predicted secondary structure of naturally occurring miRNAs to reduce or prevent pathogen-resistant toxicity based on cellular RNAi. The primary miRNAs are also designed to each contain a mature miRNA sequence for binding to different regions of the RNA transcript produced by the same natural AAT gene.

[0392] Example 2: Design of an engineered GC44 vector encoding dual miRNAs

[0393] This embodiment describes the design of a chimpanzee adenovirus vector (GC44) encoding the two primary miRNA designs of the present invention (see Example 1).

[0394] A GC44 shuttle plasmid was constructed, within which a complete gene construct was built. The shuttle plasmid was constructed using standard cloning techniques, including restriction digestion and Gibson assembly. The gene construct contained two miRNA designs, along with a miRNA-resistant wild-type AAT gene (AAT(M) variant), a liver-specific promoter, 5'UTR, and 3'UTR, inserted in reverse direction into the E1 region of the GC44 adenovirus vector. The liver-specific promoter restricted transgene expression to the liver, and the wild-type AAT gene (AAT(M) variant) was modified to include a silencing mutation sufficient to prevent reduced miRNA expression.

[0395] Example 3: Design of an engineered GC44 vector encoding a triple miRNA

[0396] This embodiment describes the design of a vector encoding the three primary miRNA designs of the present invention (see Example 1). A GC44 shuttle plasmid was constructed, in which a complete gene construct was built. The shuttle plasmid was constructed using standard cloning techniques, including restriction digestion and Gibson assembly. The gene construct contains three miRNA designs, along with a miRNA-tolerant wild-type AAT gene (AAT(M) variant), a liver-specific promoter, a 5'UTR, and a 3'UTR, inserted in reverse direction into the E1 region of the GC44 adenovirus vector. The liver-specific promoter restricts transgene expression to the liver, and the wild-type AAT gene (AAT(M) variant) is modified to include a silencing mutation sufficient to prevent reduced miRNA expression.

[0397] Example 4: Design of an engineered GC45 vector encoding dual miRNAs

[0398] This embodiment describes the design of a vector encoding the two primary miRNA designs of the present invention (see Example 1). A GC45 shuttle plasmid was constructed, in which a complete gene construct was built. The shuttle plasmid was constructed using standard cloning techniques, including restriction digestion and Gibson assembly. The gene construct contains two miRNA designs, along with a miRNA-tolerant wild-type AAT gene (AAT(M) variant), a liver-specific promoter, a 5'UTR, and a 3'UTR, inserted in reverse direction into the E1 region of the GC45 adenovirus vector. The liver-specific promoter restricts transgene expression to the liver, and the wild-type AAT gene (AAT(M) variant) is modified to include a silencing mutation sufficient to prevent reduced miRNA expression.

[0399] Example 5: Design of an engineered GC45 vector encoding a triple miRNA

[0400] This embodiment describes the design of a vector encoding the three primary miRNA designs of the present invention (see Example 1). A GC45 shuttle plasmid was constructed, in which a complete gene construct was built. The shuttle plasmid was constructed using standard cloning techniques, including restriction digestion and Gibson assembly. The gene construct contains three miRNA designs, along with a miRNA-tolerant wild-type AAT gene (AAT(M) variant), a liver-specific promoter, a 5'UTR, and a 3'UTR, inserted in reverse direction into the E1 region of the GC45 adenovirus vector. The liver-specific promoter restricts transgene expression to the liver, and the wild-type AAT gene (AAT(M) variant) is modified to include a silencing mutation sufficient to prevent reduced miRNA expression.

[0401] Example 6: In vivo assessment of liver-specific transcript expression in mice

[0402] The strength of the CMV promoter and liver-specific promoters was assessed using IVIS® imaging. BALB / C and NSG-PiZ mice were administered 1×10⁻⁶ intravenously. 11 A PU-dosage vector containing a transgenic construct (CMV:fLUC or LSP:fLUC) driven by a CMV promoter or a liver-specific promoter (LSP) to express firefly luciferase. NSG-PiZ mice express the mutant human AAT(Z) allele of SERPINA1 and are a model of AATD liver disease.

[0403] In Vitro Isosorbide Diffusion (IVIS) images of mice obtained one day after drug administration showed that the liver-specific promoter generated targeted expression of the transgene in liver tissue. Figure 1A-1CWhen the luminescence signal was quantified, it was found that liver-specific promoters could achieve similar levels of transgene expression using approximately 10 times fewer vectors compared to CMV-driven expression. Figure 2A-2B ).

[0404] In one experiment, after administering a dual miRNA construct containing miR204 and miR206 to NSG-PiZ mice, PAS-D staining showed a decrease in liver glomeruli 28 days after administration. Figures 3A-3B (and 4A-4B). In addition, the expression of adenovirus DNA in various tissues of NSG-PiZ mice was determined using GC44 and GC45 adenovirus vectors. NSG-PiZ mice were administered 1×10⁻⁶ adenovirus vectors intravenously. 11 Adenovirus vector copy number per cell in brain, heart, kidney, lung, liver, ovary, and spleen was determined using PU doses of RA-1276 (Group 3), RA-1330 (Group 4), or RA-1331 (Group 5), or as controls, administered to NSG mice (Group 1) or NSG-PiZ mice (Group 2) with an equal volume of final formulation buffer (FFB). RA-1276 is a GC44 vector whose transgenic cassette contains a liver-specific promoter-driven expression of two AAT(Z)-targeting miRNAs and an AAT(M) miRNA tolerance transgene, inserted in reverse direction with a deleted E1 region. RA-1330 is a similar vector to RA-1276 but with a transgenic cassette with reduced CpG (from promoter to 3'UTR). RA-1331 is the transgenic cassette of RA-1330 in a GC45 adenovirus vector. Adenovirus DNA was detected in all tissues, with the highest concentrations in the liver and lungs. Figure 5 The expression of the AAT(M) transcript is highest in the liver. Figure 6 ).

[0405] Example 7: In vivo dose-dependent expression of AAT(M) in mice treated with dual miRNA design

[0406] This embodiment describes how mice treated with the dual miRNA design of the present invention exhibit dose-dependent expression of the AAT(M) variant. C57BL / 6 mice and NSG mice were given an engineered GC44 chimpanzee adenovirus vector containing a liver-specific promoter and encoding a dual miRNA design and miRNA-tolerant AAT(M) variant.

[0407] Specifically, a dual miRNA design was administered to the C57BL / 6 mouse system via a single intravenous injection at the following dose levels: 1 × 10⁻⁶. 11 PU, 5×10 10 PU and 2×10 10Serum samples were collected from these mice on days 2, 9, and 17 after administration, and hAAT(M) expression in each sample was measured (see PU). Figure 7A The results showed that hAAT(M) expression in C57BL / 6 mice was dose-dependent after a single administration of the vector containing the AAT(M) variant. (1×10⁻⁶) 11 PU and 5×10 10 PU dose was associated with serum AAT concentrations above the predicted therapeutically significant threshold of 11 µM.

[0408] In addition, NSG mice were administered 1×10 via a single intravenous injection. 11 Dual miRNA design for PU. Serum samples were collected at days 2, 9, 16, 23, 30, 44, 58, 72, 79, 93, 107, 121, 128, 142, 156, 170, and 184 post-drug administration, and hAAT(M) expression in each sample was measured (see [link to study]). Figure 7B The results showed that a single administration of 1×10⁻⁶ 11 The vector containing the AAT(M) variant of PU can generate long-term expression of hAAT(M) in the serum of NSG mice, with values ​​exceeding the 11 µM threshold predicted for therapeutic benefit.

[0409] Example 8: In vivo tolerance and expression of AAT(M) in mice treated with dual miRNA design

[0410] To determine the expression of AAT(M) generated by RA-1276 ( Figure 8A In vitro experiments were conducted using HepG2 cells with the SERPINA1 gene knocked out, where, where possible, CG dinucleotides were mutated in the promoter, 5'UTR, and transgene. Eight CpG sites were retained in the promoter, located within conserved transcription factor binding regions. The serum AAT levels produced by RA-1276 with reduced CpG levels were as follows: Figure 8A As shown.

[0411] Serum AAT levels produced by the CpG-reduced GC44 adenovirus vector were evaluated in immunocompetent mice (C57BL6 / J mice). Specifically, mice were administered a single dose of 1 × 10⁻⁶ AAT via intravenous injection. 11 PU / mouse GC44 AATD vector (RA-1276) or CpG reduced transgenic cassette form (RA-1330). This treatment did not negatively affect animal body weight throughout the study compared to control mice given an equal volume of final formulation buffer (FFB). Figure 8B Both adenovirus vectors also produced high levels of AAT(M) expression in serum well above the 11 µM threshold predicted for therapeutic benefit. Figure 9 ).

[0412] Tolerance to GC44 and GC45 adenovirus vectors was further determined. NSG-PiZ mice were administered 1×10⁻⁶ doses intravenously. 11 PU doses of RA-1276 (Group 3), RA-1330 (Group 4), or RA-1331 (Group 5) were administered. NSG mice (Group 1) and NSG-PiZ mice given FFB (Group 2) served as controls, receiving an equal volume of final formulation buffer (FFB). RA-1276 is a GC44 vector whose transgenic cassette contains a liver-specific promoter-driven transgene for two AAT(Z)-targeting miRNAs and an AAT(M) miRNA tolerance transgene, inserted in reverse direction into the deleted E1 region. RA-1330 is a similar vector to RA-1276 but with a transgenic cassette containing reduced CpG (from the promoter to the 3'UTR). RA-1331 is the transgenic cassette of RA-1330 in a GC45 adenovirus vector. NSG-PiZ mice receiving the adenovirus vector (Groups 3–5) showed similar weight gain to control mice (Groups 1–2), indicating good tolerability of these vectors. Figure 10 ).

[0413] Example 9: Reduced in vivo dose-responsiveness of AAT(Z) in NSG-PiZ mice treated with dual miRNA design

[0414] This example describes how NSG-PiZ mice treated with dual miRNA design exhibit reduced expression of the AAT(Z) variant. NSG-PiZ mice were given an engineered GC44 chimpanzee adenovirus vector containing a liver-specific promoter and encoding a dual miRNA design and miRNA-resistant AAT(M) variant.

[0415] Specifically, a dual miRNA design was developed by administering the following dose levels to the NSG-PiZ mouse system via a single injection: 1 × 10⁻⁶. 11 PU, 5×10 10 1×10 10 5×10 9 and 1×10 9 PU. Hepatocyte biopsies were taken from mice 30 days after administration, and these biopsied hepatocytes were stained with PAS-D to identify mutant AAT(Z) globule formation (see [link to article]). Figure 11 Therefore, it was determined that after treatment with dual miRNAs, the formation of AAT(Z) spheres decreased with changes in the dose level of the dual miRNAs, and that the expression of the dual miRNAs played a role in reducing liver pathology.

[0416] Two other adenovirus vector constructs also showed efficacy in NSG-PiZ mice. RA-1276 is a GC44 vector whose transgenic cassette contains a liver-specific promoter-driven transgene for two AAT(Z)-targeting miRNAs and an AAT(M) miRNA tolerance transgene, inserted in reverse direction into the deleted E1 region. RA-1330 is a similar vector to RA-1276 but with a transgenic cassette containing reduced CpG (from the promoter to the 3'UTR). RA-1331 is the transgenic cassette of RA-1330 in a GC45 adenovirus vector. NSG-PiZ mice were administered 1 × 10⁻⁶ intravenously. 11 PU doses of the designated adenoviral vector were administered, or an equal volume of final formulation buffer (FFB) was given as a control. Liver tissue was collected on day 28 post-administration and treated with PAS-D staining to visualize the presence of globules, a characteristic marker of AAT(Z) protein aggregation. RNA was extracted from liver tissue on day 28 post-administration and used as input for RT-qPCR to detect AAT Z allele transcripts and miRNA-resistant AAT(M) transgenic transcripts. DNA was extracted from liver tissue on day 28 post-administration, and the adenoviral DNA copy number per cell was analyzed by qPCR. Treatment with RA-1276, RA-1330, and RA-1331 resulted in a reduction in hepatic globules ( Figure 12A Reduced expression of AAT(Z) allele transcripts Figure 12B ) and increased expression of AAT(M) allele transcripts ( Figure 12C The presence of the vector was confirmed in liver tissue after treatment with RA-1276, RA-1330, and RA-1331. Figure 12D ).

[0417] Example 10: Treatment of liver disease with GC44-dual miRNAs in patients with AAT(Z)

[0418] Patients with AAT alleles containing a defective Z-type AAT variant (“AAT(Z)”) were administered a pharmaceutical composition comprising an engineered GC44 chimpanzee adenovirus vector encoding a liver-specific promoter, a dual miRNA design (one miRNA targeting either of the AAT alleles disclosed herein, and a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant. Following administration, the patients exhibited reduced AAT(Z) globulus formation, reduced liver pathology, and enhanced AAT(M) expression.

[0419] Example 11: Treatment of liver disease with GC45-dual miRNAs in patients with AAT(Z)

[0420] Patients with AAT alleles containing a defective Z-type AAT variant (“AAT(Z)”) were administered a pharmaceutical composition comprising an engineered GC45 chimpanzee adenovirus vector encoding a liver-specific promoter, a dual miRNA design (one miRNA targeting either of the AAT alleles disclosed herein, and a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant. Following administration, the patients exhibited reduced AAT(Z) globulus formation, reduced liver pathology, and enhanced AAT(M) expression.

[0421] Example 12: Treatment of liver disease with GC46-dual miRNAs in patients with AAT(Z)

[0422] Patients with AAT alleles containing a defective Z-type AAT variant (“AAT(Z)”) were administered a pharmaceutical composition comprising an engineered GC46 chimpanzee adenovirus vector encoding a liver-specific promoter, a dual miRNA design (one miRNA targeting either of the AAT alleles disclosed herein, and a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant. Following administration, the patients exhibited reduced AAT(Z) globulus formation, reduced liver pathology, and enhanced AAT(M) expression.

[0423] Example 13: Treatment of liver disease with GC44-triple miRNA in patients with AAT(Z)

[0424] A pharmaceutical composition comprising an engineered GC44 chimpanzee adenovirus vector encoding a liver-specific promoter, a triple miRNA design (at least one miRNA targeting any of the AAT alleles disclosed herein, and at least a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant was administered to a patient with an AAT(Z) allele. Following administration, the patient exhibited reduced AAT(Z) globulus formation, reduced liver pathology, and enhanced AAT(M) expression.

[0425] Example 14: Treatment of liver disease with GC45-triple miRNA in patients with AAT(Z)

[0426] A pharmaceutical composition comprising an engineered GC45 chimpanzee adenovirus vector encoding a liver-specific promoter, a triple miRNA design (at least one miRNA targeting any of the AAT alleles disclosed herein, and at least a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant was administered to patients with AAT(Z) alleles. Following administration, the patients exhibited reduced AAT(Z) globulus formation, reduced liver pathology, and enhanced AAT(M) expression.

[0427] Example 15: Treatment of liver disease with GC46-triple miRNA in patients with AAT(Z)

[0428] A pharmaceutical composition comprising an engineered GC46 chimpanzee adenovirus vector encoding a liver-specific promoter, a triple miRNA design (at least one miRNA targeting any of the AAT alleles disclosed herein, and at least a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant was administered to patients with AAT(Z) alleles. Following administration, the patients exhibited reduced AAT(Z) globulus formation, reduced liver pathology, and enhanced AAT(M) expression.

[0429] Example 16: Treatment of lung disease with GC44-dual miRNAs in patients with AAT(Z)

[0430] A pharmaceutical composition comprising an engineered GC44 chimpanzee adenovirus vector encoding a liver-specific promoter, a dual miRNA design (one miRNA targeting either of the AAT alleles disclosed herein, and a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant was administered to a patient with an AAT allele containing a defective Z-type AAT variant (“AAT(Z)”). Following administration, the patient exhibited reduced lung pathology and enhanced AAT(M) expression.

[0431] Example 17: Treatment of lung disease with GC45-dual miRNAs in patients with AAT(Z)

[0432] A pharmaceutical composition comprising an engineered GC45 chimpanzee adenovirus vector encoding a liver-specific promoter, a dual miRNA design (one miRNA targeting either of the AAT alleles disclosed herein, and a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant was administered to a patient with an AAT allele containing a defective Z-type AAT variant (“AAT(Z)”). Following administration, the patient exhibited reduced lung pathology and enhanced AAT(M) expression.

[0433] Example 18: Treatment of lung disease with GC46-dual miRNAs in patients with AAT(Z)

[0434] A pharmaceutical composition comprising an engineered GC46 chimpanzee adenovirus vector encoding a liver-specific promoter, a dual miRNA design (one miRNA targeting either of the AAT alleles disclosed herein, and a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant was administered to a patient with an AAT allele containing a defective Z-type AAT variant (“AAT(Z)”). Following administration, the patient exhibited reduced lung pathology and enhanced AAT(M) expression.

[0435] Example 19: Design of GC44-triple miRNA therapy for lung disease in patients with AAT(Z)

[0436] A pharmaceutical composition comprising an engineered GC44 chimpanzee adenovirus vector encoding a liver-specific promoter, a triple miRNA design (at least one miRNA targeting any of the AAT alleles disclosed herein, and at least a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant was administered to a patient with an AAT allele containing a defective Z-type AAT variant (“AAT(Z)”). Following administration, the patient exhibited reduced lung pathology and enhanced AAT(M) expression.

[0437] Example 20: Design of GC45-triple miRNA therapy for lung disease in patients with AAT(Z)

[0438] A pharmaceutical composition comprising an engineered GC45 chimpanzee adenovirus vector encoding a liver-specific promoter, a triple miRNA design (at least one miRNA targeting any of the AAT alleles disclosed herein, and at least a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant was administered to a patient with an AAT allele containing a defective Z-type AAT variant (“AAT(Z)”). Following administration, the patient exhibited reduced lung pathology and enhanced AAT(M) expression.

[0439] Example 21: Treatment of lung disease with GC46-triple miRNA in patients with AAT(Z)

[0440] A pharmaceutical composition comprising an engineered GC46 chimpanzee adenovirus vector encoding a liver-specific promoter, a triple miRNA design (at least one miRNA targeting any of the AAT alleles disclosed herein, and at least a second miRNA targeting AAT(Z)), and a miRNA-resistant AAT(M) variant was administered to a patient with an AAT allele containing a defective Z-type AAT variant (“AAT(Z)”). Following administration, the patient exhibited reduced lung pathology and enhanced AAT(M) expression.

[0441] Example 22: In a system with AAT(M) Malton Using GC44-miRNA to design treatments for lung disease in patients

[0442] Towards M with defects Malton Type AAT variant (“AAT(M)”) Malton Patients with AAT alleles of )”) are given a drug composition comprising an engineered GC44 chimpanzee adenovirus vector programmed to encode a liver-specific promoter and at least two miRNAs programmed (one miRNA targeting any of the AAT alleles disclosed herein, and the second miRNA targeting AAT(M) Malton The drug was administered to the patient, along with a miRNA-resistant AAT(M) variant. Following administration, the patient exhibited reduced lung pathology and increased AAT(M) expression.

[0443] Example 23: In a system with AAT(M) Malton Using GC44-miRNA to design treatments for liver disease in patients with )

[0444] Towards M with defects Malton Type AAT variant (“AAT(M)”) MaltonPatients with AAT alleles of )”) are given a drug composition comprising an engineered GC44 chimpanzee adenovirus vector designed to encode a liver-specific promoter and a dual miRNA design (one miRNA targeting either of the AAT alleles disclosed herein, and a second miRNA targeting AAT(M) Malton The patient also exhibited AAT(M) variants of miRNA resistance. Following administration, the patient demonstrated AAT(M)... Malton Reduced globulin formation, decreased liver pathology, and increased AAT(M) expression.

[0445] Example 24: In a system with AAT(S) iiyama Using GC44-miRNA to design treatments for lung disease in patients

[0446] Towards S with inclusion defects iiyama Type AAT variant (“AAT(S)”) iiyama Patients with AAT alleles of )”) are given a drug composition comprising an engineered GC44 chimpanzee adenovirus vector programmed to encode a liver-specific promoter, at least two miRNAs programmed (at least one miRNA targeting any of the AAT alleles disclosed herein, and at least a second miRNA targeting AAT(S) iiyama The drug was administered to the patient, along with a miRNA-resistant AAT(M) variant. Following administration, the patient exhibited reduced lung pathology and increased AAT(M) expression.

[0447] Example 25: In a system with AAT(S) iiyama Using GC44-miRNA to design treatments for liver disease in patients with )

[0448] Towards S with inclusion defects iiyama Type AAT variant (“AAT(S)”) iiyama Patients with AAT alleles of )”) are given a drug composition comprising an engineered GC44 chimpanzee adenovirus vector designed to encode a liver-specific promoter and a dual miRNA design (one miRNA targeting either of the AAT alleles disclosed herein, and a second miRNA targeting AAT(S) iiyama The patient also exhibited AAT(M) variants of miRNA resistance. Following administration, the patient demonstrated AAT(S)... iiyama Reduced globulin formation, decreased liver pathology, and increased AAT(M) expression.

[0449] Example 26: Design of GC44-miRNA for the treatment of lung disease in patients with AAT(F)

[0450] A pharmaceutical composition comprising an AAT allele containing a defective F-type AAT variant (“AAT(F)”) was administered to a patient having an AAT allele comprising an engineered GC44 chimpanzee adenovirus vector programmed to encode a liver-specific promoter, at least two miRNAs programmed (at least one miRNA targeting any of the AAT alleles disclosed herein, and at least a second miRNA targeting AAT(F)), and a miRNA-resistant AAT(M) variant. Following administration, the patient exhibited reduced lung pathology and enhanced AAT(M) expression.

[0451] Example 27: Design of GC44-miRNA for the treatment of liver disease in patients with AAT(F)

[0452] Patients with AAT alleles containing a defective F-type AAT variant (“AAT(F)”) were administered a pharmaceutical composition comprising an engineered GC44 chimpanzee adenovirus vector programmed to encode a liver-specific promoter, a dual miRNA design (one miRNA targeting either of the AAT alleles disclosed herein, and a second miRNA targeting AAT(F)), and a miRNA-resistant AAT(M) variant. Following administration, the patients exhibited reduced AAT(F) globulus formation, reduced liver pathology, and enhanced AAT(M) expression.

[0453] Example 28: Design of GC44-miRNA for the treatment of lung disease in patients with AAT(I)

[0454] A pharmaceutical composition comprising an AAT allele containing a defective type I AAT variant (“AAT(I)”) was administered to a patient having an AAT allele comprising an engineered GC44 chimpanzee adenovirus vector programmed to encode a liver-specific promoter, at least two miRNAs programmed (at least one miRNA targeting any of the AAT alleles disclosed herein, and at least a second miRNA targeting AAT(I)), and a miRNA-resistant AAT(M) variant. Following administration, the patient exhibited reduced lung pathology and enhanced AAT(M) expression.

[0455] Example 29: Design of GC44-miRNA for the treatment of liver disease in patients with AAT(I)

[0456] Patients with an AAT allele containing a defective type I AAT variant (“AAT(I)”) were administered a pharmaceutical composition comprising an engineered GC44 chimpanzee adenovirus vector programmed to encode a liver-specific promoter, a dual miRNA design (one miRNA targeting either of the AAT alleles disclosed herein, and a second miRNA targeting AAT(I)), and a miRNA-resistant AAT(M) variant. Following administration, the patients exhibited reduced AAT(I) globulus formation, reduced liver pathology, and enhanced AAT(M) expression.

[0457] Example 30. In vivo efficacy of GC44-dual miRNA desig...

Claims

1. A therapeutic genetic construct encoding: (a) a first precursor miRNA; (b) a second precursor miRNA; and (c) a transgene encoding AAT(M).

2. The genetic construct of claim 1, wherein the nucleic acid encoding the first precursor miRNA and the nucleic acid encoding the second precursor miRNA are separated by at least 7 nucleotides.

3. The genetic construct of claim 1, wherein the first and second precursor miRNAs each comprise a guide miRNA that inhibits the expression of an allele encoding an α-1 antitrypsin (AAT) mutant variant.

4. The genetic construct of claim 3, wherein each guide miRNA independently inhibits the expression of an AAT mutant variant, said mutant variant being AAT(Z), AAT(S), AAT(I), AAT(S) iiyama ) or AAT(M malton ).

5. The genetic construct of claim 3, wherein each guide miRNA inhibits the expression of AAT(Z).

6. The genetic construct of claim 3, wherein the guide miRNA of the first precursor miRNA is encoded by the nucleic acid of SEQ ID NO: 64, or by a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 64 under strict hybridization conditions, and the guide miRNA of the second precursor miRNA is encoded by the nucleic acid of SEQ ID NO: 66, or by a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 66 under strict hybridization conditions.

7. The genetic construct of claim 1, wherein the first and second precursor miRNAs each independently comprise a backbone segment identical to the corresponding backbone segment of miR16, miR17, miR19, miR21, miR22, miR26a1, miR29b1, miR30a, miR122, miR126, miR133a1, miR142, miR150, miR155, miR181a, miR204, miR206, miR214, miR412, miR486, miR494, or miR1915.

8. The genetic construct of claim 1, wherein the first and second precursor miRNAs each independently comprise the same backbone segment as the corresponding backbone segment of miR16, miR17, miR21, miR22, miR26a1, miR142, miR150, miR204, or miR206.

9. The genetic construct of claim 1, wherein the first precursor miRNA comprises the same backbone segment as the corresponding backbone segment of miR204, and the second miRNA comprises the same backbone segment as the corresponding backbone segment of miR206.

10. The genetic construct of claim 1, wherein the first precursor miRNA is encoded by the nucleic acid of SEQ ID NO: 347, or by a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 347 under strict hybridization conditions, and the second precursor miRNA is encoded by the nucleic acid of SEQ ID NO: 348, or by a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO: 348 under strict hybridization conditions.

11. The genetic construct of claim 1, wherein the first and second precursor miRNAs are encoded by the nucleic acid of SEQ ID NO:267, or by a nucleic acid capable of hybridizing with the complementary sequence of SEQ ID NO:267 under strict hybridization conditions.

12. The genetic construct of claim 1, wherein the first and second precursor miRNAs are encoded by the nucleic acid of SEQ ID NO:

267.

13. The genetic construct of claim 1, wherein the transgene encoding AAT(M) is different from the wild-type gene encoding AAT(M) and is silenced by a mutation to prevent the precursor miRNA from reducing its expression.

14. The genetic construct of claim 1, wherein the transgene encoding AAT(M) comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 590 or 596.

15. The genetic construct of claim 1, wherein the transgene encoding AAT(M) comprises a nucleic acid sequence having at least about 90% sequence identity with SEQ ID NO: 590 or 596.

16. The genetic construct of claim 1, wherein the transgene encoding AAT(M) comprises a nucleic acid sequence having at least about 95% sequence identity with SEQ ID NO: 590 or 596.

17. The genetic construct of claim 1, wherein the transgene encoding AAT(M) comprises the nucleic acid sequence of SEQ ID NO: 590 or 596, or comprises a nucleic acid sequence capable of hybridizing with the complementary sequence of SEQ ID NO: 590 or 596 under strict hybridization conditions.

18. The genetic construct of claim 1, wherein the transgene encoding AAT(M) comprises the nucleic acid sequence of SEQ ID NO: 590 or 596.

19. The genetic construct of claim 1, wherein the transgene encoding AAT(M) comprises the nucleic acid sequence of SEQ ID NO:

590.

20. The genetic construct of claim 1, wherein the transgene encoding AAT(M) encodes an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

600.

21. The genetic construct of claim 1, wherein the transgene encoding AAT(M) encodes an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

600.

22. The genetic construct of claim 1, wherein the transgene encoding AAT(M) encodes an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

600.

23. The genetic construct of claim 1, wherein the transgene encoding AAT(M) encodes the amino acid sequence of SEQ ID NO: 600 or a conserved substitution variant thereof.

24. The genetic construct of claim 1, wherein the transgene encoding AAT(M) encodes the amino acid sequence of SEQ ID NO:

600.

25. The genetic construct of claim 1, further comprising a liver-specific promoter.

26. The genetic construct of claim 25, wherein the liver-specific promoter comprises the nucleic acid sequence of SEQ ID NO: 587 or 594.

27. A vector comprising the genetic construct as described in any one of claims 1-26.

28. The vector of claim 27, wherein the vector is a plasmid, a viral vector, or a non-viral vector.

29. The vector of claim 28, wherein the viral vector is an adenovirus vector.

30. The vector of claim 29, wherein the adenovirus vector is missing part or all of the E1 region and / or E4 region.

31. The vector of claim 29, wherein the adenovirus vector is a chimpanzee adenovirus vector.

32. The vector of claim 29, wherein the adenovirus vector is a GC44 gorilla adenovirus vector.

33. The vector of claim 27, comprising a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

593.

34. The vector of claim 27, comprising a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:

593.

35. The vector of claim 27, comprising a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:

593.

36. The vector of claim 27, comprising the nucleic acid sequence of SEQ ID NO: 593, or comprising a nucleic acid sequence capable of hybridizing with the complementary sequence of SEQ ID NO: 593 under strict hybridization conditions.

37. The vector of claim 27, wherein the nucleic acid sequence of SEQ ID NO: 593 is contained.

38. A method for treating a disease or disorder of a subject, the method comprising administering to the subject a therapeutically effective amount of the carrier as described in claim 27.

39. The method of claim 38, wherein the disease or disorder is a liver or lung disease associated with α-1 antitrypsin deficiency.

40. The method of claim 38, wherein the disease or disorder is COPD, bronchiectasis, neonatal cholestasis, cirrhosis and fibrosis, or hepatocellular carcinoma.

41. The method of claim 38, wherein the therapeutically effective amount comprises about 1 × 10⁻⁶. 9 To approximately 1×10 12 Particle unit (PU).

42. The method of claim 38, wherein the method further comprises administering an additional therapeutic agent.

43. The method of claim 42, wherein the additional therapeutic agent is an immunosuppressant.

44. A composition comprising the genetic construct as described in any one of claims 1-26.

45. The composition of claim 44, for treating a disease or disorder in a person who has this need.

46. ​​Use of the genetic construct according to any one of claims 1-26 in the preparation of a medicament for treating a disease or disorder of a subject in need.

47. A kit comprising the genetic construct as described in any one of claims 1-26 and instructions for use.

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