WO2025219443A1

WO2025219443A1 - Nucleic acid for a1at regulation

Info

Publication number: WO2025219443A1
Application number: PCT/EP2025/060487
Authority: WO
Inventors: Paula DE SOUSA MONTENEGRO MIRANDA; Romain Pascal GALMES; Morgane Sarah WARTEL
Original assignee: Uniqure Biopharma BV
Current assignee: Uniqure Biopharma BV
Priority date: 2024-04-19
Filing date: 2025-04-16
Publication date: 2025-10-23
Anticipated expiration: 2026-10-19

Abstract

The present invention relates to a nucleic acid that encodes both an Alpha-1 antitrypsin (A1AT or AAT) protein and an RNA molecule able to inhibit the expression of an endogenous A1AT protein. The invention further relates to associated expression cassettes, recombinant adeno-associated virus (rAAV) vectors, promoters, pharmaceutical compositions and kits for use in the treatment of Alpha-1 antitrypsin deficiency (A1ATD or AATD).

Description

NUCLEIC ACID FOR A1AT REGULATION

Field of the invention

The present invention relates to the fields of biotechnology, medicine, and gene therapy. Specifically, the invention relates to a nucleic acid comprising a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region of a human SERPINA1 gene and a sequence encoding an oxidation-resistant A1AT protein. The invention further relates to associated expression cassettes, recombinant adeno-associated virus (rAAV) vectors, promoters, pharmaceutical compositions and kits for use in the treatment of Alpha-1 antitrypsin deficiency (A1ATD or AATD).

Background of the invention

The SERPINA1 gene encodes a1 -antitrypsin (A1AT or AAT), a key serine protease inhibitor (Pi) produced primarily in the liver, but principally active in the lungs and liver. Importantly, A1AT inhibits neutrophil elastase (NE), a serine protease which is active in the lungs, thereby protecting the lungs from proteolytic damage.

Alpha-1 antitrypsin deficiency (A1ATD or AATD), caused by mutations in the SERPINA1 gene, is inherited in an autosomal co-dominant manner, meaning that each allele contributes to the phenotype. The most common allele is called M and produces normal serum levels of A1AT. The S allele, consisting of a single nucleotide mutation in the gene resulting in substitution of the amino acid glutamine for valine at position 264 in the protein (p.Glu264Val), produces lower levels of A1AT. The Z allele, which is the most common mutation, consisting of a single nucleotide mutation in the gene resulting in the substitution of the amino acid glutamine for lysine at position 342 in the protein (p.Glu342Lys), produces very low serum levels (15-20% compared to the M allele). A1ATD clinical manifestations are often correlated with the two pathogenic variants, the Z allele and the S allele, which can be combined in severe ZZ (PI*ZZ) or moderate SZ (PI*SZ) risk genotypes. Individuals affected by these most common mutations have serum A1AT concentrations in the range of 25% and 15% of normal levels, respectively, predisposing individuals to predominantly lung and/or liver disease (Barjaktarevic and Miravitlles, 2021) (Fregonese et al. 2008).

In patients with the ZZ mutation, the mutant protein accumulates and aggregates within hepatocytes. This protein aggregation causes liver diseases such as cirrhosis, fibrosis, and hepatocellular carcinoma (Crowther et al. 2004). The resulting decrease of circulating A1AT protein leads to reduced uptake of A1AT by the lungs, which then leads to reduced NE inhibition (Taggart et al. 2000). As a consequence, there is an imbalance between NE and antiprotease activity, eventually leading to lung diseases (Crowther et al. 2004), such as panacinar emphysema and chronic obstructive pulmonary disease (COPD).

There is no cure for A1ATD. Current therapy focuses on treating the symptoms of the separately affected organs. For example, COPD therapeutics (inhaled bronchodilators for symptomatic treatment) and enzyme replacement therapy, which requires recurrent (often weekly) intravenous infusions, for the lungs and, in severe cases, liver transplantation for treatment of symptoms related to the liver. There is thus a clear need for a treatment for A1ATD which treats both the liver and lungs.

Summary of the Invention

The present invention solves the problem by providing novel nucleic acid and gene therapy technologies.

In a first aspect, the present invention provides a nucleic acid that comprises a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region of a human SERPINA1 gene;

In one embodiment, the present invention provides a nucleic acid comprising a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region within an mRNA encoded by the human SERPINA1 gene.

In one embodiment, the present invention provides a nucleic acid that comprises a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region of a human SERPINA1 gene and/or a target region within an mRNA encoded by the human SERPINA1 gene, and a sequence encoding an oxidation-resistant A1AT protein.

A second aspect of the invention relates to a promoter as defined herein comprising: a. a sequence comprising SEQ ID NO. 171 , SEQ ID NO. 172, SEQ ID NO. 173, SEQ ID NO. 174 and SEQ ID NO. 176 or a variant thereof; b. a minimal promoter, preferably wherein the minimal promoter comprises SEQ ID NO. 177; and c. an intronic sequence, preferably comprising one of SEQ ID NOs. 164, 165, or 166, more preferably comprising SEQ ID NO. 164.

A third aspect of the invention relates to an expression cassette comprising a nucleic acid according to the invention, preferably wherein the expression cassette is flanked by Inverted Terminal Repeats (ITRs).

A fourth aspect of the invention relates to a recombinant adeno-associated virus (rAAV) vector comprising the expression cassette according to the invention, preferably wherein the rAAV vector comprises AAV5 capsid proteins.

In one embodiment, the rAAV has a serotype selected from AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, variants thereof and hybrid serotypes A fifth aspect of the invention relates to the use of a nucleic acid molecule, an expression cassette, or an rAAV vector according to the invention in a treatment, preferably for use in the treatment of Alpha- 1 Antitrypsin Deficiency.

Detailed description of the invention

The present invention relates to gene therapy, in particular to nucleic acids comprising a sequence encoding an RNA that reduces or silences endogenous A1AT expression, and a sequence encoding an oxidation-resistant A1AT protein. This approach allows targeting of both the liver and lungs of patients by, respectively, reducing the accumulation and aggregation of the endogenous (nonfunctional) mutant A1 AT in the liver, and restoring the expression of functional A1AT in the circulation to protect the lung. To further optimize this strategy, the present invention provides a functional A1 AT that is more resistant to oxidative stress.

General definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In contrast, the verb "to consist of and its conjugations are used in a more limiting sense to mean that items not specifically mentioned are excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

As used herein, the term "and/or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

As used herein, the term "at least" followed by a particular value n means n or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14,15, ... ,etc.

The word "approximately" when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (10, for the previous example) more or less 10% of the value (a value of between 9 - 11 for the previous example).

"Sequence identity" is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, when it comes to amino acid or nucleic acid sequences, "identity" means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Sequence identity" can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using global alignment algorithms (e.g. Needleman Wunsch) which align the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using local alignment algorithms (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical" when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). Alternatively, percentage identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.

Within the context of the present invention, the term "variant thereof, when referring to a given sequence (SEQ ID NO.) may include any nucleic acids with at least 75, 80, 90, 95 or 98% sequence identity with that given sequence.

The term "substantially complementary" in this context means that a given nucleic acid sequence is at least 85 %, for instance at least 90 %, and in some embodiments, at least 95 % complementary to another nucleic acid sequence. It is not required to have all the nucleotides of the guide sequence and the target sequence to be base paired, i.e. to be fully complementary.

The term “coding sequence” as used herein includes both the sense and the antisense strand of a gene, as well as intronic sequences.

Where herein reference is made to the gene “SERPINA1", reference is made to the human serpin family A member 1 gene as referenced by HGNC (HUGO Gene Nomenclature Committee) accession code:8941 and variants thereof. Synonyms for SERPINA1 are “AAT” “PI1 ” and “PI”.

Where herein reference is made to variants of the SERPINA1 gene, such variants are intended to include mutations, point mutations, deletions and/or insertions in the coding sequence of the human SERPINA1 gene, resulting in decreased serum levels of the alpha-1 -antitrypsin (A1AT) protein. Variants of the SERPINA1 gene include but are not limited to the Glu264Val and Glu342Lys amino acid substitutions, also referred to as “S allele” and “Z allele” respectively.

The terms “target region within a human SERPINA1 gene” or “target region within the coding sequence a human SERPINA1 gene” refer to a DNA sequence present in the human SERPINA1 gene, including the sense and antisense strands, as well as any intronic sequences. The term “target region within an mRNA encoded by the human SERPINA1 gene” refers to an RNA sequence present in the mRNA that is transcribed from the SERPINA1 gene as defined herein.

The term "complementary" or "complement" refers to two nucleotides that can form multiple favorable interactions with one another. Such favorable interactions include and may exclusively be Watson-Crick base pairing.

As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter is "operably linked" to a nucleotide sequence when the promoter controls and regulates the transcription of a coding sequence.

The use of a substance as a medicament as described in this document can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment. Products for use as a medicament described herein can be used in methods of treatments, wherein such methods of treatment comprise the administration of the product for use.

Nucleic acid

The present invention provides in a first aspect a nucleic acid comprising a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region within a human SERPINA 1 gene, or a variant thereof.

In one embodiment, the present invention provides a nucleic acid comprising a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region within an mRNA encoded by the human SERPINA1 gene and/or variants thereof.

In one embodiment, the target region within an mRNA encoded by the human SERPINA1 gene is selected from SEQ ID NOs: 206, 207, and/or 208.

The terms "nucleic acid molecule" or "nucleic acid" or “nucleotide sequence”, as used herein, takes its regular meaning in the art and refers to a string of nucleotides, which can either be ribonucleotides or deoxyribonucleotides. Thus, the terms "RNA" or "RNA molecule" or "ribonucleic acid molecule" as used herein refers to a polymer of ribonucleotides (e.g. 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides) and the terms "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" as used herein refers to a polymer of deoxyribonucleotides (e.g. 2, 3, 4, 5, 10, 15, 20, 25, 30, or more deoxyribonucleotides). Ribonucleotides and deoxyribonucleotides are both nucleotides that are composed of three subunit molecules: a nucleobase, a five-carbon sugar (ribose or deoxyribose), and a phosphate group consisting of one to three phosphates. The four nucleobases in DNA are guanine, adenine, cytosine and thymine; in RNA, uracil is used instead of thymine.

DNA and RNA can be synthesized naturally, e.g. by DNA replication or transcription of DNA, respectively. RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (/.e. ssRNA and ssDNA, respectively) or multistranded (e.g. double stranded, i.e. dsRNA and dsDNA, respectively).

Where herein reference is made to a guide sequence and this guide sequence includes “T” for the thymine base, the skilled person knows that the same sequence integrated in an miRNA scaffold will include “U” for the uracil base.

RNA molecules as described herein include RNA interference (RNAi) molecules.

RNAi occurs in cells naturally to remove foreign RNAs (e.g. viral RNAs) and to regulate gene expression. Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be induced, for example, to silence the expression of target genes. RNAi molecules are RNA molecules that are capable of RNA interference such as, siRNA (short or small interfering RNA) or micro RNAs (miRNAs) or short-hairpin RNAs (shRNAs).

In general, RNA molecules as used herein serve to silence, or negatively influence, or inhibit gene expression in a sequence-specific way. For example, RNA molecules, such as RNAi molecules, can be applied to suppress (or silence) the process of gene expression leading towards the production of the pathogenic or dysfunctional protein.

As used herein, the term "RNA interference" ("RNAi") refers to a selective intracellular degradation of RNA, pre-mRNA or mRNA. "mRNA" or "messenger RNA" is a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA. The pre-mRNA is the mRNA precursor that becomes a messenger RNA (mRNA) after processing. It contains both introns and exons. Pre-mRNA requires splicing (removal) of introns to produce the final mRNA molecule containing only exons. RNA molecules that are suitable for used in RNAi include siRNAs, shRNA miRNAs.

An siRNA is a double stranded RNA that comprises two separate RNA strands, one strand comprising a first RNA sequence and the other strand comprising a second RNA sequence. An siRNA design that may be used involves consecutive base pairs with a 3' overhang. The first and/or second strand(s) may comprise a 3'-overhang. The 3'-overhang preferably is a dinucleotide overhang on both strands of the siRNA. Such a design is based on observed Dicer processing of larger double stranded RNAs that results in siRNAs having these features (Vermeulen et al. 2005). The 3'-overhang may be comprised in the first strand. The 3'- overhang may be in addition to the first strand. The length of each of the two strands of which an siRNA is composed may, independently, be 19, 20, 21 , 22, 23, 24, 25, 26 or 27 nucleotides or more.

An shRNA, like the siRNA Dicer substrate described above, can be processed by e.g. Dicer to provide for an siRNA having a design such as described above, having e.g. 19 consecutive base pairs and 2 nucleotide overhangs at both 3'-ends. In case the shRNA is to be processed by Dicer, it is preferred to have the first and second strands at the end of the shRNA, i.e. such that the putative strands of the siRNA are linked via a stem loop sequence: 5' - first strand - stem loop sequence - second strand - optional 2 nt overhang sequence - 3'. Or, conversely, 5' - second strand - stem loop sequence - first strand - optional 2 nt overhang sequence - 3'. Another shRNA design may be a shRNA structure that is processed by the RNAi machinery to provide for an activated RNA-induced silencing complex (RISC) that does not require Dicer processing (Liu et al. 2013 Nucleic Acids Res. 41 (6):3723-33, incorporated herein by reference), so called AgoshRNAs, which are based on a structure very similar to the miR-451 scaffold as described below. Such an shRNA structure comprises in its loop sequence part of the first RNA sequence. Such an shRNA structure may also consist of the first strand, followed immediately by the second strand.

A microRNA, i.e. miRNA, is a guide strand that originates from double stranded RNA molecules that are expressed e.g. in mammalian cells. A miRNA is processed from a pre-miRNA precursor molecule, similar to the processing of an shRNA or an extended siRNA as described above, by the RNAi machinery and incorporated in an activated RNA-induced silencing complex (RISC) (Tijsterman M, Plasterk RH. Dicers at RISC; the mechanism of RNAi. Cell. 2004 Apr 2;117(1):1-3). Without being bound by theory, a pre-miRNA is a hairpin molecule that can be part of a larger RNA molecule (pri- miRNA), e.g. comprised in an intron, which is first processed by Drosha to form a pre-miRNA hairpin molecule (Lin et al. 2003). The pre-miRNA molecule is an shRNA-like molecule that can subsequently be processed by Dicer to result in an siRNA-like double stranded duplex. The miRNA, i.e. the guide strand, that is part of the double stranded RNA duplex is subsequently incorporated in RISC.

An RNA molecule such as present in nature, i.e. a pri-miRNA, a pre-miRNA or a miRNA duplex, may be used as a scaffold for producing an artificial miRNA that specifically targets a gene of choice, by binding to a specific region on the pre-mRNA or the mRNA derived from that gene of choice. Based on the predicted RNA structure, e.g. as predicted using e.g. m-fold software, the natural miRNA sequence as it is present in the RNA structure (i.e. duplex, pre-miRNA or pri-miRNA), and the sequence present in the structure that is complementary therewith are removed and replaced with a guide sequence and a complementary passenger sequence. The guide sequence and the complementary passenger sequence may be selected such that the RNA structures that are formed, i.e. pre-miRNA, pri-miRNA and/or miRNA duplex, resemble the corresponding predicted original sequences. pre-miRNA, pri- miRNA and miRNA duplexes (that consist of two separate RNA strands that are hybridized via complementary base pairing), as found in nature often are not fully base paired, i.e. not all nucleotides that correspond with the first and second strand as defined above are base paired, and the first and second strand are often not of the same length. How to use miRNA precursor molecules as scaffolds for any selected target sequence and substantially complementary first RNA sequence is described e.g. in Liu YP Nucleic Acids Res. 2008 May;36(9):2811-2. As defined herein, the guide and the passenger RNA sequence might be incorporated in a pri-miRNA hairpin or pre-miRNA.

In one embodiment, the nucleic acid comprises a sequence encoding an RNA, preferably wherein the RNA is selected from siRNA, shRNA or miRNA, more preferably the RNA is a miRNA.

In one embodiment, the sequence encoding the RNA according to the invention comprises in a 5’ to 3’ direction (/.e. the direction of the coding strand in case of a double stranded (ds) nucleic acid): a 28 nucleotides 5’ flanking region (SEQ ID NO. 184), miR-144 helper scaffold (SEQ ID NO. 181), a spacer (SEQ ID NO. 185), one miR-451 scaffold comprising a guide sequence of Table 1 with the characteristics described below, and a 205 nucleotides 3’ flanking region (SEQ ID NO. 186).

In some embodiments, the spacer comprises at least: 15; 20; 25; 30; 35; 40; 45; 50; 55; 60; 65; 70; 75; 80; 85; or 90 nucleotides. In some embodiments, the spacer comprises at least 75 nucleotides. In a specific embodiment of the invention, the spacer comprises a sequence having SEQ ID NO. 185; or variants thereof.

In one embodiment, the nucleotide sequence encoding the miR-144 hairpin comprises SEQ ID NO. 183, or a variant thereof and the nucleotide sequence encoding the miR-451 hairpin comprises SEQ ID NO. 182 or a variant thereof.

In one embodiment, the pri-miRNA scaffold comprises, or is based on SEQ ID NO: 182.

In some embodiments, the nucleotide sequence encoding the miR-144 hairpin has at least one mutation. In some other embodiments, the mutation is a single point mutation to reduce processing and/or expression of the miR-144. As a consequence, any mismatch, bulge or G-U-wobble introduced within positions 4-8 of the Drosha cleavage site may impair the enzymatic activity of Drosha. Double and triple mismatches, bulges or wobbles within said positions further decrease the activity of Drosha. Therefore, any of the following single nucleotide polymorphisms (SNPs) and combinations thereof within the 4-8 nucleotide stretch of miR-144 may alter (pre-)miR-144 expression. In some embodiments, the nucleic acid encoding for the miR-144 hairpin comprises at least one mutation selected from the group consisting of: T>G at position 4; A>T or G at position 5; T>A at position 6; C>G or T at position 7; and A>T or G at position 8. In preferred embodiments, the nucleic acid encoding for the miR-144 hairpin comprises: a single point mutation A>T at position 5. Thus, in one embodiment the nucleotide sequence encoding the miR-144 comprises SEQ ID NO. 181 or a variant thereof, and reduces or eliminates processing and/or expression of the miR-144 hairpin. The skilled person can easily determine whether this is the case by using standard assays and appropriate controls such as described in the examples and as known in the art. The miR-451 scaffold is found to be particularly useful within the present invention as it can induce RNA interference that can result in mainly guide strand induced RNA interference. The miR-451 scaffold does not result in a passenger strand because the processing is different from the canonical miRNA processing pathway (Cheloufi et al. 2010 Nature 465(7298):584-9 and Yang et al., 2010 Proc Natl Acad Sci USA 107(34):15163-8). The miR-451 scaffold represents an excellent backbone to develop a gene therapy product as unwanted potential off-targeting by passenger strands can be largely, if not completely, avoided. As the passenger strand (corresponding to the second strand) may result in targeting other transcripts, using such scaffolds may prevent such unwanted targeting. Hence, it is preferred that selected scaffolds produce less than 15%; less than 10%; less than 5%; less than 4%; or less than 3% of passenger strands, when compared to the production levels of the guide sequence.

A miR-451 hairpin preferably comprises from 5' to 3', firstly 5'-CUUGGGAAUGGCAAGG-3' (SEQ ID NO. 187), followed by a sequence of 22 nucleotides, comprising or consisting of the first strand (guide sequence), followed by a sequence of 17 nucleotides, which can be regarded as the second strand, which is complementary over its entire length with nucleotides 2-18 of said sequence of 22 nucleotides, subsequently followed by sequence 5'-VWCUUGCUAUACCCAGA-3' (SEQ ID NO. 188, wherein V is an A or a G or a C and W is an A or a U). Preferably the first 5'-G/C/A nucleotide of the latter sequence is not to base pair with the first nucleotide of the first strand of the RNA.

Alternatively, the 5'-CUUGGGAAUGGCAAGG-3' (SEQ ID NO. 187) and 5'-VWCUUGCUAUCCCAGA- 3' (SEQ ID NO. 188) may be replaced by flanking sequences of other pri-miRNA structures.

As is clear from the above, the sequence of the hairpin may differ not only with regard to the (putative) guide strand sequence, and sequence complementary thereto, as present in the wild type hairpin, but may also comprise additional mutations in the 5’, loop and 3’ sequence as well, as additional mutations may be required to provide for an RNA structure that is predicted to mimic the secondary structure of the wild type hairpin.

Such a hairpin may be comprised in a larger sequence such as an intron or an RNA transcript, e.g. a pol II expressed transcript, comprising e.g. a 5'UTR and a 3' UTR and a polyA tail. Flanking structures may also be absent.

Guide sequences

The terms "guide strand" or "guide sequence" may also be referred to as "antisense strand" as it is complementary ("anti") to a target RNA sequence, i.e. the sense target RNA sequence that is comprised in a pre-mRNA encoded by a human gene. The RNA comprising a guide sequence also comprises a "sense strand or sequence" also called "passenger sequence" or "passenger strand", that may have substantial sequence identity with, or be identical to, the target RNA sequence. Therefore, the RNA comprising a guide and a passenger sequence can be described as a hairpin or a double stranded RNA as it is substantially complementary to itself. Such double stranded RNA is to induce RNA interference, thereby reducing expression of transcripts of a human gene. Transcripts that may be targeted may include mis-spliced, unspliced and spliced RNA. The double stranded RNA may also induce transcriptional silencing.

In one embodiment, the guide sequence is substantially complementary to a target RNA sequence within the mature RNA transcript encoded from the human SERPINA1 gene. A mature RNA transcript is known in the art to refer to mature mRNA consisting exclusively of exons. Therefore, in one embodiment, guide sequence is substantially complementary to a target region within the coding sequence of a human SERPINA1 gene.

The substantial complementarity between the guide sequence and the target RNA sequence preferably consists of at most three mismatched nucleotides, more preferably two or one mismatched nucleotide. It is understood that having three mismatches over the entire length of the guide sequence when base paired with the target RNA sequence, means that three nucleotides do not base pair with the target RNA sequence. Having two mismatches means that two nucleotides of the guide sequence do not base pair with the target RNA sequence. One mismatched nucleotide means that one nucleotide of the guide sequence does not base pair with the target RNA sequence. Having no mismatches means that all nucleotides of the guide sequence do base pair with the target RNA sequence.

The guide sequence or the target sequence may also comprise additional nucleotides that do not have complementarity to the target RNA sequence or the guide sequence, respectively. In such a scenario, the substantial complementarity is determined over the entire length of the target RNA sequence. This means that, if there are at most three additional nucleotides in either sequence, the target RNA sequence in this embodiment has either no, one, two or three mismatches over its entire length when base paired with the guide sequence.

Non-complementary, or mismatch, base pairs encompass the following nucleotide base pairs: A and A, G and G, C and C, U and U, A and C, C and U, or A and G. A mismatch may also result from a deletion of a nucleotide, or an insertion of a nucleotide. When the mismatch is a deletion in the strand sequence, this means that a nucleotide of the target RNA sequence is not base paired with the sequence when compared with the entire length of the guide sequence. Nucleotides that can align as a base pair are A-U, G-C and G-U which is also referred to as a G-U wobble, or wobble base pair. In one embodiment the number of G-U base pairs between the strand sequence and the target RNA sequence is 0, 1 or 2.

As long as the guide sequence is capable of inducing RNA interference by sequence-specifically targeting a sequence comprising the target RNA sequence, such substantial complementarity is contemplated in accordance with the invention.

Most preferably the guide sequence and the target RNA sequence have no mismatches. In some embodiments, there are no mismatches between the strand RNA sequence and the target RNA sequence and a G-U base pair or G-U base pairs is allowed. Preferably, there may be no G-U base pairs between the strand sequence and the target RNA sequence, or the strand sequence and the target RNA sequence only have base pairs that are A- U or G-C. Preferably, there are no G-U base pairs and no mismatches between the RNA strand sequence and the target RNA sequence. The strand sequence of the double stranded RNA as defined herein preferably is fully complementary to the target RNA sequence, said complementarity consisting of G-U, G-C and A-U base pairs. The strand sequence of the double stranded RNA as defined herein more preferably is fully complementary to the target RNA sequence, said complementarity consisting of G-C and A-U base pairs.

As said, it may not be required to have full complementarity (/.e. full base pairing (no mismatches) and no G-U base pairs) between the first or second strand RNA, and the target RNA sequence as such a guide strand can still allow for sufficient suppression of gene expression. Also, not having full complementarity may be contemplated for example to avoid or reduce off-target RNA sequence specific gene suppression while maintaining sequence specific inhibition of transcripts comprising the target RNA sequence. However, it may be preferred to have full complementarity as it may result in more potent inhibition.

Amongst others, exemplified guide sequences that target a region within the coding sequence of a human SERPINA1 gene include those included in Table 1 .

Table 1. A selection of guide sequences targeting a coding region within the coding sequence of the human SERPINA1 gene. The skilled person will be aware that below sequences when expressed as guide sequences according to the invention include U (uracil) where here T (thymine) is indicated.

In one embodiment, the present invention provides a nucleic acid comprising a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region within an mRNA encoded by the human SERPINA1 gene, wherein the guide sequence is selected from SEQ ID Nos: 3-115, or variants thereof.

In one embodiment, the present invention provides a nucleic acid comprising a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region within an mRNA encoded by the human SERPINA1 gene, wherein the guide sequence is selected from SEQ ID Nos: 209-321 , and/or variants thereof.

In one embodiment, the guide sequence has a length of 19 nucleotides, preferably 20 nucleotides, more preferably 21 or 22 nucleotides. In one embodiment, the guide sequence is substantially complementary to a target region within the coding sequence of a human SERPINA1 gene, preferably the guide sequence is selected from one of SEQ ID NOs. 3-115, or a variant thereof. In a preferred embodiment the guide sequence is substantially complementary to a target region within the coding sequence of a human SERPINA1 gene, preferably the guide sequence is selected from one of SEQ ID NOs. 3, 7, 8, 9, 11, 13, 31, 38, 39, 49, 50, 51, 52, 53, 54, 55, 56, 60, 61, 72, 74, 75, 76, 77, 78, 80, 81, 82, 83, 85, 89, 90, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 109, 110, and 114, or a variant thereof. In a more preferred embodiment, the guide sequence is substantially complementary to a target region within the coding sequence of a human SERPINA1 gene, preferably the guide sequence is selected from one of SEQ ID NOs. 55, 99, and 105, or a variant thereof.

In a preferred embodiment, the guide sequence is fully complementary to the target region within an mRNA encoded by the human SERPINA1 gene.

In one embodiment, the target region within an mRNA encoded by the human SERPINA1 gene is SEQ ID NO: 206, and the guide sequence is SEQ ID NO: 55, and/or the target region within an mRNA encoded by the human SERPINA1 gene is SEQ ID NO: 207, and the guide sequence is SEQ ID NO: 99, and/or the target region within an mRNA encoded by the human SERPINA1 gene is SEQ ID NO: 208, and the guide sequence is SEQ ID NO: 105.

In a preferred embodiment, the target region within an mRNA encoded by the human SERPINA1 gene is SEQ ID NO: 207, and the guide sequence is SEQ ID NO: 99.

In one embodiment, the target region within an mRNA encoded by the human SERPINA1 gene is SEQ ID NO: 206, and the guide sequence is SEQ ID NO: 261 , and/or the target region within an mRNA encoded by the human SERPINA1 gene is SEQ ID NO: 207, and the guide sequence is SEQ ID NO: 305, and/or the target region within an mRNA encoded by the human SERPINA1 gene is SEQ ID NO: 208, and the guide sequence is SEQ ID NO: 311 .

In a preferred embodiment, the target region within an mRNA encoded by the human SERPINA1 gene is SEQ ID NO: 207, and the guide sequence is SEQ ID NO: 305.

In one embodiment the guide sequence reduces endogenous A1 AT, or alpha-1 -antitrypsin expression by at least 40 % to 100 %. In one embodiment the guide sequence reduces endogenous A1AT expression by at least 50 % to 100 %. In one embodiment the guide sequence reduces endogenous A1AT expression by at least 60 % to 100 %. In one embodiment the guide sequence reduces endogenous A1AT expression by at least 70 % to 100 %. In a preferred embodiment the guide sequence reduces endogenous A1AT expression by at least 80 % to 100 %.

It will be understood that the complementarity between the guide sequence and the target region, when discussing RNAi, may have an effect on target gene expression levels. The skilled person is able to determine by how much the guide sequence reduces endogenous A1ATD expression by using gene expression assays which are known in the art, such as, but not limited to, standard luciferase reporter assays, qPCR assays, which incorporate appropriate controls (Zhuang et al. 2006 Methods Mol Biol.342:181-7). In some embodiments, the complementarity between the guide sequence and the target region within the coding sequence of a human SERPINA1 gene leads to a reduction of endogenous A1 AT expression of at least 40 %, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100%.

In a further embodiment, the nucleic acid as defined herein further comprises a sequence encoding an oxidation-resistant A1 AT protein.

A1AT

The SERPINA1 gene as described herein above encodes the protein Alpha-1 -antitrypsin, abbreviated as A1AT, which has alternative names Alpha-1 -protease inhibitor, Alpha-1 -antiprotease and SerpinAI . The wild-type A1AT protein sequence includes a methionine at both positions 351 (M351) and 358 (M358) (SEQ ID NO. 135). M351 and M358 are localized in the site that binds serine proteases through an irreversible interaction that inactivates both the protease and A1AT (Sosulski et al. 2020). Wild-type A1 AT protein is sensitive to oxidation, where exposure to oxidants results in the oxidation of M351 and M358. This oxidation reduces the ability of A1AT to inhibit neutrophil elastase (NE) (Taggart et al. 2000).

In one embodiment, the nucleic acid comprising a sequence encoding an oxidation-resistant A1AT protein comprises one or more oxidation-resistant amino acids. In a further embodiment, the nucleic acid according to the invention comprises a sequence encoding an oxidation-resistant A1AT protein, which comprises one or more oxidation-resistant amino acids. Oxidation-resistant amino acids include but are not limited to leucine, valine, glycine, isoleucine, alanine, threonine, asparagine, serine, aspartic acid and glutamic acid (Taggart et al. 2000, Sandoval et al. 2002, Sosulski et al. 2020, WO2013003641 A2). In one embodiment, the one or more oxidation-resistant amino acids are selected from leucine, valine, glycine, isoleucine, alanine, threonine, asparagine, serine, aspartic acid and glutamic acid. In one embodiment, the oxidation-resistant amino acid is at least at position 351 or at least at position 358. In one embodiment, the oxidation-resistant amino acid at least at position 351 . In one embodiment, the oxidation-resistant amino acid is leucine (M351 L), valine (M351V), glycine (M351 G), isoleucine (M351 I), alanine (M351A), threonine (M351T), asparagine (M351 N), serine (M351 S), aspartic acid (M351 D) or glutamic acid (M351 E). In a preferred embodiment, the oxidationresistant amino acid at position 351 , is one of leucine (M351 L), valine (M351V) and glutamic acid (M351 E).

In one embodiment, the oxidation-resistant amino acid is at least at position 358. In one embodiment, the oxidation-resistant amino acid is leucine (M358L), valine (M358V), glycine (M358G), isoleucine (M358I), alanine (M358A), threonine (M358T), asparagine (M358N), serine (M358S), aspartic acid (M358D) or glutamic acid (M358E). In a preferred embodiment, the oxidation-resistant amino acid is leucine (M358L) or valine (M358V). In one embodiment, the nucleic acid comprising a sequence encoding an oxidation-resistant A1AT protein comprises two or more oxidation-resistant amino acids. In one embodiment, the oxidationresistant amino acid is at least at position 351 and 358.

In one embodiment, the oxidation-resistant A1 AT protein comprises leucine at position 351 and leucine at position 358 (M351 L/M358L). In one embodiment, the oxidation-resistant A1AT protein comprises valine at position 351 and valine at position 358 (M351 V/M358V). In one embodiment, the oxidationresistant A1AT protein comprises glutamic acid at position 351 and valine at position 358 (M351 E/M358V). In a preferred embodiment, the oxidation-resistant A1 AT protein comprises valine at position 351 and leucine at position 358 (M351 V/M358L). In a more preferred embodiment, the oxidation-resistant A1AT protein comprises glutamic acid at position 351 and leucine at position 358 (M351 E/M358L). An A1AT protein that is more resistant to oxidative stress, could be beneficial in the lung microenvironment where there is a high level of pollutants inflammatory factors (Ciencewicki et al. 2008).

In one embodiment, the sequence encoding an oxidation-resistant A1AT protein is located between the promoter and the sequence encoding the polyAdenylation tail.

In one embodiment, the sequence encoding an oxidation-resistant A1AT protein does not comprise a sequence that is substantially complementary to the guide sequence.

In a preferred embodiment, the sequence encoding an oxidation resistant A1AT protein does not encode a sequence that is substantially complementary to the guide sequence.

In one embodiment, the sequence encoding an oxidation-resistant A1 AT protein is codon optimized. In one embodiment, the sequence encoding an oxidation-resistant A1 AT protein is codon optimized with reference to the full-length of the sequence.

The term “codon optimization” or “codon optimized” as used herein refers to the process or result of improvement of DNA or nucleotide codon composition of a recombinant gene without altering the amino acid sequence that is encoded by the recombinant gene.

Codon optimization may be carried out across the whole or only in part, or in multiple parts, of the sequence length. In one embodiment, codon optimization may be for the purpose of increasing transgene expression in relation to the host cell. In a further embodiment, codon optimization may be for the purpose of evading recognition of any co-expressed or resident inhibitory sequences. Examples of codon optimized sequences of the invention encoding a wild-type A1AT protein include, but are not limited to SEQ ID NOs. 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 126 and 127. In one embodiment, the sequence encoding an oxidation-resistant A1AT protein is codon optimized over the full-length of the sequence. In another embodiment, the sequence encoding an oxidationresistant A1 AT protein is codon optimized in one or more regions targeted by the guide sequences of the RNA as defined herein. Thus, the A1AT transcripts of such sequences differ sufficiently from the endogenous A1AT mRNA sequence such that it would not be recognized by mRNA targeting guide sequences. In one embodiment, the sequence encoding an oxidation-resistant A1 AT protein is codon optimized by reducing or completely removing the CG dinucleotides from the nucleotide sequences.

In one embodiment, the sequence encoding an oxidation-resistant A1 AT protein comprises one of SEQ ID NOs. 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 120, 122, 128 or 129 or a variant thereof. In a preferred embodiment, the sequence encoding an oxidation-resistant A1AT protein comprises SEQ ID NOs. 147, 120 or 122 or a variant thereof. In one embodiment, the amino acid sequence of the oxidation-resistant A1AT protein comprises one of SEQ ID NOs. 130 or 131 or a variant thereof, preferably SEQ ID NO. 131.

In one embodiment, the sequence encoding an oxidation resiatnt A1AT protein comprises SEQ ID NO: 120, 122, 128, or 129, or a variant thereof.

In one embodiment, the sequence encoding an oxidation-resistant A1AT protein further comprises a sequence encoding a hinge and sequence encoding a human Ig Fc, or a variant thereof. Thus, the sequence encoding an oxidation-resistant A1 AT protein further encodes a hinge and CH2/CH3 domains (Fc region (Fragment crystallizable)) of human immunoglobulins (Ig or antibodies), which consist or five main classes: IgA, IgD, IgE, IgG, an IgM, and the Fc (fragment crystallizable) region is the portion of an antibody that interacts with the immune system, or a variant thereof. The term “hinge” as used herein identifies a region that connects two proteins, generating an A1 AT-Fc fusion protein. A hinge is often a short sequence that acts as a flexible linker or region between two proteins or peptides. The Fc region extends the A1AT protein half-life via the protective binding to the neonatal Fc receptor (FcRn) (Liu et al. 2018). Indeed, the protein expressed from the sequence encoding an oxidation-resistant A1 AT fused to a human Ig Fc dimerizes forming a traditional Fc portion with two functional A1AT proteins, and by following the hFcRn recycling pathway, the half-life of the A1AT protein in the blood is increased.

In one embodiment, the human Ig Fc is an IgA Fc, an IgD Fc, an IgE Fc, an IgG Fc or an IgM Fc. In a preferred embodiment, the human Ig Fc is IgG Fc. In some embodiments, the Fc region is a human IgG 1 Fc. In some embodiments, the Fc region is a human lgG2 Fc. In some embodiments, the Fc region is a human lgG3 Fc. In a preferred embodiment, the Fc region is a human lgG4 Fc. In one embodiment, the sequence encoding an oxidation-resistant A1AT protein fused with an IgG Fc comprises one of SEQ ID NOs. 121 , 123, 124 or 125 or a variant thereof. Thus, In one embodiment, the sequence encoding an oxidation-resistant A1AT protein fused with an lgG1 Fc comprises SEQ ID NO. 123 or a variant thereof. In one embodiment, the sequence encoding an oxidation-resistant A1 AT protein fused with an lgG2 Fc comprises SEQ ID NO. 124 or a variant thereof. In one embodiment, the sequence encoding an oxidation-resistant A1AT protein fused with an lgG4 Fc comprises one of SEQ ID NOs.

125 or 121 or a variant thereof.

In one embodiment, the amino acid sequence of the oxidation-resistant A1AT-Fc fusion protein comprises one of SEQ ID NOs. 132, 133 or 134, or a variant thereof.

Expression construct

A nucleic acid for expression is commonly comprised within an expression construct, often comprising a promoter and/or further gene regulators. Therefore, the nucleic acid as defined herein may further comprise a promoter and a sequence encoding a polyA tail. Therefore, in one embodiment, the nucleic acid as defined herein further comprises a promoter and a sequence encoding a polyA tail. In one embodiment, the sequence encoding an A1AT protein is located between the promoter and the sequence encoding the polyA tail. The terms “expression construct” and “expression cassette” may be used interchangeably herein.

Wild type AAV particles carry a single stranded DNA genome, which consists of two open reading frames carrying the replicase genes and the capsid genes, Rep and Cap, and is flanked by two inverted terminal repeats (ITRs). In recombinant AAV particles, the single stranded DNA genome is replaced with an expression cassette.

The term “expression cassette” as used herein describes the functional unit capable of affecting expression of a transgene, or sequence encoding a product to be expressed. The coding sequence is operably linked to the appropriate expression control sequences, which at least comprises a suitable transcription regulatory sequence and optionally, 3' transcription termination signals. In addition, the term “expression cassette” as used herein described the functional unit capable of affecting expression of a transgene, or sequence encoding one or more products to be expressed. The sequence encoding one or more products to be expressed may be operably linked to the appropriate expression control sequences, which may comprise a suitable transcription regulatory sequence and optionally, 3' transcription termination signals. Additional factors necessary or helpful in affecting expression may also be present, such as expression enhancer elements, and would be known to the skilled person.

In one embodiment, the present invention provides an expression cassette comprising a nucleic acid comprising:

- a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region within an mRNA encoded by the human SERPINA1 gene;

- a sequence encoding an A1AT protein, preferably wherein the A1AT protein is oxidation-resistant;

- a promoter; and

- a sequence encoding a polyAdenylation tail. An expression cassette as used herein may be flanked by at least one ITR. In one embodiment, the expression cassette is flanked by two AAV ITR nucleotide sequences, preferably whereby the expression cassette is located in between the two AAV ITR nucleotide sequences. In another embodiment, the expression cassette is flanked by one ITR engineered with two D regions, wherein preferably the expression cassette is located on either side of the engineered ITR. In a preferred embodiment, the expression cassette is flanked by inverted terminal repeats.

Typically, the nucleic acid molecule comprising an expression cassette and at least one ITR as used herein is 5,000 nucleotides (nt) or less in length. The skilled person is aware that the maximum AAV packaging limit is understood to be 5.5 kilobasepairs (kbp). However, in other embodiments, wherein oversized nucleic acid constructs are used, e.g. more than 5,000 nt in length, or even more than the maximum AAV packaging limit of 5.5 kbp may also still allow the generation of rAAV particles and are therefore not excluded.

The terms “polyA tail”, “polyAdenylation tail” or “poly(A) tail” as used herein are used to define the chain or sequence of adenine nucleotides that is added to the 3’ end of a messenger RNA (mRNA) sequence after transcription in order to increase the stability of the mRNA molecule. The synthesis of a polyA tail requires a polyAdenylation signal and a polyAdenylation site. In some embodiments the sequence encoding a polyA tail includes a sequence encoding a polyadenylation signal and a sequence encoding a polyAdenylation site.

In one embodiment, there is provided a nucleic acid according to the invention, further comprising a promoter and a sequence encoding a polyAdenylation tail.

The term “promoter” as used herein, refers to sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter. Typically, promoters are structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. The promoter and the coding sequence may be operably linked.

A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer or biological entity (cyclespecific). A "tissue-specific" or a "cell-type-specific" promoter shows activity in a specific cell or tissue. Many such promoters are known in the art (see Sambrook and Russel, 2001 , supra). Constitutive promoters that are broadly active in many cell types, include but are not limited to the CMV promoter (Foecking et al. 1986) or CAG promoter (Miyazaki et al. 1989). Examples of liver-specific promoters include but are not limited to the LP1 promoter (Nathwani et al. 2006, included herein by reference, as are all other cited references) and the P5 promoter (SEQ ID NO. 163; W02020104424A1 , which is included herein by reference).

In one embodiment, the promoter comprises SEQ ID NO. 162 or 168. Thus, the promoter of the invention is capable of driving transcription in a liver cell, such as in hepatocytes. In a more preferred embodiment, the promoter comprises SEQ ID NO. 168. Thus, the promoter of the invention may be a liver-specific promoter.

In one embodiment, the promoter comprises an intronic sequence. In a preferred embodiment, the intronic sequence comprises SEQ ID NOs. 164, 165 or 166, or the intronic sequence is selected from SEQ ID NOs. 164, 165 or 166. In a more preferred embodiment, the intronic sequence comprises SEQ ID NO. 164.

The sequence encoding the RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region within a human SERPINA1 gene, may be comprised within the promoter as defined herein.

Therefore, in one embodiment, the promoter comprises the sequence encoding the RNA.

In one embodiment, the promoter comprises SEQ ID NO. 164 and the sequence encoding the RNA.

By incorporating the sequences encoding the RNA within the promoter, the inventors identified the beneficial effect of deriving a smaller construct for expression of both the RNA and the protein of the invention. This may have significant benefit in downstream processing, such as in the application of viral vector technology.

The nucleic acid, as described above, may further comprise a sequence encoding a polyA tail operably linked to the 3’ end of the sequence encoding an oxidation-resistant A1AT protein. Examples of a suitable polyA tails include, but are not limited to those derived from the simian virus 40 polyadenylation signal (SV40 polyA), a synthetic polyadenylation signal or the Bovine Growth Hormone polyadenylation signal (BGH polyA). In one embodiment, the sequence encoding the polyA comprises the SV40 polyA. In a further embodiment, the sequence encoding for the polyA comprises SEQ ID NO. 180.

The term "expression cassette" or “expression construct” as used herein describes the functional unit capable of affecting expression of a transgene, or sequence encoding a gene product of interest to be expressed. The expression cassette contains elements that are operably linked. The coding sequence is operably linked to the promoter and, optionally, 3' transcription termination signals. The expression cassette may comprise a suitable transcription regulatory sequence. Additional factors necessary or helpful in affecting expression may also be present, such as expression enhancer elements. The expression cassette may further contain leader sequences and fusion partner sequences. The expression cassette can also include the design of the nucleic acid sequence such that undesirable, potential initiation codons in and out of frame, are removed from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. Thus, an expression cassette can include promoters comprising enhancers and at least one intronic sequence, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a proteinencoding gene, splicing signal for introns, and stop codons.

A novel promoter

A further aspect of the invention relates to a promoter comprising: a) a sequence comprising SEQ ID NO. 171 , SEQ ID NO. 172, SEQ ID NO. 173, SEQ ID NO. 174 and SEQ ID NO. 176 or variants thereof; b) a minimal promoter and c) an intronic sequence.

In some embodiments, the minimal promoter comprises SEQ ID NO. 177 or 178. In a preferred embodiment, the minimal promoter comprises SEQ ID NO.177.

In a further embodiment, the minimal promoter comprises SEQ ID NO: 322.

In one embodiment, the intronic sequence comprises or consists of SEQ ID Nos: 164, 165 or 165. In a preferred embodiment the intronic sequence comprises or consists of SEQ ID NO: 164.

The inventors have fortuitously identified a promoter that drives efficient expression of a transgene in a liver cell.

The term “minimal promoter” as used herein is also known as a core promoter, as refers to is a short DNA sequence that allows for the formation of the initiation complex. Previously identified minimal promoters include but are not limited to those defined in WQ2020104424A1. In some embodiments, a variant of SEQ ID NO. 176 is selected from SEQ ID NOs. 175 or 205.

In some embodiments, the intronic sequence is derived from the SV40 promoter. In another embodiments, the intronic sequence derived from the LP1 promoter. In one embodiment, the intronic sequence comprises one of SEQ ID NOs. 164, 165, or 166, more preferably comprises SEQ ID NO. 164. In one embodiment, the intronic sequence is adjacent to the minimal promoter sequence. In one embodiment, the intronic sequence is comprised within the minimal promoter sequence. In a preferred embodiment, the promoter comprises SEQ ID NO. 168.

The promoter, as defined above, may drive expression of a transgene at least 1.5 fold greater than the same promoter without the intronic sequence. In some embodiments, the promoter drives the expression of a transgene encoding for a protein, such as Alpha-1 -antitrypsin (SERPINA 1), Alanineglyoxylate aminotransferase AGXT), Alpha Galactosidase (GL4), Arginase-1 (ARGT), Argininosuccinate lyase (ASL), ATPase Copper Transporting Beta (ATP7B), 2-oxoisovalerate dehydrogenase subunit alpha (BCKDHA), 2-oxoisovalerate dehydrogenase subunit beta (BCKDHB), Complement Factor H (CFH), Lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex (DBT), Alpha-galactosidase A (GLA), Fumarylacetoacetase (FAH), Coagulation Factor IX (FIX), Coagulation Factor VIII (FVIII), Hepcidin (HAMP), Hereditary hemochromatosis protein (HFE), N-Acetylglutamate Synthase (NAGS), Ornithine Transcarbamylase (OTC), Propionyl-CoA carboxylase alpha chain (PCCA), Propionyl-CoA carboxylase beta chain (PCCB), Glutathione S-transferase P (GSTP1), Solute carrier family 40 member 1 (SLC40A1), Transferrin receptor protein 2 (TFR2), Transthyretin (TTR), UDP-glucuronosyltransferase 1A1 (UGT1A1), or variants, derivatives or equivalents thereof.

Vectors

Expression cassettes according to the invention can be transferred to a cell, using e.g. transfection or transduction methods. Any suitable means may suffice to transfer an expression cassette according to the invention. Preferably, gene therapy vectors are used that stably transfer the expression cassette to the cells such that stable expression ofthe RNA and/orthe protein as described above can be achieved. Suitable gene therapy vectors may be lentiviral vectors, retrotransposon-based vector systems, or recombinant adeno-associated virus (rAAV) vectors.

Recombinant parvoviruses, in particular dependoviruses such as infectious human or simian adeno- associated virus (AAV), and the components thereof (e.g. a parvovirus genome), may be used as vectors for introduction and/or expression of nucleic acids in mammalian cells, preferably human cells. An AAV vector is defined as a recombinantly produced AAV or AAV particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.

A preferred viral vector used is an rAAV vector. Therefore, a further aspect of the present invention relates to rAAV vectors comprising the nucleic acid or the expression cassette of the invention.

In a preferred embodiment, the expression cassette as disclosed herein is flanked by at least one Inverted Terminal Repeats (ITR), preferably one on each side. The expression cassette flanked by at least one ITR might be incorporated into a larger nucleic acid construct (e.g. in a chromosome or in another vector such as a plasmid or baculovirus used for cloning or transfection), this is typically referred to as a "pro-vector" which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions.

In one embodiment, the AAV ITR sequences for use in the context of the present invention are derived from AAV1 , AAV2, and/or AAV5. More preferably, the AAV ITR sequences are derived from AAV2.

In one embodiment, the present invention provides an rAAV vector comprising an expression cassette, wherein the expression cassette comprises a nucleic acid comprising: - a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region within an mRNA encoded by the human SERPINA1 gene;

- a promoter; and

- a sequence encoding a polyAdenylation tail.

Preferably, the rAAV vector that is used is a rAAV vector of serotype 5, i.e. an AAV comprising AAV5 capsid proteins. Thus, in one embodiment the rAAV vector that is used is a rAAV vector of serotype 5. rAAV5 vector may be particularly useful for transducing liver cells. The capsid proteins are three (VP1- VP2-VP3) and are the structural proteins that define the serotype of an AAV. The capsid proteins are combined to form the viral capsid in a ratio of VP1 :VP2:VP3 of 1 :1 :10. In another embodiment, the rAAV vector that is used is a rAAV vector of serotype 8 or rAAV vector of serotype 6.

The sequences coding for the capsid viral proteins derived from the baculoviral cap gene: VP1 , VP2, and VP3, for use in the context of the present invention may be taken from any of the known 42 serotypes, more preferably from AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 or newly developed AAV-like particles obtained by e.g. capsid shuffling techniques and AAV capsid libraries. AAV capsids may consist of VP1 , VP2 and VP3, but may also consist of VP1 and VP3.

In some embodiments, the rAAV vector that is used is a chimeric rAAV vector, preferably the rAAV vector that is used is rAAV5-2. A chimeric rAAV vector has a capsid composed of capsid proteins that have been modified by domain or amino acid swapping between different serotypes. A rAAV5-2, for example, has the N-terminus of the original AAV5 VP1 capsid protein replaced by the AAV2 VP1 (Urabe et al. 2006). Such modification allows high titer production of recombinant AAV5. Other modifications include additions, deletions, or substitutions of amino acids in the capsid proteins. Most preferably, the viral vector is of the AAV5 serotype and of the AAV2 serotype, which can also be referred to as an AAV2/5 serotype. Herein the first 136 residues of the AAV5 VP1 protein are replaced with the first 137 residues of the AAV2 VP1 protein.

In a preferred embodiment, the rAAV vector has a serotype selected from AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, variants thereof and hybrid serotypes.

In some embodiments, the rAAV vector that is used is a mosaic rAAV vector. Such vector a rAAV vector has a capsid composed of a mixture of capsid subunits from different serotypes.

Compositions and kits

In another aspect, the invention relates to a pharmaceutical composition comprising a nucleic acid or an expression cassette according to the invention or a rAAV vector comprising the nucleic acid according to the inventions, or the expression cassette according to the inventions. In one embodiment, the invention provides a composition comprising a rAAV vector according to the invention and suitable excipients, such as buffers and stabilizers. In one particular embodiment, these compositions are used to transduce cells with the rAAV vector in vitro or ex vivo, in which case the excipients will need to be compatible with cell culture.

In other embodiments, the compositions are administered to an animal subject. In other preferred embodiments, the compositions are used for treatment of (human) subjects. For that purpose, the invention provides a pharmaceutical composition comprising a rAAV vector according to the invention and at least one pharmaceutically acceptable carrier. In the case of AAV gene delivery viral vectors, a pharmaceutical composition typically comprises physiological buffers, such as e.g. PBS, comprising further stabilizing agents such as e.g. sucrose. Such compositions are compatible with and suitable and intended for use in an administration which is performed intravascularly, intravenously, intraperitoneally, intramuscularly, subcutaneously, intrathecally, intravitreally, sub-retinally, intranasally, or injection directly into the hepatic portal vein or the intracoronary system or perfusion of the isolated limb. In preferred embodiments, the administration is performed intravenously. In some embodiments, one administration is followed at least by a second administration. In preferred embodiments, the administration is only given one single time. In further preferred embodiments, the composition is administered intravenously.

In one embodiment, the composition comprising the rAAV vector according to the invention is substantially isotonic to human blood. Tonicity is a measure for the effective osmotic pressure that a liquid formulation can exert and depends primarily on the number of dissolved particles in solution. Osmotic pressure is an important factor affecting biological cells. Hypertonicity is the presence of a solution that causes cells to shrink. Hypotonicity is the presence of a solution that causes cells to swell. Isotonicity is the presence of a solution that produces no change in cell volume. When a biological cell is in a hypotonic environment, the cell interior accumulates water, water flows across the cell membrane into the cell, causing it to expand. For mammalian cells this can lead to cytolysis, and tonicity is therefore important when fragile cells are to be exposed to a composition. Tonicity agents can therefore be added to preparations such as injectable preparations to prevent osmotic shock at the site of injection upon administration and thereby reduce local irritation or even damage to tissues or blood cells.

In a preferred embodiment, the isotonic composition as described herein is substantially isotonic to human blood, which has a tonicity or osmotic concentration of about 290 mOsm/kg. In particular, the composition as described herein may have an osmolality, also referred to as tonicity or osmotic concentration, of from 250 to 330 mOsm/kg, such as 260 to 310 mOsm/kg. In some preferred embodiments, the isotonic composition as described herein has an osmolality of from 260 to 320 mOsm/kg, more preferably from 270 to 315 mOsm/kg, most preferably from 274 to 310 mOsm/kg. For example, the isotonic composition as described herein can have an osmolality of 290 mOsm/kg. Typical tonicity agents are excipients used for tonicity adjustment and are known in the art. Tonicity agents can include dextrose, glycerin, mannitol, and metal salts. Metal salts are preferred, and preferred metal salts are pharmaceutically acceptable metal salts. Thus in preferred embodiments the isotonic composition as described herein further comprises a pharmaceutically acceptable salt at a concentration of at least 50 mM, wherein the salt is preferably NaCI, KCI, CaCh, MgCh, or combinations thereof.

Pharmaceutically acceptable metal salts may comprise a periodic group 1 or group 2 metal salt, preferably a periodic group 1 or group 2 metal chloride salt, preferably selected from the group consisting of NaCI, KCI, CaCh, MgCh, and combinations thereof. NaCI is particularly preferred.

The isotonic composition as described herein preferably comprises the pharmaceutically acceptable metal salt at a concentration greater than about 55, 60, or 65 mM, particularly 75 mM, which has been found beneficial for the stability of the drug product, as measured through absorbance and visual inspection per the Examples disclosed herein. Preferably, the concentration of pharmaceutically acceptable salt may be about 75 mM to about 200 mM, preferably about 80 mM to about 175 mM, more preferably about 85 mM to about 160 mM, more preferably about 90 to about 155 mM, more preferably about 95 to about 150 mM, more preferably about 100 to about 145 mM, more preferably about 105 to about 140 mM, more preferably about 115 to about 135 mM, more preferably about 120 to about 130 mM, such as most preferably about 125 mM. Thus, in some embodiments, the isotonic composition as described herein comprises a pharmaceutically acceptable salt selected from the group consisting of NaCI, KCI, CaCh, MgCh, and combinations thereof, at a concentration of about 100 mM to about 150 mM, preferably of about 115 mM to about 135 mM, more preferably about 120 to about 130 mM, such as most preferably about 125 mM.

In some specific embodiments, the pharmaceutically acceptable salt comprises NaCI, present in the composition as described herein at a concentration of about 75 mM or higher, preferably about 75 mM to about 150 mM, more preferably about 100 mM to about 150 mM, preferably of about 115 mM to about 135 mM, most preferably of about 125 mM.

The isotonic composition as described herein may have a pH value compatible with human blood. For example, the isotonic composition as described herein may have a pH of about 6.5 or higher, preferably about 7 or higher. In some embodiments, the composition as described herein may have a pH value of from 6.5 to 8.5, preferably from 7 to 8, more preferably from 7.3 to 7.7. In some embodiments, the isotonic composition as described herein has a pH value of 7.5. In some embodiments, the isotonic composition as described herein has a pH value of from 6.5 to 8.5, preferably of 7.5 to 8, most preferably of 7.5. In a preferred embodiment, the isotonic composition as described herein has a pH value of 7.2 to 7.8, most preferably of 7.3 to 7.5. 1

In one embodiment, the isotonic composition as described herein comprises a buffer. Buffering agents are known in the art and help maintain the pH of the composition stable within a given range. A buffering agent is often a buffer salt. Thus, the isotonic composition as described herein may comprise a buffer selected from acetate, citrate, phosphate, Tris (tris(hydroxymethyl)aminomethane or tromethamine), and derivatives (e.g. Tris hydrochloride) and combinations thereof, including tromethamine in combination with Tris hydrochloride. In a preferred embodiment, Tris is tromethamine. The isotonic buffer may be a Tris buffer at a pH of about 7.5 to about 8.0, a citrate buffer at a pH of about 5.5 to about 6.5, or a phosphate buffer at a pH of about 7.0 to about 7.5. Preferably, the buffer is a T ris buffer. In some embodiments, the isotonic composition as described herein comprises a Tris buffer at a pH of about 7.5 to about 8.0. In some specific embodiments, the buffer is a Tris buffer at a pH of 7.5. The buffering agent is preferably present at about 5 to about 50 mM, more preferably about 10 to about 40 mM, still more preferably about 12 to about 35 mM, still more preferably about 14 to about 30 mM, most preferably about 15 to about 25 mM. The buffering agent can also be present at about 16 to about 24 mM, more preferably about 17 to about 23 mM, still more preferably about 18 to about 22 mM, most preferably at about 19 to 21 mM, such as at 20 mM. In some preferred embodiments, the buffer is a 20 mM T ris buffer at a pH of 7.5.

The isotonic as described herein comprises a cyclodextrin or a derivative thereof. It has been found that cyclodextrins can contribute to an improvement in the preservation of the stability of recombinant adeno- associated viruses, and/or are particularly advantageous in providing a stable drug product that substantially reduces the formation of aggregates or agglomerates. Cyclodextrins are a family of cyclic oligosaccharides, consisting of a macrocyclic ring of glucose subunits joined by a-1 ,4 glycosidic bonds. Cyclodextrins can be a (alpha)-cyclodextrin having 6 glucose subunits, p (beta)-cyclodextrin having 7 glucose subunits, or y (gamma)-cyclodextrin having 8 glucose subunits. Preferred cyclodextrins are p- cyclodextrins. Combinations of cyclodextrins can also be used.

Cyclodextrins can be substituted or unsubstituted. Preferably, the cyclodextrin is an unsubstituted or substituted p-cyclodextrin. Substituted cyclodextrins are generally modified at their hydroxyl moieties, preferably at all of them, preferably having the same modification at all of them. Examples of substitutions are methylation, acetylation, and hydroxypropylation such as 2-hydroxypropylation (having for instance derivatized the hydroxyl moieties using propylene oxide). A preferred cyclodextrin is a substituted cyclodextrin, particularly a substituted p-cyclodextrin. A preferred substituted cyclodextrin is hydroxypropyl-cyclodextrin, and 2-hydroxypropyl-p-cyclodextrin is particularly preferred (CAS number 128446-35-5).

In some embodiments, the cyclodextrin is present in the isotonic composition as described herein in an amount less than about 4% w/v (weight per volume percent), preferably less than about 3% w/v, preferably about 0.05% w/v to about 4% w/v, preferably about 0.1 % w/v to about 3.5% w/v, preferably about 0.5% w/v to about 3.2% w/v, more preferably about 1 % w/v to about 3.1 % w/v, more preferably about 1 .2% w/v to about 3% w/v, more preferably about 1 .4% w/v to about 2.8% w/v, more preferably about 1 .6% w/v to about 2.6% w/v, more preferably about 1 .8% w/v to about 2.4% w/v, most preferably about 1.9% w/v to about 2.2% w/v such as about 2% w/v. In some specific embodiments, the cyclodextrin is a substituted p-cyclodextrin at a concentration of from 1.5% w/v to 2.5% w/v.

In some embodiments, the isotonic composition as described herein further comprises a sugar or sugar alcohol, preferably a monosaccharide, disaccharide, or sugar alcohol. Preferably, the sugar or sugar alcohol may be selected from the group consisting of trehalose, sucrose, maltose, mannitol, and derivatives and combinations thereof. For example, the sugar or sugar alcohol is mannitol. It has been found that, sugar or sugar alcohols may act as cryoprotectants and may be particularly advantageous in providing a stable pharmaceutical formulation. In preferred embodiments, the composition as described herein does not further comprise a sugar or sugar alcohol.

In some embodiments, when present, the sugar or sugar alcohol is present in the isotonic composition as described herein in an amount of about 0.01 % w/v to about 4% w/v, preferably about 0.05% w/v to about 2% w/v, preferably about 0.1 % w/v to about 1 % w/v. Thus, in some embodiments, the isotonic composition as described herein comprises a sugar or sugar alcohol at a concentration of from about 0.05% w/v to about 2% w/v. In some specific embodiments, the isotonic composition as described herein comprises a sugar or sugar alcohol selected from the group consisting of trehalose, sucrose, maltose, mannitol, and derivatives and combinations thereof, at a concentration of from about 0.05% w/v to about 2% w/v.

Where the sugar or sugar alcohol is mannitol, it may be present in the isotonic composition as described herein in an amount less than about 2% w/v, preferably about 1 % w/v or less, preferably about 0.05% w/v to about 1 % w/v, preferably about 0.1 % w/v. In some embodiments, the isotonic composition as described herein comprises mannitol at a concentration of about 0.1 % w/v.

In some embodiments, the isotonic composition as described herein comprises the combination of 2- hydroxypropyl-p-cyclodextrin and mannitol, preferably in the amount of about 0.1 w/v% 2- hydroxypropyl-p-cyclodextrin and about 0.1 w/v% mannitol.

In some embodiments, the isotonic composition as described herein further comprises an amino acid, which were found to be beneficial for the stability of the drug product. Amino acids are natural osmolytes that can stabilize proteins when in solution. For example, the amino acid may be selected from cysteine, arginine, histidine, glycine, and derivatives and combinations thereof. Preferably, the amino acid may comprise histidine and/or glycine. Where the amino acid is histidine and/or glycine, it may be present at a concentration of about 2 mM to about 3 mM, preferably about 2.5 mM. In preferred embodiments the isotonic composition as described herein does not further comprise an amino acid.

When present, in some embodiments, the isotonic composition comprises an amino acid selected from the group consisting of cysteine, arginine, histidine, glycine, and derivatives and combinations thereof, at a concentration of from about 2 mM to about 3 mM. In some specific embodiments, the isotonic composition comprises histidine, or a derivative thereof, at a concentration of about 2.5 mM.

Preferably, the isotonic composition as described herein does not comprise a surfactant. Surfactants and their characteristics are well known; surfactants generally comprise at least one polar head group and at least one apolar or hydrophobic tail and are preferably charge neutral, i.e., they do not have a net charge at the conditions for their use. For example, the isotonic composition as described herein does not comprise Polysorbate 20, Polysorbate 80, or Poloxamer 188.

The following are some preferred embodiments of the composition:

• The isotonic composition having an osmolality of from 250 to 330 mOsm/kg and a pH value of from 6.5 to 8.

• The isotonic composition wherein it is substantially isotonic to human blood. For example, the composition has an osmolality of 260 to 310 mOsm/kg and a pH value of 7.1 to 7.7.

• The isotonic composition comprising about 15-25 mM buffer; a recombinant adeno-associated viral vector; about 110-140 mM pharmaceutically acceptable salt; about 1 .5-2.5% (w/v) cyclodextrin or a derivative thereof; wherein the composition has a pH of about 7 to 8.

• The isotonic composition comprising about 15-25 mM Tris; a recombinant adeno-associated viral vector comprising an AAV5 serotype; about 110-140 mM NaCI; about 1.5-2.5% (w/v) hydroxypropyl-beta-cyclodextrin; wherein the composition has a pH of about 7 to 8.

• The isotonic composition comprising about 18-22 mM buffer; a recombinant adeno-associated viral vector; about 120-130 mM pharmaceutically acceptable salt; about 1 .8-2.2% (w/v) cyclodextrin or a derivative thereof; wherein the composition has a pH of about 7.2 to 8.

• The isotonic composition comprising about 20 mM buffer; a recombinant adeno-associated viral vector; about 125 mM pharmaceutically acceptable salt; about 2% (w/v) cyclodextrin or a derivative thereof; wherein the composition has a pH of about 7 to 8 such as 7.5 to 8 such as 7.5.

• The isotonic composition comprising about 18-22 mM Tris; a recombinant adeno-associated viral vector comprising an AAV5 serotype; about 120-130 mM NaCI; about 1.8-2.2% (w/v) hydroxypropyl-beta-cyclodextrin; wherein the composition has a pH of about 7.2 to 8.

• The isotonic composition comprising about 20 mM Tris; a recombinant adeno-associated viral vector comprising an AAV5 serotype; about 125 mM NaCI; about 2% (w/v) hydroxypropyl-beta-cyclodextrin; wherein the composition has a pH of about 7 to 8 such as 7.5 to 8 such as 7.5.

In one embodiment, the isotonic composition as described herein comprising a recombinant adeno- associated viral vector comprising the nucleic acid according to the invention is administered at a dosage regime from 1 E12 to 1 E15 genome copies per kilogram (gc/kg), for example from 6E12 to 6E14 genome copies per kilogram. For example, when the isotonic composition is administered intravenously, the injection volume may be 100 - 800 mL at a concentration of the recombinant adeno- associated viral vector of 2E12 or 2E13 gc/ml.

In preferred embodiments the isotonic composition as described herein comprising a recombinant adeno-associated viral vector comprising the nucleic acid according to the invention is administered at a dosage regime from 1 E13 to 1 E15 gc/kg, preferably from 5E13 to 5E14 gc/kg, more preferably from 6E13 to 3E14 gc/kg, most preferably at 6.0E13 gc/kg or 3.0E14 gc/kg.

In preferred embodiments the isotonic composition as described herein comprising a recombinant adeno-associated viral vector comprising the nucleic acid according to the invention is administered at a dosage from 1 E13 to 1 E15 gc/kg, preferably from 4E13 to 8E14 gc/kg, more preferably from 6E13 to 5E14 gc/kg, most preferably at 6.0E13 gc/kg, 3. OEM gc/kg. The range of 1 E13 to 1 E15 gc/kg as described above includes, but is not limited to, doses of 4E13 gc/kg, 6E13 gc/kg, 2E14 gc/kg, 3E14 gc/kg, 5E14 gc/kg, and 7.3E14 gc/kg.

In a further aspect, there is provided a kit comprising a nucleic acid according to the invention, an expression cassette according to the invention, a rAAV vector according to the invention, and/or a pharmaceutical composition according to the invention, wherein optionally the kit further comprises an immunosuppressive agent. In certain embodiments of the invention, the immunosuppressive compound may reduce and/or prevent an immune response induced by administration of the nucleic acid, the rAAV vector, or the pharmaceutical composition of the invention. In some embodiments, the immunosuppression can be a regime of glucocorticosteroids, rituximab and sirolimus (Prasad et al. 2022, Corti et al. 2015) aiming to reduce antibody-mediated transgene neutralization and clearance. Corticosteroid treatments have been shown to exert general anti-inflammatory effects through suppression of pro-inflammatory cytokines and upregulation of anti-inflammatory cytokines. Rituximab induces CD20+ B cell apoptosis thus reduces a major pathway for antibody-mediated clearance. Sirolimus (rapamycin) suppresses cytotoxic T cell proliferation, T helper cell differentiation, and at higher doses, B cell proliferation and differentiation.

Uses

Another aspect of the invention relates to the use in a medical treatment of the nucleic acid or the expression cassette or a rAAV vector according to the invention. In one embodiment, there is provided a nucleic acid according to the invention, an expression cassette according to the invention, a rAAV vector according to the invention, and/or a pharmaceutical composition according to the invention for use in a medical treatment.

In a further embodiment, there is provided a nucleic acid according to the invention, an expression cassette according to the invention, a rAAV vector according to the invention, and/or a pharmaceutical composition according to the invention for use in the treatment and/or prevention of Alpha-1 antitrypsin (A1AT) deficiency (A1ATD).

In other embodiments, there is provided a nucleic acid according to the invention, an expression cassette according to the invention, a rAAV vector according to the invention, and/or a pharmaceutical composition according to the invention for use in the treatment and/or prevention of diseases associated with a deficiency of A1 AT, such as chronic obstructive pulmonary disease (emphysema), bronchiectasis or asthma affecting the lung; cirrhosis, neonatal hepatitis or hepatocellular carcinoma affecting the liver; proliferative glomerulonephritis, IgA nephropathy or nephrotic syndrome affecting the skin; necrotizing panniculitis, systemic vasculitis, psoriasis, urticaria or angioedema affecting the vascular system; inflammatory bowel disease affecting the intestines. These diseases can be caused by the inability to upregulate A1AT during acute illness or by a lack in activity, i.e. the levels of A1 AT are comparable to the levels found in healthy patients, however the protein shows lower activity. During acute illness in patients that do not suffer from A1 ATD, A1 AT levels go from a production of 2 g/ml per day to 6 g/ml per day.

In one embodiment, there is provided a method of treatment comprising administering the nucleic acid according to the invention, the expression cassette according to the invention, or the rAAV vector according to the invention, or a pharmaceutical formulation according to the invention to a person in need thereof, preferably for the treatment of Alpha-1 Antitrypsin Deficiency.

In another aspect, the present invention relates to a method of treating or preventing a disorder, wherein the method comprises administering a nucleic acid of the invention orthe expression cassette according to the invention, or the rAAV vector according to the invention, or a pharmaceutical formulation according to the invention to a subject, preferably wherein the disorder is chosen from A1ATD or a disease associated with a deficiency of A1AT.

In another aspect, the invention relates to a nucleic acid according to the invention or an AAV vector according to the invention for use in the manufacture of a medicament for the treatment of A1ATD or diseases associated with a deficiency of A1AT.

Host cells and methods for producing a rAAV vector

In a further aspect, the invention relates to a host cell comprising the expression cassette according to the invention. Thus, a further aspect of the invention relates to a method for producing a rAAV vector comprising an expression cassette according to the invention. There are several methods to produce rAAV vectors. Preferably, the method of production allows for scale-up and increased production yields.

In some embodiments, the method to produce rAAV vectors involves insect cells such as Spodoptera frugiperda (Sf9), Drosophila, or mosquito, e.g. Aedes albopictus derived cells. Preferably, the insect cells are susceptible to baculovirus infection. Examples of insect cells include but are not limited to S2 (CRL-1963, ATCC), Se301 , SelZD2109, SeUCRI , Sf9, Sf900+, Sf21 , BTI-TN- 5B1-4, MG-1 , Tn368, HzAml , Ha2302, Hz2E5, High Five (Invitrogen, CA, USA) and expresSF+® (US 6,103,526; Protein Sciences Corp., CT, USA). In general, suitable methods for producing rAAV vectors according to the invention in insect host cells are described in: Urabe et al. (2002, Hum. Gene Ther. 13:1935-1943), WG2007/046703, WG2007/148971 , WG2009/014445, WG2009/104964, WO2011/122950, WO2013/036118, WO2015/137802, WO2019/016349 and in co-pending applications EP21177449.2, PCT/EP2021/058794 and PCT/EP2021/058798, all of which are incorporated herein in their entirety. Typically, insect cells are infected with recombinant baculovirus, known as baculovirus expression vectors (BEVs), which serve to deliver the essential components to produce rAAV vectors containing the expression cassette flanked by ITRs. Indeed, recombinant baculovirus provides the cap gene, the rep gene and the expression cassette. As explained herein the cap gene encodes for the three structural capsid proteins. The rep genes encode four proteins required for viral replication that are named after their molecular masses: Rep78, Rep68, Rep52 and Rep40.

In some embodiments, the method to produce rAAV vectors involves mammalian cells such as HEK293 cells (Gao et al. 2012) or HeLa cells (Clark et al. 1995). The selection of the mammalian species providing the cells is not a limitation of this invention, nor is the type of mammalian cells. In general, suitable methods for producing rAAV vectors according to the invention in mammalian host cells are described in: Clark et al. (1995, Hum. Gene Ther. 6, 1329-134), Gao et al. (1998, Hum. Gene Ther. 9, 2353-2362), Inoue and Russell (1998, J. Virol. 72, 7024-7031), Grimm et al. (1998, Hum. Gene Ther. 9, 2745-2760), Xiao et al. (1998, J. Virol. 72, 2224-2232) and Judd et al. (Mol Ther Nucleic Acids. 2012; 1 : e54).

Typically, HEK293 cells, which harbor constitutively expressed adenovirus (AdV) E1 a and E1 b genes, are transfected with 3 plasmids: a plasmid expressing rep and cap genes, a plasmid with the expression cassette flanked by ITRs, and helper plasmid containing other AdV genes that serve helper function, such as the E2A, E4 and VA RNA genes that are essential for replication, mRNA processing and translation, respectively. When the host cells have produced the rAAV vectors different steps are necessary for recovery, purification and formulation of the rAAV vectors using suitable techniques which are known to those of skill in the art.

In one embodiment, monolith columns (e.g. in ion exchange, affinity or IMAC mode), chromatography (e.g. capture chromatography, fixed method chromatography, and expanded bed chromatography), centrifugation, filtration and precipitation, can be used for purification and concentration of the rAAV. These methods may be used alone or in combination. In one embodiment, capture chromatography methods, including column-based or membrane-based systems, are utilized in combination with filtration and precipitation. Suitable precipitation methods, e.g. utilizing polyethylene glycol (PEG) 8000 and NH3SO4, can be readily selected by one of skill in the art. Thereafter, the precipitate can be treated with benzonase and purified using suitable techniques. In addition, recovery may preferably comprise the step of affinity-purification of the virions comprising the AAV vector using an anti-AAV antibody, preferably an immobilised antibody. The anti-AAV antibody preferably is a monoclonal antibody. A particularly suitable antibody is a single chain camelid antibody or a fragment thereof as, e.g. obtainable from camels or llamas (see e.g. Muyldermans, 2001 , Biotechnol. 74: 277-302). The antibody for affinitypurification of AAV preferably is an antibody that specifically binds an epitope on an AAV capsid protein, whereby preferably the epitope is an epitope that is present on capsid protein of more than one AAV serotype, e.g. the antibody may be raised or selected on the basis of specific binding to AAV5 capsid but at the same time also it may also specifically bind to AAV1 , AAV3 and AAV2 capsids.

The present invention has been described above with reference to a number of exemplary embodiments as shown in the drawings. Modifications and alternative implementations of some parts or elements are possible and are included in the scope of protection as defined in the appended claims.

Embodiments

Embodiment 1 : A nucleic acid comprising: a. a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region within a human SERPINA1 gene; and b.a sequence encoding an oxidation-resistant A1 AT protein.

Embodiment 2: A nucleic acid according to Embodiment 1 , wherein the guide sequence is substantially complementary to a target region within the coding sequence of a human SERPINA1 gene, preferably the guide sequence is selected from one of SEQ ID Nos. 55, 99, and 105, or a variant thereof.

Embodiment 3: A nucleic acid according to any one of Embodiment 1 or 2, wherein the sequence encoding an oxidation-resistant A1AT protein does not comprise a sequence that is substantially complementary to the guide sequence.

Embodiment 4: A nucleic acid according to any one of Embodiment 1 to 3, wherein the sequence encoding an oxidation-resistant A1 AT protein comprises one of SEQ ID NOs. 120, 122, 128, or 129, or a variant thereof.

Embodiment 5: A nucleic acid according to any one of Embodiment 1 to 4, wherein the sequence encoding an oxidation-resistant A1AT protein further comprises a sequence encoding a hinge and a sequence encoding a human Ig Fc, preferably an IgG Fc.

Embodiment 6: A nucleic acid according to any one of Embodiment 1 to 5, further comprising a promoter and a sequence encoding a polyA tail.

Embodiment 7: A nucleic acid according to any one of Embodiment 1 to 6, wherein the promoter comprises SEQ ID NO. 162 or 168.

Embodiment 8: A nucleic acid according to any one of Embodiment 6 or 7, wherein the promoter comprises an intronic sequence, preferably wherein the intronic sequence is selected from SEQ ID NOs. 164, 165 and 166.

Embodiment 9: A nucleic acid according to Embodiment 8, wherein the intronic sequence comprises the sequence encoding the RNA.

Embodiment 10: A nucleic acid according to any one of Embodiment 6 to 9, wherein the sequence encoding an oxidation-resistant A1AT protein is located between the promoter and the sequence encoding the polyA tail.

Embodiment 11 : A promoter as defined in Embodiment 6 to 8 comprising: a. a sequence comprising SEQ ID NO. 171 , SEQ ID NO. 172, SEQ ID NO. 173, SEQ ID NO. 174 and SEQ ID NO. 176 or a variant thereof; b. a minimal promoter, preferably wherein the minimal promoter comprises SEQ ID NO. 177 or 178; and c. an intronic sequence, preferably comprising one of SEQ ID NOs. 164, 165, or 166, more preferably comprising SEQ ID NO. 164.

Embodiment 12: An expression cassette comprising a nucleic acid according to any one of Embodiment 1 to 10, preferably wherein the expression cassette is flanked by Inverted Terminal Repeats (ITRs).

Embodiment 13: A recombinant adeno-associated virus (rAAV) vector comprising the expression cassette according to Embodiment 12, preferably wherein the rAAV vector comprises AAV5 capsid proteins.

Embodiment 14: A nucleic acid according to any one of Embodiment 1-10 or an expression cassette according to Embodiment 12, or an rAAV vector according to Embodiment 13 for use in a medical treatment, preferably for use in the treatment of Alpha-1 Antitrypsin Deficiency.

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Description of the Figures

Figure 1. Dual luciferase assay in vitro. A) Knockdown of z-AIAT Luc reporter by miAIAT constructs in vitro after co-transfection with 20 nM miAIAT constructs and 4nM of z-AIAT luc reporter construct in HEK293T cells. Relative renilla expression (RL/FL) of the control (miSCR) was set at 100%. Striped bars below the line represent miAIAT constructs that induced at least an 80% decrease in luciferase levels. B) Dose-dependent knockdown of z-AIAT Luc reporter by a selection of miAIAT constructs in vitro after co-transfection 10, 5 or 1 nM miAIAT construct with a z-AIAT luciferase reporter construct in HEK293T cells. Relative renilla expression (RL/FL) of the miSCR was set at 100%. Figure 2. Endogenous mRNA lowering in vitro. Endogenous A1AT mRNA expression levels in vitro after transfection with miAIAT constructs or control miRNA (miSCR) in Huh7 cells. Non-transfected mRNA level was set at 100%. Striped bars below the line indicate A1AT mRNA levels below 50% of the non-transfected cells.

Figure 3. Effect of miAIATs on A1AT protein expression in vitro. A1AT protein expression levels in supernatant after transfection with 20nM miAIAT constructs or control miRNA (miSCR) in Huh7 cells. Striped bars below the line indicate a decrease in A1AT protein levels of at least 30% compared to the control condition.

Figure 4. Effect of codon optimization on A1AT protein expression in vitro. A1AT protein expression levels in the supernatant of Huh7 cells transfected with 20 different A1 AT constructs, each with an A1 AT codon optimized transgene encoding for either an oxidation-sensitive (wt) or for an oxidation-resistant M351V/M358L.

Figure 5. A1AT protein expression in vitro using different promoters. A1AT protein expression levels in supernatant of Huh7 cells transfected with wild type A1 AT under modified liver-specific promoters.

Figure 6. A1AT mRNA and protein expression in vitro. A1AT mRNA expression (A) and protein expression (B) of transfected Huh7 cells with the construct carrying the miAIAT in P1-derived intron within the P5 promoter s’ of A1AT, the P5-miA1AT construct or the P5-A1AT construct.

Figure 7. A1AT protein expression levels in vitro. A1AT protein expression levels in supernatant of Huh7 cells which were transduced with an rAAV5 vector containing either P5, P2, or P3 liver-specific promoter. Cells were transduced at three different multiplicities of infection for each of the three constructs.

Figure 8. Vector DNA and vector DNA biodistribution in Piz mice. A) The rAAV transduction of Piz mice with different dual function constructs was assessed at two doses, 1 xi o¹³ gc/kg and 4x10¹³ gc/kg, using qPCR. The results are expressed as vector DNA copies per pg of genomic DNA. The black dots represent groups with the P5 promoter, while the transparent dots represent the P1 promoter. B) Biodistribution of vector DNA at the high dose of 4x10¹³ gc/kg. The black bars indicate vDNA transduction of animals that received a construct with the P5 promoter, while the gray bars represent animals that received a construct with the P1 promoter. *Adrenal from P1 group has only one animal due to insufficient tissue availability.

LLOQ: lower limit of quantification (667 copies/ pg DNA).

Figure 9. Mutant endogenous A1AT knockdown % in PiZ mice liver. Relative expression level of mutant endogenous A1AT in PiZ mice liver was analyzed by normalizing to averaged levels of non-treated (vehicle group) by RT-qPCR. Black bars represent constructs with a P5 promoter while the gray bars represent constructs with an P1 promoter. The different constructs were assessed at two doses, 1 xi o¹³ gc/kg and 4x10¹³ gc/kg, represented on the top of the bars. Bars represent the means and standard errors of the different animals (n=6).

*Dataset is incomplete due to the presence of poor RNA quality in some samples, resulting in only a few animals for each group.

Figure 10. miA1AT209 quantification in PiZ mice liver. Mature miA1AT209 concentration in liver tissues was determined using a specific custom-made Taqman RT-qPCR assay. The results are shown for 6 animals per condition, except for miA1AT209-A1AT with marked with an asterisk. Due to poor RNA quality, it was decided to exclude 4 animals from the analysis. Each dot represents an animal, hence, an average of duplicate qPCR data.

Figure 11. Expression level A1AT in PiZ mice liver. A) Relative expression level of transgenic A1 AT in PiZ mice liver was analyzed by normalizing to averaged levels of non-treated (vehicle group) animals by RT-qPCR. The dual function constructs were assessed at two doses, 1 xi o¹³ gc/kg and 4X10¹³ gc/kg, which are represented on the top of the bars. Bars represent the means and standard errors from different animals (n=6).

Figure 12. Expression level A1AT in serum of PiZ mice. Total A1AT levels in serum (ug/mL) of PiZ mice at 1 week pre-injection and 8 weeks post-injection (striped bars). Bars represent the means and standard errors from different animals (n=6). The therapeutic level is shown by the dotted line at 570 ug/uL.

Figure 13. Mutant A1 AT polymers reduction in serum of PiZ mice. Relative expression of mutant A1 AT polymers in serum of PiZ mice at 1 week pre-injection and 8 weeks post-injection. Relative expression of mutant A1 AT polymers was normalized to average levels of non-treated (vehicle) animals.

Figure 14. Mutant A1AT globules quantification in of PiZ mice liver. A) Representative images of the liver of mice injected with the vehicle or rAAV5-P2-miA1 AT209-A1 AT. The liver sections were stained with PAS-D staining. B) Quantification of the number and size of mutant-A1AT globules from 3 liver sections per animal (n=6).

Figure 15. In vivo test of dual function vectors. A) The rAAV transduction of different constructs was assessed at doses of 1 xi o¹⁴ gc/kg, using qPCR. The results are expressed as vector DNA copies per pg of genomic DNA. Bars represent the means and standard errors of the different animals (n=9). B) Expression level of mutant endogenous A1 AT in PiZ mice liver by RT-qPCR. The results are expressed as endogenous mRNA copies per pg of DNA (n=9). C) Relative expression of mutant A1 AT polymers in serum of PiZ mice at 1 week pre-injection and 2- and 4-weeks post-injection. The data are normalized to average levels at 1week pre injection of each animal with standard deviation of the different animals (n=9) per group.

Figure 16. Expression levels of A1AT in vivo using dual function vectors. A) Expression level of codon optimized A1AT transgene in PiZ mice liver by RT-qPCR. The results are expressed as transgene mRNA copies per pg of DNA (n=9). B) Total A1 AT levels in serum of PiZ mice at 1 week pre-injection and 2-, 4-, 6- and 8-weeks post-injection. Standard deviation represents the deviation of the different animals (n=9) per group.

Figure 17. In vitro test of dual function vectors expressing A1ATox-Fc proteins. A) A1ATox-Fc proteins expression in the supernatant of Huh7 cells transfected by Western blot. B) Inhibition of Neutrophil Elastase activity by Huh7 supernatant expressing A1 ATox-Fc variant. NC is a negative control missing the Elastase.

Figure 18. In vivo test of dual function vectors expressing A1ATox-Fc proteins. A) The rAAV transduction of different constructs was assessed at doses of 1 x10¹⁴ gc/kg, using qPCR. The results are expressed as vector DNA copies per pg of genomic DNA. Bars represent the means and standard errors of the different animals (n=12). B) Expression level of A1AT transgene mRNA in hFcRN TG32 mice liver by RT-qPCR. The results are expressed as transgene mRNA copies per pg of DNA (n=9). C) Western blot confirming A1 ATox-Fc protein expression in serum of injected hFcRn TG32 mice.

Figure 19. vDNA, human A1AT mRNA and miA1AT209 expression in C57BL/6 mice-derived liver tissue. C57BL/6 mice were dosed with 1x10¹⁴ gc/kg of rAAVs comprising transgene 1 , 2, 3 or 4 (Table 3) or 2x10¹³ gc/kg of rAAVs comprising transgene 4. A) vDNA copies expressed per pg of genomic DNA. B) miA1AT209 copies expressed per pg of RNA. C) Human A1AT mRNA copiesexpressed per pg of RNA. D) Serum protein levels of human A1 AT protein. N=9 animals per group.

Figure 20. vDNA, human A1AT mRNA and miA1AT209 expression in NHP-derived liver tissue. NHP were dosed with 4x10¹⁴ gc/kg of rAAVs comprising transgene 2, 3 or 4 A) vDNA copies expressed per pg of genomic DNA . B) Human A1AT mRNA copies expressed per pg of host RNA. C) miA1AT209 copies expressed per pg of host RNA. N=3 animals per group.

Figure 21. vDNA, human A1AT mRNA and miA1AT209 expression in NHP-derived lung tissue. NHP were dosed with 4x10¹⁴ gc/kg of rAAVs comprising transgene 2, 3 or 4. A) vDNA copies expressed as per pg genomic DNA. B) Human A1AT mRNA copies expressed per pg host RNA. C) miA1AT209 copies expressed per pg host RNA. N=3 animals per group.

Figure 22. Human A1AT serum protein levels in NHP. NHP were dosed with 4x10¹⁴ gc/kg of rAAVs comprising transgene 2, 3 or 4. A) Human A1AT serum protein levels determined using a Meso Scale Discovery (MSD) assay. B) Human A1AT serum protein levels determined using a Parallel Reaction Monitoring-Mass Spectrometry (PRM-MS) assay.. Results are indicated as the mean and standard error of 3 animals.

Figure 23. Liver and lung tissue levels of human A1AT protein in NHP. dosed with 4x10¹⁴ gc/kg rAAVs comprising transgene 2, 3 or 4. Results are indicated as the mean and standard error of 3 animals.

Figure 24. Liver and lung tissue levels of endogenous NHP A1 AT mRNA. NHP were dosed with vehicle control or 4x10¹⁴ gc/kg of rAAVs comprising transgene 2, 3 or 4. A) Knockdown of endogenous NHP A1AT mRNA by miA1AT209 in liver. B) Knockdown of endogenous NHP A1AT mRNA miA1AT209 in lung.

Figure 25. Serum proteins levels of endogenous NHP A1AT. NHP were dosed with vehicle control or 4x10¹⁴ gc/kg of rAAVs comprising transgene 2, 3 or 4. Serum protein levels were quantified by detection of digested peptides (by PRM-MS over the course of the study. Results are indicated as the mean and standard error of 3 animals.

Figure 26. Human A1 AT protein levels in lung-derived fluids. NHP were dosed with vehicle control or 4x10¹⁴ gc/kg of rAAVs comprising transgene 2, 3 or 4. A) Human A1AT protein detected in Broncho Alveolar Lavage (BAL) using an MSD assay. B) Extrapolation of human A1AT protein detected in BAL (A) to Epithelial Lining Fluid (ELF). The expected concentration of human A1AT in ELF was set at 100 mg/ml. Results are indicated as the mean and standard error of 3 animals.

EXAMPLES

Materials and Methods

DNA constructs miRNA guide sequence design

113 individual artificial miRNA guide sequences (miAIATs) (SEQ ID NOs. 3-115) were designed to target various coding regions of the SERPINA1 gene, i.e. to target sequences within the endogenous human A1AT mRNA with high specificity. The miAIATs did not discriminate between wild-type and mutant A1AT mRNA. Of note, the A1AT mRNA target sequences were identified based on their conservation between non-human primate (SEQ ID NO. 2) and human (SEQ ID NO. 1) coding sequences, with the aim to have full complementary sequence throughout all our pharmacological studies. As a control, scrambled miRNA guide sequences were used (SEQ ID NOs. 116-117). The miAIATs and the scrambled controls were incorporated into a scaffold which comprises the miR-144 scaffold and the miR-451 downstream scaffold. Precisely, the miAIAT and the scrambled control guide sequences were embedded in the human pri-miR-451 -scaffold. The pri-miA1AT cassettes were expressed from the CMV immediate-early enhancer fused to chicken p-actin promoter CAG promoter (SEQ ID NO. 167) or P5 promoter (SEQ ID NO. 163) and terminated by the simian virus 40 polyadenylation signal (SV40 polyA, SEQ ID NO. 180).

A1AT transgene design

To enhance the potency of the A1AT transgene and to render the A1AT mRNA expressed from the transgene resistant to the selected miRNAs, the A1 AT sequence was codon optimized for expression in humans using the IDTA codon optimization tool. This was done either with or without CG dinucleotide removal. Of note, different types of codon optimization were designed, e.g. codon optimization was applied either to the entire length of the A1AT sequence (the transgene is then identified as coAIAT) or only to the target regions of the miA1AT165, miA1AT209 and miA1AT215 [the transgene is then identified as mrA1 AT (miRNA resistant A1 AT)]. The A1 AT transgene used encodes for an A1 AT wildtype protein or for an A1 AT protein variant that is more resistant to oxidative stress (Taggart et al. 2000). Based on literature, different double mutant potentially oxidation-resistant A1AT variants were designed, such as M351 V/M358L or M351 E/M358L (Jallat, Carvallo et al. 1986; Jallat, Tessier et al. 1986; Sandoval et al. 2002). The codon optimized transgene encoding for oxidation-resistant A1AT variants are identified as coAIATox. Finally, A1AT lgG1 , lgG2 and lgG4 Fc-fusion proteins (SEQ ID NOs. 123, 124 or 125) were created by cloning in frame the coAIATox sequence with a sequence encoding the hinge region (SEQ ID NOs. 157,159 or 161), followed by a sequence encoding for a CH2 domain, and a CH3 domain of human lgG1 , lgG2 or lgG4. The immunoglobulin G (IgG) fragment region (Fc) and the neonatal Fc receptor (fcRN) interaction is involved in increasing the half-life of circulating proteins through pH-dependent intracellular trafficking and recycling. Various codon optimized A1AT lgG1 , lgG2 and lgG4 Fc-fusion proteins were designed, wherein the codon optimized and M351 E/M358L oxidation-resistant mutated A1AT encoding gene was cloned in frame with a gene encoding the hinge region, followed by a CH2 domain, and a CH3 domain of human lgG1 , lgG2 or lgG4. In the lgG4 Fc polypeptide, the lower region has been mutated at position 235 from leucine to glutamic acid (L235E) in order to reduce immune-effector interaction. In addition, the hinge region has been mutated at position 228 from serine to proline (S228P) in orderto enhance the core-hinge stability. Additional mutations at position 252 (M252I), 256 (T256D) and 428 (M428L) were made to enhance half-life through disruption of the binding site with FcRn. In further variants two molecules of A1AT protein were fused to one Fc fragment.

The A1AT transgenes were expressed using P5 (SEQ ID NO. 163), P1 (SEQ ID NO. 162), P2 (SEQ ID NO. 168), P3 (SEQ ID NO. 169) or P4 (SEQ ID NO. 170) promoters and terminated by SV40 polyA (SEQ ID NO. 180). The P2 promoter comprises a P1 promoter-derived intron, the P3 promoter comprises an SV40 promoter-derived intron, and the P4 promoter comprises an LP1 promoter-derived intron.

Dual function construct design

Selected miAIAT (SEQ ID NOs. 3, 55, 99, 105) or scrambled negative control (SED ID NO. 116) were combined with an A1AT transgene (SED ID NOs. 120-129) to facilitate simultaneous knock-down of A1AT and overexpression of a miRNA-resistant A1AT variant. The dual expression of the miRNA and transgene was driven by the P1 promoter (SEQ ID NO. 162), P2 promoter (SEQ ID NO.168) or P3 promoter (SEQ ID NO.169) containing an intronic element, and terminated by the SV40 polyA signal. Each of the construct identities was confirmed by sequencing of the construct.

A Z-A1 AT luciferase reporter containing the Renilla luciferase (RL) gene transcriptionally fused to the human Z-A1AT(E366K) protein coding sequence under the control of the SV40 promoter and the HSV- TK driven Firefly Luciferase (FL) was generated (SED ID NO. 189). The human Z-A1AT(E366K) protein coding sequence was synthesized with added 5' (Xhol) and 3' (Notl) restriction site sequences and cloned in the 3’UTR of the RL gene of the pDualluc by GeneWiz (Azenta Life Sciences). rAA V5 vectors

Recombinant AAV5 particles were produced by various methods. The rAAVs were produced by transfecting HEK293T cells (Sirion Biotech, Germany). The rAAVs were also produced in an insect cell based systems. For instance, rAAVs harboring the expression cassettes were produced in SF+ insect cells (Protein Sciences Corporation, Meriden, Connecticut, USA) using baculoviruses. Following standard protein purification procedures using a fast protein liquid chromatography system (AKTA Avant 150, GE 30 Healthcare) and AVB sepharose (GE Healthcare) the titer of the purified rAAV particles was determined using QPCR. For rAAV production, ITRs were placed at the 3’ and 5’ end.

Transfection experiments

Human hepatocellular carcinoma (Huh7) or human embryonic kidney 293T (HEK293T) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum without antibiotics. Cells were seeded in 24-well plates at a density of 1 E+05 cells per well or in 96-well plate at a density of 2E+04 cells per well the day before transfection or transduction experiments. Transfections were performed using Lipofectamine® 3000 according to the manufacturers’ protocol.

Luciferase assays

HEK293T cells were co-transfected with the miAIAT constructs, scramble construct or pBluescript together with the luciferase reporters containing both the RL gene fused to human Z-A1AT sequences and the Firefly luciferase (FL) gene. Transfected cells were harvested 48 hours post-transfection in 100 pl 1x passive lysis buffer (Promega, Thermo Fisher Scientific) by gentle rocking for 15 minutes at room temperature. The cell lysates were centrifuged for 5 minutes at 4,000 rpm and 10 pl of the supernatant was used to measure FL and RL activities with the Dual-Luciferase Reporter Assay System (Promega, Thermo Fisher Scientific). Relative luciferase activity was calculated as the ratio between RL and FL activities.

Transduction experiments

For transduction Huh7 cells were seeded in 24-wells plates at a density of 1 E+05 cells per well 1 day prior to transduction. The next day cells were incubated with rAAV vectors at a multiplicity of infection (MOI) of 1 E+05, 1 E+06 and 1 E+07 gc/cell. Cells and culture supernatants were harvested 2 days post- transduction. In some experiments, the medium of the cells was replaced 2 days post-transduction and the cells harvested 3 days post-transduction for isolation of DNA and RNA.

Pulverization of mouse tissues using a cryogenic pulverization system

Frozen tissue samples were pulverized using an automated cryogenic sample pulverization system. Snap frozen tissues were crushed by exerting one or several punches of varying impacts with the CryoPREP system type CP02 (Covaris). Before and after each impact the tissueTUBE (Covaris) with tissue was dipped into liquid nitrogen, the procedure was repeated until the sample was pulverized. Powder was stored at -80°C in cryovials (Covaris or Corning) until further use.

Vector DNA quantification

DNA was isolated using The MagMAX™ DNA Multi-Sample Ultra 2.0 Kit with the KingFisher Flex system. The isolated and quantified DNA samples were further diluted to a final concentration of 150 ng per qPCR reaction. Specific primers and probes were designed to target and amplify the sequence specific to SV40 polyA region of the constructs, e.g. forward primer (SED ID NO. 190), reverse primer (SED ID NO. 191) and the probe (SED ID NO. 192) labeled with FAM dye. Specific primers and probes were designed to target and amplify the sequence specific to the codon optimized A1 AT sequence, e.g. forward primer (SED ID NO. 202), reverse primer (SED ID NO. 203), and the probe (SED ID NO. 204) labeled with FAM dye. In addition, ACTB locus present in genomic DNA were also taken along and amplified as an inhibition and loading control. The primers and probe used to target and amplify ACTB locus were, e.g. forward primer (SED ID NO. 193), reverse primer (SED ID NO. 194) and the probe (SED ID NO. 195), also labeled with FAM dye. qPCR reactions were performed using TaqMan™ Universal PCR Master Mix, ThermoFisher scientific. The run was performed in Quantstudio™ 5 Real- Time PCR System, ThermoFisher Scientific).

As a quantification standard, the particles DNA plasmid was used. For quantification, serial dilutions of this plasmid containing the SV40 target sequence were subjected to qPCR in parallel to the samples. From the results of the plasmid dilutions, a calibrator curve was established by linear regression. The range of the plasmid standard line was 1 E8 to 50 copies per reaction. The vector DNA copies in the samples were calculated by interpolation from the calibrator curve.

Quantification of endogenous A1AT knockdown & expression of transgene AlATcDNA synthesis of mRNA

Total RNA was isolated using The MagMAX™ mirVana™ Total RNA Isolation Kit with the KingFisher Flex system. cDNA from purified RNA samples was conducted using the Maxima RT-PCR kit (Thermo Fisher Scientific, Waltham, USA). cDNA was synthesized during a 10 min incubation at 25°C followed by 15 min at 50°C. After synthesis cDNA samples were further diluted in nuclease free water. qPCR The cDNA samples were subjected to RT-qPCR to assess the knock down of endogenous A1 AT and quantify the expression of A1AT transgene. The primers and probes stock of 100 pM used in RT-qPCR are listed below in the Table 2:

Table 2: Primers and probes

To ensure the normalization of gene expression in the context of endogenous expression knockdown analysis, Proteasome subunit beta 4 (PSMB4), Hs00160598_m1 , ThermoFisher Scientific, was included as housekeeping gene. qPCR reactions were performed using TaqMan™ Universal PCR Master Mix, ThermoFisher scientific in a total volume of 10 pL. The run was performed in Quantstudio™ 5 Real-Time PCR System, ThermoFisher Scientific).

The serial dilution of DNA plasmid containing the A1 AT target sequence was used as the quantification standard for transgene analysis. From the results of the plasmid dilutions, a calibrator curve was established by linear regression. The range of the plasmid standard line was 1 E8 to 100 copies per reaction. The transgene RNA copies in the samples were calculated by interpolation from the calibrator curve.

To analyze the endogenous A1AT, delta delta CT (AACt), also known as the Livak method, was applied. In brief, the AACT compared the difference of expression between A1AT gene and the reference gene, in this case, PSMB4. Subsequently, the difference between the experimental and the negative (vehicle) group samples was determined. The relative changes in gene expression between the two groups were analyzed by using the formula 2-AACt, where 2 is the efficiency set at 100%. miRNA quantification: cDNA synthesis of miRN A The total RNA was isolated using The MagMAX™ mirVana™ Total RNA Isolation Kit with the KingFisher Flex system. To examine miA1AT209 expression, 150 ng cDNA was synthesized from isolated total RNA with custom made gene-specific RT primers targeting mature miA1AT209 of 24nt using TaqMan microRNA Reverse Transcription kit (Applied Biosystems, ThermoFisher Scientific). miRNA quantification: qPCR

The cDNA samples were further analyzed by qPCR. A single stranded 24-nt long miA1AT209 oligo standard line was taken along for the quantification of miA1AT209 in total mice liver total RNA. Next, gene-specific Taman qPCR was performed with mature miA1 AT209-24 nt specific primers and probes using Taqman fast Universal PCR master Mix (Applied Biosystems, ThermoFisher Scientific). Using the mature miA1AT209-24nt standard line, miA1AT209 copies per reaction were determined, and the number of miA1 AT209 copies per total /pg RNA was subsequently calculated.

Total A1 AT quantification

Total A1AT protein concentration in mice serum samples was determined by enzyme-linked immunosorbent assay (ELISA). Serum samples were collected on a weekly basis, commencing from week -1 (pre-treatment group) until week +8 after treatment. The analysis of A1AT content was conducted using the Human alpha 1 Antitrypsin ELISA Kit (SERPINA1) (ab108799, Abeam pic, Cambridge, UK) in accordance with the manufacturer's instructions, using a Glomax Discover microplate reader (Promega).

A 1A T polymer reduction

The analysis of polymer reduction in PiZ mice serum was performed using Meso Scale Discovery (MSD) technology, specifically the MESO QuickPlex SQ 12 instrument. Briefly, capture antibody 2C1 (HycultBiotech) was immobilized on plates and incubated overnight at 4 °C. Subsequently, the plates were treated with 5% blocking buffer A for one hour and washed. The serum samples were added to the plate and incubated on a shaker at room temperature for one hour. Following the incubation, the plates were washed, and a detection antibody (GOLD-SULFO-TAG conjugated Alpha-1 -antitrypsin, mAb 2C1) was introduced to the plate for an additional hour at room temperature. After another round of washing, 150 pL of MSD GOLDTM Read Buffer B was added to the plate, and immediate reading was performed using the MESO QuickPlex SQ 12 instrument. The electrochemiluminescent labels (SULFO-TAG) attached to the detection antibody were then detected using MSD, enabling the highly sensitive detection of mutant A1 AT polymers in the mice serum samples.

SDS-PAGE and Western Blot

Samples were prepared in 1x Laemmli sample buffer (1610747; Bio-Rad) and heated to 95°C for 5 minutes. Proteins were separated on a Mini-PROTEAN TGX Stain-Free Protein Gel 4-20% (4568093; Bio-Rad) and transferred to PVDF-membrane using Trans-blot Turbo Mini PVDF Transfer packs (1704156; Bio-Rad) and the Trans-Blot Turbo™ Transfer System (1704150; Bio-Rad). The PVDF- membrane was blocked with SuperBlock T20 (PBS) Blocking Buffer (37516; Bio-Rad) and stained with the primary human A1AT polyclonal antibody conjugated with HRP(A80-122P, Thermo Scientific) in blocking buffer. After washing with 0.5% Tween-20 in phosphate buffered saline (PBS), the proteins were visualized using SuperSignalTM West Pico PLUS Chemiluminescent Substrate (34580; Thermo Scientific) and the ChemiDoc Touch Gel Imaging System (1708370; Bio-Rad).

Liver tissue embedding and PAS-D staining

After sacrificing the mice, the liver tissues were cut into pieces, which were fixed in a 4% formaldehyde solution. Four hours after collection, the fixative was replaced with fresh formaldehyde solution and fixation continued for 48 hours. After fixation, samples were maintained in 70% ethanol at 4 degrees until tissue processing and paraffin embedding at StageBio (USA). Liver blocks were microtomed and stained with PAS with Diastase (PAS-D). The slides were scanned in brightfield using a whole slide scanner (AxioScan, Zeiss, Germany) with a LD Plan-Neofluar 40x objective with 0.6NA. The scanned slides were then analyzed using the HALO Software from IndicaLab with the Al module. The image analyzing was performed in the following steps. An Al-classifier was trained to detect the tissue and ignore artefacts such as folds, bubbles etc. Within this annotation layer an additional classifier was trained to detect each PAS-D positive object individually and measure their area. This single object data was then used to calculate the average size of the Pas-D objects and to plot the size distribution within the different groups.

Animal studies

Different mouse models were used. The PiZ mouse-model were obtained from Prof. Jeffrey Teckman, St Louis university or from our own colony, derived from Prof. Teckman colony.

The SCID FcRn-/- hFcRn (32) Tg mice were also used. The SCID FcRN-/-hFcRn (32) Tg were purchased from Jackson Laboratory (#018441).

In vivo assessment of CG optimized transgene in C57BL/6 mice

As described above, A1AT transgenes were optimized by the removal of the CG dinucleotides (SEQ ID NOs. 120-121). Such transgenes were used to generate new dual function expression vectors [P2- miA1AT209-coA1ATox(CG) (#3 of Table 3), P2-miA1AT209-coA1ATox(CG)-Fc4 (#4 of Table 3)]. rAAV5 viral vectors containing such dual function vectors were tested in vivo. The rAAV5 viral vectors [P2-coA1Atox (#1), P1-miA1AT209-coA1ATox (#2), P2-miA1AT209-coA1ATox(CG) (#3), P2- miA1AT209-coA1ATox(CG)-Fc4 (#4)] were administered to 3 months old C57BL/6 mice via intravenous tail injection at the dose of 1 E14gc/kg or 2E13gc/kg. The mice were followed up for 8 weeks. Liver samples were collected to assess vDNA, miAIAT and transgene mRNA expression. A1AT protein levels were assessed in the serum using an Meso Scale Discovery assay.

Study in non-human primates rAAV5 dual function transgenes P2-miA1AT209-coA1ATox(CG), P2-miA1AT209-coA1ATox(CG)-Fc4, P1-miA1AT209-coA1ATox were administered to male Macaca fascicularis (cynomolgus macaque), aged at least 20 months old. Three animals were dosed per test condition (i.e. different transgenes) as well as for the vehicle control group. Animals were dosed through intravenous injection by infusion over a duration of 30 min. For each candidate vector, plus the vehicle control group, 3 animals were treated at 4. OEM gc/kg and followed in life for a duration of 2 months.

All animals were immunosuppressed to dampen the innate immune system and to prevent any antibody- and T cell-mediated reactivity formation against the human A1AT transgene. The immunosuppression treatment protocol included Rituximab (Ruxience) administration through intravenous injection (IV) infusion over 2 hours, 20 mg/kg (concentration at 10 mg/mL). Rituximab was administered 2 times: on pre-dose Day-7 and pre-dose Day -1. In addition, Prednisone (1 mg Tablet) was orally administered daily at a concentration of 0.5 mg/kg. Prednisone was administered from predose day-1 up to the day before necropsy. Furthermore, Tacrolimus (1 mg Tablet) was orally administered daily at a concentration of 1 mg/kg from pre-dose day-1 up to the day before necropsy.

During the study, the animals were observed for clinical signs of tolerability to the treatment and blood samples were taken weekly to assess human A1AT protein levels. After either 2 or 3 months, the animals were sacrificed and a bronchoalveolar lavage was performed using 25 ml saline solution, followed by macroscopic examinations. Microscopy examinations were performed on the liver and the dorsal root ganglia. Both the liver and lung were sampled for genomic and protein analyses.

RESULTS

In vitro testing of mi A1 AT constructs on z-AIAT Luc reporter system

To evaluate the knockdown efficiency of the 113 designed microRNAs targeting A1AT (miAIATs; SEQ ID NOs. 3-115), a dual luciferase assay was performed. In short, HEK293T cells were co-transfected with miAIAT construct or non-targeting control construct miSCR, and the z-AIAT luciferase reporter bearing the complementary A1 AT target regions.

Two days post-transfection, cells were harvested to measure luciferase expression. Several miAIAT candidates (represented by striped bars below the line, Figure 1A-B) presented more than 80 % lowering of luciferase activity levels compared to the control miSCR (Figure 1).

To accurately determine the potency of the miAIAT constructs further, six miAIAT constructs, having more >80 % knockdown efficiency, were tested in different concentrations. All selected miAIAT constructs induced dose-dependent reporter knockdown (Figure 1 C).

In vitro testing of miAIAT constructs on endogenous A1 AT

Next, the ability of miAIAT candidates to silence endogenously expressed A1AT mRNA and reduce A1 AT protein levels was tested in in vitro. Huh7 cells were transfected with either miAIAT construct or control (miSCR) construct. Expression of A1AT mRNA was determined using RT-QPCR with GAPDH gene expression as reference gene. Most of the miAIAT constructs reduced A1AT mRNA levels with more than 50% (represented by striped bars below the line, Figure 2 A-B) compared to non-transfected cells representing 100% of A1AT mRNA levels (Figure 2 A-B). In addition, the A1AT protein levels in the culture supernatants showed a decrease of more than 30% of endogenous secreted A1AT after 48h (Figure 3). In vitro testing of A1 AT overexpression constructs

Twenty different transgenes (SEQ ID NOs. 136-155) encoding for an A1AT protein were designed and tested in vitro. Ten of those transgenes encoded for an oxidation-sensitive A1AT protein. Such transgenes had different types of codon optimization. Such transgenes were further modified to be able to encode for an oxidation-resistant A1 AT protein, a variant with two points mutations (M351 V/ M358L). Subsequently, each of these transgenes was transfected in Huh7 cells and expressed under a liverspecific promoter (P5; SEQ ID NO. 163). Protein expression levels were analyzed by performing an ELISA on the supernatant (Figure 4). Surprisingly, only one sequence (co#2) showed higher expression levels in supernatant compared to the wild-type sequence both in oxidation-sensitive or oxidationresistant forms.

Generation of a strong liver-specific promoter by incorporation of an intronic sequence within the P5 promoter

With the aim to identify a new promoter able to further burst a liver-specific expression, the intronic sequences derived from the P1 promoter (SEQ ID NO. 164), or the intronic sequences derived from the SV40 promoter (SEQ ID NO. 165) or the intronic sequences derived from the LP1 promoter (SEQ ID NO. 166) were incorporated within the P5 promoter (SEQ ID NO. 163), generating, respectively, the P2 (SEQ ID NO.168), P3 (SEQ ID NO. 169) or P4 (SEQ ID NO. 170) promoter. To test the activity of such promoters, four expression vectors were generated by adding downstream of each promoter a sequence encoding for a wild-type A1AT protein (AlATwt) and an SV40 polyadenylation signal sequence. Such expression vectors were used to transfect Huh7 cells. 24h post-transfection, medium was refreshed. After 48 hours, the supernatant from the transfected Huh7 cells was collected and A1AT protein concentration was quantified using an ELISA assay. Surprisingly, only cells transfected with the P2 promoter (SEQ ID NO.168), i.e. P5 promoter modified with the P1 -promoter-derived intronic sequences, showed higher A1AT expression levels in supernatant compared to the P5 promoter (without the additional intronic sequence; SEQ ID NO. 163) (Figure 5). Introduction of SV40- or LP1- promoter-derived intronic sequences in the P5 promoter did not result in a significant change in protein levels.

Generation and in vitro testing of a dual function expression vector

In order to simultaneously express both a miAIAT and an A1AT protein, a sequence encoding for a miA1 AT was introduced within the P1 promoter-derived intronic sequence of the P2 promoter (SEQ ID NO.168), followed by an A1AT transgene and the SV40 poly(A) signal. This design identifies an expression cassette having a double function, also called dual function expression vector. Huh7 cells were transfected with a dual function expression vector (P2-miA1AT-coA1AT), or with a miAIAT construct (P5-miA1AT), or with a AlATwt (P5-A1ATwt) construct, or a control (miSCR) construct. Approximately 24h after transfection, the medium was refreshed. After 48 hours the supernatant was collected, and RNA was isolated from the cells. The level of mRNA A1 AT expression levels and A1 AT protein levels were assessed using RT-qPCR and ELISA, respectively. Endogenous A1AT mRNA levels were found to be similarly decreased in both vectors containing the miAIAT, irrespective of its location (Figure 6A). Interestingly, cells transfected with the dual function expression vector expressing both an miAIAT and a codon-optimized sequence encoding human A1AT protein showed higher A1 AT protein secreted in supernatant, despite having lower endogenous expression, as compared to cell transfected with a sequencing encoding human wild-type A1 AT only (Figure 6B). This surprising finding shows that using this approach, endogenous A1 AT can be downregulated by the miAIAT present within an P1 promoter-derived intron in the P5 promoter, while codon-optimized A1AT is expressed very efficiently from the same promoter. rAAV5-mediated delivery of a microRNA and codon-optimized A1 AT in Huh7 cells

After the selection of both miAIAT and A1AT transgene candidates, several dual function viral vectors were developed. In summary, the expression cassettes were generated by incorporating the selected miAIAT candidates within the P1 promoter-derived intron or the SV40 promoter-derived intron within the P5 promoter, which has downstream an A1AT transgene and the SV40 poly(A) signal. The expression cassette was flanked with wild-type AAV2 ITRs. Subsequently, rAAV5 particles comprising the above-described expression vector (rAAV5-miA1AT-A1AT) were produced in HEK293T cells. To provide in vitro proof of mechanism, Huh 7 cells were transduced with 3 different multiplicities of infection. After 48h, supernatant was collected, and RNA was obtained from the cells. The protein concentration in the supernatant was quantified by using an ELISA assay (Figure 7). Surprisingly, it was found that A1AT expression was higher in a dual function construct driven by the P2 promoter (SEQ ID NO. 168) compared to the P5 promoter (SEQ ID NO. 163) expressing only A1AT protein. Furthermore, A1AT expression levels were also found to be lower when using a design comprising the P5 promoter with miAIAT in an SV40 intron instead of an P1 promoter-derived intron (Figure 7). All three constructs showed dose-dependent expression of A1AT transgene (Figure 7). vDNA levels in PiZ mice

To provide in vivo proof of concept, 3-month-old PiZ male mice were intravenously injected with rAAV5 vectors comprising different dual function vectors expressing both miAIAT and A1AT protein and followed up for 8 weeks. All mice that were transduced with rAAV5 exhibited detectable levels of vector DNA (Figure 8A). The PiZ mouse-model is a gain-of-function transgenic mouse, which expresses a well-established human PiZ A1AT mutant that recapitulates many features of human liver injury. The expression of the human PiZ allele results in a misfolded and aggregated protein in the ER of the hepatocytes, presenting intracellular globular inclusions of misfolded Z-a1AT and a low level of secretion of the human protein, like the human PiZ deficiency. Besides the intracellular globular accumulations, which are a histological hallmark of the disease, PiZ mice present with a low-grade inflammation, regeneration, progressively developing hepatic fibrosis and hyperplasia or even hepatocellular carcinoma (Marcus, Brunt et al. 2010; Hidvegi, Ewing et al. 2010). vDNA transduction was observed across all organs, with the highest levels detected in the liver, lower levels in kidney and spleen, still lower levels in the lung and adrenals, and the lowest levels in the testis (Figure 8B). This analysis reveals that the administration of rAAV5 comprising the construct at a dose of 4x10¹³ gc/kg via intravenous injection leads to a vDNA distribution ranging from 1x10⁴ to 1x10⁶ vDNA copies/pg DNA, apart from testis samples, which exhibit an approximate count of 5x10³ vDNA copies/pg DNA.

Lowering of endogenous A1 AT mRNA levels in liver of PiZ mice

To evaluate the effect of the different miA1 AT on the mRNA-levels of mutant endogenous human A1 AT (Z-A1AT) in PiZ mice liver, RNA expression of endogenous A1AT was assessed. All miAIAT, with the exception of miA1AT113, induced a significant decrease of Z-A1AT levels in the liver, with at least 50% (Figure 9). Of note, the knockdown efficacy between the two different doses appears to be similar, as both the 1 E13 gc/kg and 4E13 dose of P2-miA1AT165-mrA1AT results in approximately 60-80% decrease in Z-A1AT mRNA levels. miA1AT209 expression level in liver tissue of PiZ mice

Among the three best performing miAIATs, miA1AT209 expression level was assessed in murine liver tissue (Figure 10). Consistent with the observed rAAV transduction pattern, mature miA1AT209 copies were detected in the liver samples of all animals treated with rAAVs expressing miA1AT209, both groups showed comparable miA1AT209 copy numbers, approaching approximately 1x10⁶ copies/pg of total RNA.

A1AT expression level in liver tissue of PiZ mice

Similar to vDNA levels, as shown in Figure 8A, A1AT transgene levels show a dose response relationship between the two doses of 1 xi o¹³ gc/kg and 4X10¹³ gc/kg for P2-miA1AT165-mrA1AT (Figure 11A). This seems to be the case in general, as both other constructs dosed at 4X10¹³ gc/kg show similar transgene expression levels (Figure 11 A). This correlation is strongly positive, as evidenced by a linear relationship between transduction levels and transgene expression (vDNA copy numbers and transgene mRNA expression) (Figure 11 B). This suggests a robust association between the efficiency of rAAV transduction and the resulting expression levels of the transgene.

Total A1 AT protein serum levels and mutant A1 AT serum levels of PiZ mice

To assess the impact of the dual function viral vectors on protein levels, murine serum samples were subjected to ELISA analysis. Both a pre-treatment group (week -1) and a post-treatment group (week +8) were evaluated to determine the changes in total A1AT protein levels before and after treatment (Figure 12). The therapeutic threshold for A1AT is established at 570 pg/mL (Wall M et al. 1990). Such required protein level was not achieved at the lower dose of 1 xi o¹³ gc/kg. The higher dose, i.e. 4x10¹³ gc/kg, allowed to reach the required protein level. Of note, the construct with the P1 promoter seemed to show comparatively higher expression at both doses. However, this is not surprising as the P1 promoter is not a liver-specific promoter, and therefore drives expression of the A1AT protein also in the peripheral organs. In addition, codon optimization over the full length of the transgene encoding for A1AT seemed to result in higher protein expression compared to non-codon optimized A1AT transgenes. In addition to total A1AT serum levels, Z-A1AT serum levels were also analyzed using ELISA (Figure 13). After 8 weeks of treatment, all miAIATs but one (miA1AT113) established a significant reduction in mutant A1AT polymers across the majority of groups injected with a viral dual function vector expressing a miRNA (Figure 13). Notably, the exception observed for mil 13 aligns with prior findings from RT-qPCR data (Figure 9), in which the level of reduction of the mutant A1 AT gene was determined. z-AIAT globules size in liver of PIZ mice

To assess phenotypic correction, liver sections of animals injected with miA1AT209-A1AT or vehicle were stained with Periodic Acid-Schiff (PAS)-D and the number and size of z-AIAT globules was quantified. A significant reduction in the PAS-D staining in animals treated with miA1 AT209 was found. While the number of globules remained unchanged, the average size of the globules was significantly decreased in animals treated with miA1AT209 (Figure 14).

Liver-specific promoter assessment in PiZ mice

To assess promoter-efficiency, 3-month-old PiZ male mice were intravenously injected with rAAV5 carrying one of three different expression vectors: P5-coA1AT, P2-miA1AT209-coA1AT or P3- miA1AT209-coA1AT at an higher dose of 1x10¹⁴ gc/kg, or vehicle. The mice were followed-up until 8 weeks post-injection. Vector DNA and endogenous/mutant A1AT mRNA levels in the liver were quantified by RT-qPCR to analyze rAAV transduction efficiency and z-AIAT mRNA lowering. All mice injected with rAAV5 showed a significant increase in vector DNA levels of up to 8.8E+06 gc/pg DNA (Figure 15A). Vehicle-treated or rAAV5- P5-coA1AT mice showed endogenous/mutant mRNA levels around 1 E+08 copies per pg mRNA, whereas mice treated with rAAV5-P2-miA1AT209-coA1AT showed on average 8E+06 copies/pg mRNA and mice treated with rAAV5-P3-miA1AT209-coA1AT showed on average 2E+07copies/pg mRNA (Figure 15B). These results indicate that the miAIAT present in the construct is functional and reduces endogenous A1AT levels in PiZ mice. To examine whether the decreased mRNA copy levels in the miA1AT209 treated groups are in line with decreased in serum A1AT expression, an MSD was performed on samples taken 1 week prior to injection, as well as 2- and 4-weeks post injection. It was found that in animals injected with a construct containing miA1AT209, serum A1AT levels were decreased with 97% on average 4 weeks post injection (Figure 15C). These results further confirm functionality of miA1AT209. Next, A1AT transgene mRNA and serum protein levels were quantified to assess transgene expression. Mice treated with rAAV5-P5- coAIAT, AAV5-P2-miA1AT209-coA1AT and rAAV5-P3-miA1AT209-coA1AT showed a similar and significant increase in coAIAT mRNA expression up to 1.3E+08 copies/pg mRNA (Figure 16A). To assess transgene expression, total A1AT levels (detecting both z-AIAT and coAIAT) were quantified by ELISA at different time points prior to and post injection. Vehicle-treated mice showed baseline z- A1AT levels of around 350 pg/mL which remained constant over time. Mice treated with rAAV5- P5- coAIAT showed an A1AT expression level of 305ug/mL 1 week prior to injection, increasing to and 1171 ug/mL at week +8 after injection. Surprisingly, despite similar mRNA levels, mice treated with rAAV5-P2-miA1AT209-coA1AT and rAAV5-P3-miA1AT209-coA1AT showed different A1 AT expression levels. More interestingly, mice treated with rAAV5-P2-miA1AT209-coA1AT and rAAV5-P3- miA1AT209-coA1AT showed similar decrease in mutant A1AT but resulted in different transgene expression. Mice treated with rAAV5-P2-miA1AT209-coA1 AT showed an average of 784 ug/mL while mice treated with rAAV5-P3-miA1AT209-coA1AT showed an average of 503 ug/mL of total A1AT at week 8 after injection.

In vitro testing of A1AT-lgG Fc fusion transgene constructs

The dual function constructs encoding for miA1AT209 and for an A1AT protein fused with lgG1 , lgG2 and lgG4 Fc protein (A1 AT-Fc variants) were tested in vitro for evaluating their potency in expressing A1AT. All the A1AT transgenes were codon optimized over the entire length of the sequence and encoded for an A1AT wild-type (coAIATwt) protein or for an oxidation-resistant protein variant M351 E/M358L (coAIATox). The dual function constructs (P2-miA1AT209-coA1ATox-Fc- coAIATox; P2-miA1AT209-coA1ATox-Fc1 ; P2-miA1AT209-coA1ATox-Fc2; P2-miA1AT209-coA1ATwt) and the P5-coA1ATwt construct were used to transfect Huh7 cells. The supernatant was collected 48 hours after transfection and used to assess the protein level by western blot and the protein activity by an Elastase inhibition assay. The supernatant collected from non-transfected cells was used as control for endogenous A1 AT expression. The data showed a high A1 AT expression. All the supernatant of Huh7 cells transfected with the different A1 AT constructs (Figure 17A). Furthermore, A1AT was able to inhibit Elastase protein, as shown in Figure 17B.

In vivo studies of the A 1A T-IgG Fc-fusion transgene constructs - Humanized hFcRn Tg32 mouse model Tg32 mice (also called hFcRn Tg32 or FcRn-/- hFcRn line 32 Tg) carry a knock-out mutation for the mouse Fcgrt (Fc receptor, IgG, alpha chain transporter) gene and a transgene expressing the human FCGRT gene underthe control of its own native promoter (hTg32). The homozygous Tg32 mice have the highest, most human-like protection of humanized IgG and are the best model for use when maximum half-life data is required. Therefore, this model is useful in evaluating the pharmacokinetics and pharmacodynamics of human immunoglobulin G (IgG) and Fc-domain based therapeutics.

To study the expression of the A1 AT-Fc variants compared to the non-Fc A1AT in Tg32 mice, 12 mice per group were injected with rAAV5 dual function vectors at the single dose 1x10¹⁴ gc/kg. At week 6 post injection, animals were sacrificed, and the vector DNA levels and mRNA levels in the liver were determined (Fig 18 A-B). On average, animals injected with rAAV5-P2-miA1AT209-coA1ATox and rAAV5-P2-miA1AT209-coA1ATox-Fc1 expressed 1 E+06 genomic copies per ug of DNA. Animals injected with rrAAV5-P2-miA1AT209-coA1ATox-Fc2 expressed on average 6.6E+05 gc/ug DNA, animals injected with rAAV5-P2-miA1AT209-coA1ATox-Fc4 expressed on average 3E+05 gc/ug DNA and animals injected with AAV5-P1-miA1AT209-coA1ATox-Fc4 expressed on average 4E+05 gc/ug DNA. Surprisingly, mRNA data showed that animals injected with rAAV5P2-miA1AT209-coA1ATox expressed on average higher transgene mRNA copies with 2.5E+07 per ug mRNA while all the other groups resulted in average of 3 to 5 E+06 copies/ug mRNA.

To compare expression levels of the different A1 AT variants, at week 6 post injection, mice serum from 1 or 2 random mice in each group were analyzed by Western blot (Figure 18C). This resulted in similar or higher expression of A1ATox-Fc fusion proteins compared to AlATox protein levels despite lower transgene mRNA levels. This showed that Fc-fusion proteins have an extended serum half-life resulting in higher expression.

From Figure 19 to Figure 26 the transgenes are numerically identified as shown in Table 3:

In vivo assessment of CG optimized transgene in C57BL/6 mice

In order to study expression of CG optimized transgene in non-diseased mice, C57/BL6 mice were dosed with rAAV comprising either transgene #1 , 2, 3, or 4 (Table 3). Generally, there was no clear difference in liver tissue vector DNA levels between the 4 transgenes (Figure 19A). Indeed the lower dose (2E13 gc/kg) resulted in lower vector DNA levels as compared to the higher dose (1 E14 gc/kg). Of note, expression of miA1AT209 in the liver did vary between the different transgenes, with #2 reaching the highest copy number per pg RNA, followed by #3, and #4 (#1 did not encode miA1 AT209). Similarly, human A1AT mRNA expression in liver tissue differed between the transgenes, with transgene #2 reaching highest expression, followed by #2, and lowest levels of mRNA expression were determined in liver tissue from mice that received transgenes #3 and #4. Furthermore, all transgenes resulted in A1AT serum levels that surpassed the established therapeutic efficacy threshold of 570 pg/mL A1AT when provided in the high dose (1 E14 gc/kg) (Figure 19D).

In vivo assessment of target engagement and biodistribution in cynomolgus macaque rAAV5 dual function vectors P2-miA1AT209-coA1ATox(CG) [#3], P2-miA1AT209-coA1ATox(CG)-Fc4 [#4], P1-miA1AT209-coA1ATox [#2] were administered to male Macaca fascicularis (cynomolgus macaque) of at least 20 months of age through intravenous injection. All three vectors were well tolerated. No macroscopic findings were seen at necropsy for any animals, and no differences were observed in hematology, coagulation parameters, or clinical chemistry. No adverse reactions were observed microscopically in the liver. However, minimal neuronal degeneration/necrosis was observed in the cervical dorsal root ganglia of 2 of the 3 monkeys in the P1-miA1AT209-coA1Atox group (#2) but not in any other animals.

All three rAAV5 vectors transduced well in the liver (Figure 20A) and the lung (Figure 21 A). In the liver, vDNA transduction reached greater than 1 E6 copies of vector DNA per mg of host DNA, with transgene #3 performing slightly better than the other two transgenes (Figure 20 A). The mRNA levels of the human A1AT transgene were greater than 1 E7 copies per mg of host RNA for the group that received transgene #3 (Figure 20B). The expression level of miA1AT209 was also higher in the same group (Figure 20C). In the lung, the three rAAV5 vectors produced similar vDNA levels (Figure 21 A) in all groups. Transgene #2 induced the highest level of the human A1AT mRNA per mg of host RNA (Figure 21 B), and a similar result was observed with the expression level of the miA1AT209 (Figure 21 C).

Serum protein levels of human A1AT were measured using Meso Scale Discovery (MSD) and via Parallel Reaction Monitoring-Mass Spectrometry (PRM-MS) of peptides digested from the total serum samples. Results were generally similar between the two methods though absolute quantities differed (Figure 22A-B). The PRM-MS method appears to underestimate the quantities of endogenous and human A1AT compared to MSD. Within the liver and lung tissues, the level of A1AT protein was highest in the lung and, in particular in the group that received transgene 3# as measured by MSD (Figure 23).

Expression of miA1AT209 significantly reduced the absolute and relative levels of endogenous cynomolgus A1 AT mRNA (Figure 24). The knockdown efficiency of endogenous A1 AT mRNA with P2- miA1 AT209-coA1 ATox(CG) was about 99% in the liver (Figure 24A) and 40% in the lung (Figure 24B). The knockdown of endogenous A1AT mRNA resulted in a significant lowering of serum A1AT protein levels over time, as revealed by PRM-MS (Figure 25).

Human A1AT protein levels were also measured in the bronchoalveolar lavage (BAL) fluids by MSD and extrapolated back to epithelial lining fluid (ELF) levels based on total protein levels observed in the BAL and what is expected in the ELF (with 100 mg/ml as the expected concentration) (Figure 26 A-B). Similarly, also here the group that received transgene #3 reached the highest concentrations of A1AT protein.

In conclusion, all three tested transgenes (#2, #3 and #4) performed very well in non-human primates with good levels of liver transduction and transgene expression. miA1AT209 knockdown of the endogenous NHP A1AT gene was surprisingly successful with up to 99% lowering in the liver which then resulted in a significant lowering of A1AT in the serum, especially for the animals that received transgene #3. In addition, the vectors also transduced the lung tissue to satisfactory levels, with good vDNA levels and transgene expression. Of note, the expression level of human A1AT protein in the lung far exceeded that of the liver. This may at least in part be due to the tissue specificity of the promoter, which tends to vary between species. Of note, knockdown of NHP A1 AT by the miA1 AT209 reached a desirable level (60% lowering). Lung NHP A1AT protein knockdown data is pending, but is expected to reach similar levels. The human transgene protein of A1AT penetrated to the lung epithelial lining fluid (ELF) which is the target tissue for A1 AT treatment and may have originated from both the lung tissue and A1AT produced in the liver. The levels detected in the ELF of NHP are similar to those reported for SS allele patients who have no symptoms of A1AT deficiency.

In vivo dose-response in PiZ mice

3-month-old PiZ male mice are intravenously injected with 3 different doses of rAAV5 expressing both miAIAT and A1AT protein (Table 4). The mice are followed up for 12 weeks. Two groups are followed up for 6 months, to test the vector durability. Liver samples are collected to assess vDNA, mRNA (endogenous mRNA, and transgenic mRNA) and miRNA levels. Liver slices are prepared to assess z- A1AT globules number and size. A1AT protein levels are assessed in the serum. This study aims to show a dose response in both miRNA (miA1AT209) and A1AT protein levels over three months and to demonstrate target engagement for miRNA (miA1 AT209) at lower doses.

Table 4: study design of dose-response study in PiZ mice

Efficacy in disease PiZ mice with manifested fibrosis

6-month-old PiZ male mice are intravenously injected with 3 different doses of a rAAV5 expressing both miAIAT and A1AT protein. The mice are followed up for 6 months. Liver samples are collected to assess vDNA, mRNA (endogenous mRNA, and transgenic mRNA) and miRNA levels. Liver slices are prepared to assess z-A1 AT globules number and size. A1 AT protein levels are assessed in the serum. The two studies with PiZ animals (3M and 6M of age) focus on simulating patients with advanced liver fibrosis, considering both male and female populations with differing disease progression. It also evaluates potential safety concerns related to high hepatocellular PiZ-AAT accumulation and Tg-A1AT production. The proof of concept for the miRNA candidate will demonstrate the potential to halt or reduce fibrosis, while the study also assesses vector durability in a disease model characterized by high hepatocyte proliferation.

Assessment of A1 AT expression level in an A1 AT (or AAT) knockout (KO) mouse model

An A1AT (or AAT) knockout (KO) mouse model is used to assess A1AT expression levels upon administration of an rAAV5 vector comprising either transgene #3 or #4 (Table 3). In this animal model, the secretion of functional A1AT protein upon the rAAV-mediated delivery of transgene #3 or #4 can be measured directly. Functional expression of A1AT is a requirement for developing a successful treatment for A1ATD that not only relies on lowering of toxic (PiZ variant of) A1AT, but also supplementation of functional A1AT. These studies in AAT-KO mice are expected to confirm the finding that the rAAV vectors comprising transgenes as described herein, are able to induce therapeutic A1 AT serum levels in the lungs in addition to restoring liver function. Read outs for this study include the functional presence of transgene-derived (Fc-)A1AT in bronchoalveolar lavage (BAL) fluid as well as the potential matrix effect of A1 AT.

Claims

1. A nucleic acid comprising a sequence encoding an RNA, wherein the RNA comprises a guide sequence of at least 19 nucleotides substantially complementary to a target region within an mRNA encoded by the human SERPINA1 gene.

2. The nucleic acid of claim 1 , wherein the target region within an mRNA encoded by the human SERPINA1 gene is selected from SEQ ID NOs: 206, 207, and/or 208.

3. The nucleic acid of claim 1 or 2, wherein the guide sequence is selected from: SEQ ID Nos 55, 99, and/or 105, or a variant thereof.

4. The nucleic acid according to any one of claims 1-3, wherein the nucleic acid further comprises a sequence encoding an A1 AT protein, preferably wherein the A1 AT protein is oxidation-resistant.

5. The nucleic acid according to claim 4, wherein the sequence encoding an oxidation-resistant A1 AT protein does not encode a sequence that is substantially complementary to the guide sequence.

6. The nucleic acid according to claim 4 or 5, wherein the sequence encoding an oxidation resistant A1AT protein comprises SEQ ID NO: 120, 122, 128, or 129, or a variant thereof.

7. The nucleic acid according to any one of claims 1-6, further comprising a promoter and a sequence encoding a polyAdenylation tail.

8. The nucleic acid according to any one of claims 1 -7, wherein the promoter comprises SEQ ID NO. 162 or 168.

9. The nucleic acid according to claim 7 or 8, wherein the promoter comprises an intronic sequence, preferably wherein the intronic sequence comprises SEQ ID NO. 164, 165 or 166.

10. The nucleic acid according to claim 9, wherein the intronic sequence further comprises the sequence encoding the RNA.

11. The nucleic acid according to any one of claims 7 to 10, wherein the sequence encoding an oxidation-resistant A1AT protein is located between the promoter and the sequence encoding the polyAdenylation tail.

12. The promoter as defined in claims 8 and 9 comprising:

- a sequence comprising the nucleic acid of SEQ ID NO. 171 , 172, 173, 174 and 176, or variants thereof;

- a minimal promoter, preferably wherein the minimal promoter comprises SEQ ID NO. 177 or 178, or variants thereof; and - an intronic sequence, preferably an intronic sequence comprising SEQ ID NO. 164, 165, or 166, more preferably SEQ ID NO. 164.

13. An expression cassette comprising a nucleic acid according to any one of claims 1 to 11 , preferably wherein the expression cassette is flanked by Inverted Terminal Repeats (ITRs).

14. A recombinant adeno-associated virus (rAAV) vector comprising the expression cassette according to claim 13, preferably wherein the rAAV has a serotype selected from AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, variants thereof and hybrid serotypes.

15. A pharmaceutical formulation comprising an rAAV vector according to claim 14 and a pharmaceutically acceptable excipient.

16. The nucleic acid according to any one of claims 1-11 or the expression cassette according to claim 13, or an rAAV vector according to claim 14, or the pharmaceutical formulation according to claim 15 for use in a medical treatment, preferably for use in the treatment of Alpha-1 Antitrypsin Deficiency.

17. A method of treatment comprising administering the nucleic acid according to any one of claims 1- 11 , the expression cassette according to claim 13, or the rAAV vector according to claim 14, or the pharmaceutical formulation according to claim 15 to a person in need thereof, preferably for the treatment of Alpha-1 Antitrypsin Deficiency.

18. A host cell comprising the expression cassette according to claim 13.

19. A method for producing an rAAV vector according to claim 14.