ADA2 proteins and uses thereof

Abstract

The present invention is inter alia directed to a fusion protein comprising (i) an adenosine deaminase 2 (ADA2) domain and (ii) a DNA binding domain, wherein the DNA binding domain is capable of binding to a target DNA sequence; a pharmaceutical composition comprising adenosine deaminase 2 (ADA2) or a functional variant thereof and at least one pharmaceutically suitable excipient, wherein the pharmaceutical composition is targeted to the lysosome; and a pharmaceutical composition comprising adenosine deaminase 2 (ADA2) or a functional variant thereof and at least one pharmaceutically suitable excipient, wherein the pharmaceutical composition is targeted to a macrophage.

Description

[0001] ADA2 PROTEINS AND USES THEREOF

[0002] Field of the Invention

[0003] The present invention lies in the field of DNA editing, enzyme replacement therapy and treatment of TLR9-linked diseases.

[0004] Background of the Invention

[0005] As outlined in Pacesa et al. (Cell 187, February 29, 2024, 10.1016 / j. cell.2024.01.042), genome editing has been a transformative force in the life sciences and human medicine, offering unprecedent opportunities to dissect complex biological processes and treat the underlying causes of many genetic diseases.

[0006] There is the need to provide further tools for gene editing, in particular tools for gene editing relying on human enzymes in order to minimize undesired side effects associated with the use of non-human enzymes in the human body. Furthermore, there is the need to provide an effective treatment for ADA2 deficiency diseases (DADA2) as well as forTLR9-linked diseases, including cancer and inflammation.

[0007] Summary of the Invention

[0008] The present invention solves the above needs. It is based on the inventors’ surprising finding that ADA2 is a lysosomal protein that binds DNA and that is capable of deaminating deoxyadenosine (dA) of the bound DNA to deoxyinosine (dl). The inventors also surprisingly found that dA-to-dl editing of DNA by ADA2 facilitates immune detection of DNA mediated by TLR9, thus increasing TLR9 activity.

[0009] The invention described herein inter alia provides an ADA2-fusion protein suitable for DNA editing, as well as pharmaceutical compositions comprising lysosome-targeted ADA2 or macrophage-targeted ADA2, respectively, for medical uses in the field of treating DADA2 as well as TLR9-linked diseases.

[0010] DNA editing

[0011] In a first aspect, the invention relates to a fusion protein comprising (i) an adenosine deaminase 2 (ADA2) domain and (ii) a DNA binding domain, wherein the DNA binding domain is capable of binding to a target DNA sequence. It is preferred that, from the N- to the C-terminus, the fusion protein comprises the adenosine deaminase 2 (ADA2) domain, followed by the DNA binding domain. A target-specific DNA-binding domain recognizes and binds to a specific DNA sequence or a specific DNA sequence pattern, respectively, such as a recognition sequence or motif. A target-specific DNA-binding domain can generally be derived from DNA binding molecules such as transcription factors, restriction enzymes (including DNA endonucleases including Cas-proteins including Cas-nickases), zinc finger proteins or Transcription Activator-Like Effectors (TALEs). Thus, the binding to a target DNA sequence is to be understood as binding to a pre-defined DNA sequence (the “recognition sequence or motif’), which is ideally present only once in a genome to exclude an undesired effect at a further location in the genome. In some embodiments, the ADA2 domain comprises ADA2 or a functional variant thereof. In some embodiments, the ADA2 is a primate ADA2 or a functional variant thereof. The primate ADA2 may be selected from the group consisting of human, chimpanzee, bonobo, gorilla, orangutan, macaque and new word monkey (Platyrrhini) ADA2. In preferred embodiments thereof, the ADA2 or functional variant thereof is a human ADA2 or a functional variant thereof.

[0012] ADA2 is a homodimer. Homodimers are assembled from two monomers. Each monomer may comprise several active domains. Typically, when expressing monomers of ADA2 in a cell, the monomers will assemble into a homodimer via molecular interactions such as hydrogen bonds, hydrophobic interactions, ionic bonds or van der Waals forces.

[0013] In some embodiments, the ADA2 domain comprises two ADA2 monomers, optionally wherein at least one monomer is a functional variant of ADA2. Thus, the first ADA2 monomer may be ADA2 and the second ADA2 monomer may be a functional variant of ADA2. Alternatively, the first ADA2 monomer may be a functional variant of ADA2 and the second ADA2 monomer may be ADA2. Yet alternatively, the first and the second ADA2 monomers may be ADA2. Still alternatively, both ADA2 monomers may be functional variants of ADA2. It is preferred in these options that the ADA2 or functional variant thereof is human ADA2 or a functional variant thereof.

[0014] In some embodiments, the ADA2 domain comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0015] In some embodiments, the ADA2 domain comprises two amino acid sequences of SEQ ID NO: 1 or two amino acid sequences having independently at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0016] In some embodiments, the ADA2 domain comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0017] In some embodiments, the ADA2 domain comprises two amino acid sequences of SEQ ID NO: 2 or two amino acid sequences having independently at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0018] In some embodiments, each of the two ADA2 monomers is individually selected from an ADA2 species of the group consisting of human, chimpanzee, bonobo, gorilla, orangutan, macaque, new word monkey (Platyrrhini). In some embodiments, the two monomers or amino acid sequences, respectively, are connected by a linker and / or by a cleavable peptide domain. Suitable linkers are for example disclosed in Chen et al.

[0019] 2012, Adv Drug DelivRev. 65(10):1357-1369, hereby incorporated by reference. In some embodiments, the linker comprises 4-50 consecutive amino acids. In some embodiments, the linker comprises 4-40, 4-30, 4-20, 4-10, 10-40, 20-40, 30-40, 10-30, or 20-30 consecutive amino acids.

[0020] In some embodiments, the DNA binding domain and the ADA2 domain are connected by a linker, preferably a linker as defined above linking the two monomers. In a preferred embodiment, the linker is a flexible linker. In some embodiments, the linker comprises glycine and / or serine residues (“GS” linker). In some embodiments, the linker is selected from the group consisting of (GGGGS)n(SEQ ID NO: 3), (GGS)n(SEQ ID NO: 4), and (SGSG)n(SEQ ID NO: 5), wherein n is from 1 to 20; and Gn, wherein n is 4-20 (SEQ ID NO: 6). In some embodiments, the linker is a flexible “GS-rich” linker, including a linker comprising or consisting of the amino acid sequence GSAGSAAGSGEF (SEQ ID NO: 7), KESGSVSSEQLAQFRSLD (SEQ ID NO: 8), EGKSSGSGSESKST (SEQ ID NO: 9), SGSETPGTSESATPES (SEQ ID NO: 10) or SGGSPKKKRKVGSSGS (SEQ ID NO: 11).

[0021] In some embodiments, the linker is selected from:

[0022] i) rigid alpha-helix forming linkers, such as (EAAAK)n, wherein n is 1-5 (SEQ ID NO: 26);

[0023] A(EAAAK)n, wherein n is 1-5 (SEQ ID NO: 27); A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 28); or AEAAAKEAAAKA (SEQ ID NO: 29);

[0024] ii) rigid Pro-rich linkers, such as (XP)n, wherein X is any amino acid (SEQ ID NO: 30), preferably Ala (SEQ ID NO: 31), Lys (SEQ ID NO: 32) or Gin (SEQ ID NO: 33), and wherein n is 2-20); such as (AP)7(SEQ ID NO: 34); or PAPAP (SEQ ID NO: 35); or

[0025] iii) mixed flexible-rigid linkers comprising GS-rich and alpha-helix forming elements, such as (EAAAK)n(GGGGS)m, wherein n is 1-2 and m is 2-3 (SEQ ID NO: 36).

[0026] In other embodiments, the linker is selected from the group consisting of, (EAAAK)2(GGGGS)3(SEQ ID NO: 37), (EAAAK)1(GGGGS)3(SEQ ID NO: 38), (EAAAK)1(GGGGS)2(SEQ ID NO: 39), and (EAAAK)2(GGGGS)2(SEQ ID NO: 40).

[0027] In some embodiments, the cleavable peptide domain is a 2A peptide domain, preferably selected from the group consisting of a T2A, P2A, E2A or F2A peptide domain. In this case, once the cleavable peptide has been cleaved, a monomer located previously C-terminally to the cleavage peptide will assemble with the monomer located previously N-terminally to the cleavage peptide to form a homodimer, wherein the monomer located previously N-terminally to the cleavage peptide is and remains fused to the DNA binding domain.

[0028] In some embodiments, the fusion protein further comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is a signaling peptide derived from a nuclear protein. In some embodiments, the NLS is about 4 to 30 amino acids in length and acts as a ligand for a nuclear import transporter. An NLS can be located N- or C-terminally or at an internal position within a protein. In some embodiments, the NLS is located at the N-terminus of the fusion protein. In some embodiments, the NLS is derived from an Adeno-associated virus (AAV) viral protein 1 (VP1). Suitable NLS are described in the literature, for example in Lu et al. Cell Commun Signal 19, 60 (2021), doi 10.1186 / s 12964-021-00741-y or Hoad et al., J Virol 99:e01345-24, doi 10.1128 / jvi.01345-24, both of which are hereby incorporated by reference.

[0029] The NLS may comprise or consist of an amino acid sequence selected from the group consisting of PKKKRKV (SEQ ID NO: 12), KRPAATKKAGQAKKKK (SEQ ID NO: 13), GKRKLITSEEERSPAKRGRKS (SEQ ID NO: 14), KGKKGRTQKEKKAARARSKGKN (SEQ ID NO: 15), AVKRPAATKKAGQAKKKKLD (SEQ ID NO: 16), MSRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 17), PAAKRVKLD (SEQ ID NO: 18), KLKIKRPVK (SEQ ID NO: 19), RKRCAAGVGGGPAGCPAPGSTPLKKPRR (SEQ ID NO: 20), RKPVTAQERQREREEKRRRRQERAKEREKRRQERER (SEQ ID NO: 21), FGNYNNQSSNFGPMKGGNFGGRSSGPY (SEQ ID NO: 22), TLLLRETMNNLGVSDHAVLSRKTPQPY (SEQ ID NO: 23), PGKMDKGEHRQERRDRPY (SEQ ID NO: 24), and GRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFG (SEQ ID NO: 25). It is noted that a NLS can generally be comprised anywhere in the fusion protein and thus not mandatorily e.g. at the N- or C-terminus. It is further noted that it is preferred that the NLS is exposed at the surface of the fusion protein.

[0030] In some embodiments, the DNA binding domain is an RNA-guided DNA binding domain.

[0031] In some embodiments, the RNA-guided DNA binding domain is capable of forming a complex with an RNA, preferably with a guide RNA (gRNA).

[0032] In some embodiments, the fusion protein further comprises (iii) a DNA endonuclease domain. In some embodiments, the DNA endonuclease domain produces a target-specific double strand break or a target-specific single-strand break. It is preferred that the target-specific double strand break or the target-specific single-strand break is produced at a deoxyadenosine. The double strand break or the single-strand break may be produced 3’ or 5’ to the deoxyadenosine (in other words before or after the deoxyadenosine). Most preferably, the target-specific break is a target-specific single-strand break. Enzymes resulting in a single-strand break are typically referred to as nickases.

[0033] A fusion protein according to the first aspect may therefore comprise a DNA binding domain, wherein the DNA binding domain is capable of binding to a target DNA sequence (which is typically the first activity of the fusion protein, i.e., DNA binding); a DNA endonuclease domain, wherein the DNA endonuclease domain is capable of producing a target-specific double strand break or a target-specific single-strand break at a deoxyadenosine (which is typically the second activity of the fusion protein after DNA binding, i.e., DNA single- or double-strand cleavage at a deoxyadenosine); and an ADA2 domain capable of converting dA to dl (which is typically the third activity of the fusion protein after DNA binding and DNA cleavage at a deoxyadenosine). Most preferably, the target-specific break is a target-specific single-strand break.

[0034] In some embodiments, the (i) DNA binding domain and (iii) the DNA endonuclease domain are comprised in a single polypeptide. In some embodiments, the DNA binding domain and the DNA endonuclease domain are derived from the same polypeptide, i.e. from the same enzyme. Hence, in some embodiments, the DNA binding domain and the DNA endonuclease domain are both comprised in one polypeptide, and this polypeptide belongs to the class of RNA-guided DNA endonucleases. Hence, in some embodiments, the fusion protein according to the first aspect comprises an ADA2 domain and an RNA-guided DNA endonuclease. In some embodiments, the RNA-guided DNA endonuclease is suitable for genome editing, CRISPR genome editing, or transposon-based recombination.

[0035] In some embodiments, the RNA-guided DNA endonuclease is selected from Cas enzymes, such as Cas9, Cas3 and Cas12, transposon-associated enzymes such as IscB, IsrB and TnpB, and the eukaryotic RNA-guided DNA endonuclease enzyme Fanzor. Hence, the RNA-guided DNA endonuclease may be selected from the group consisting of Cas9, Cas3, Cas12, IscB, IsrB, TnpB and Fanzor; or a functional mutant or variant of any of the foregoing.

[0036] In some embodiments, the RNA-guided DNA endonuclease is suitable for CRISPR genome editing and OMEGA genome editing. In some embodiments, the RNA-guided DNA endonuclease is suitable for CRISPR genome editing. In some embodiments, the RNA-guided DNA endonuclease is suitable for OMEGA genome editing.

[0037] In a preferred embodiment, the RNA-guided DNA endonuclease is a Cas domain.

[0038] Hence, in some embodiments, the fusion protein according to the first aspect comprises an ADA2 domain and a Cas domain.

[0039] It can be preferred that the single polypeptide comprising the DNA binding domain and the endonuclease domain is selected from a Cas9 endonuclease or a functional variant thereof, including a Cas9 nickase; and a Cas12 endonuclease or a functional variant thereof, including a Cas12 nickase. The single polypeptide may in particular be a Cas9 nickase or a Cas12 nickase, or a functional variant of such a nickase.

[0040] In some embodiments, Cas9 is from Streptococcus pyogenes. In some embodiments, Cas12 includes Cas12a. In some embodiments, Cas12a is from Eubacterium rectale.

[0041] Hence, in some embodiments, the fusion protein according to the first aspect comprises an ADA2 domain and a Cas9 nickase domain. In some embodiments, the Cas9 nickase carries a D10 or a H840 mutation. In some embodiments, the Cas9 nickase carries a D10A or a H840A mutation.

[0042] In some embodiments, the RNA-guided DNA endonuclease comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, ora functional variant of any of the foregoing.

[0043] In some embodiments, the fusion protein according to the first aspect comprises an ADA2 domain comprising an amino acid sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 95% identity to the amino acid sequence depicted in SEQ ID NO: 1; and a Cas9 domain comprising an amino acid sequence with at least about 80%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence depicted in SEQ ID NO: 41. In some embodiments, the fusion protein according to the first aspect comprises an ADA2 domain comprising an amino acid sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 95% identity to the amino acid sequence depicted in SEQ ID NO: 1; and a Cas9 domain comprising an amino acid sequence with at least about 80, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence depicted in SEQ ID NO: 42.

[0044] In some embodiments, the fusion protein according to the first aspect comprises an ADA2 domain comprising an amino acid sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 95% identity to the amino acid sequence depicted in SEQ ID NO: 1; and a Cas9 domain comprising an amino acid sequence with at least about 80, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence depicted in SEQ ID NO: 43.

[0045] In some embodiments, the DNA binding domain and the ADA2 domain are connected by a linker, preferably a linker as defined above linking the two monomers. In a preferred embodiment, the linker is a flexible linker. In some embodiments, the linker comprises glycine and / or serine residues (“GS” linker). In some embodiments, the linker is selected from the group consisting of (GGGGS)n(SEQ ID NO: 3), (GGS)n(SEQ ID NO: 4), and (SGSG)n(SEQ ID NO: 5), wherein n is from 1 to 20; and Gn, wherein n is 4-20 (SEQ ID NO: 6). In some embodiments, the linker is a flexible “GS-rich” linker, including a linker comprising or consisting of the amino acid sequence GSAGSAAGSGEF (SEQ ID NO: 7), KESGSVSSEQLAQFRSLD (SEQ ID NO: 8), EGKSSGSGSESKST (SEQ ID NO: 9), SGSETPGTSESATPES (SEQ ID NO: 10) or SGGSPKKKRKVGSSGS (SEQ ID NO: 11).

[0046] In some embodiments, the linker is selected from:

[0047] i) rigid alpha-helix forming linkers, such as (EAAAK)n, wherein n is 1-5 (SEQ ID NO: 26);

[0048] A(EAAAK)n, wherein n is 1-5 (SEQ ID NO: 27); A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 28); or AEAAAKEAAAKA (SEQ ID NO: 29);

[0049] ii) rigid Pro-rich linkers, such as (XP)n, wherein X is any amino acid (SEQ ID NO: 30), preferably Ala (SEQ ID NO: 31), Lys (SEQ ID NO: 32) or Gin (SEQ ID NO: 33), and wherein n is 2-20); such as (AP)7(SEQ ID NO: 34); or PAPAP (SEQ ID NO: 35); or

[0050] iii) mixed flexible-rigid linkers comprising GS-rich and alpha-helix forming elements, such as (EAAAK)n(GGGGS)m, wherein n is 1-2 and m is 2-3 (SEQ ID NO: 36).

[0051] In other embodiments, the linker is selected from the group consisting of, (EAAAK)2(GGGGS)3(SEQ ID NO: 37), (EAAAK)1(GGGGS)3(SEQ ID NO: 38), (EAAAK)1(GGGGS)2(SEQ ID NO: 39), and (EAAAK)2(GGGGS)2(SEQ ID NO: 40).

[0052] In some embodiments, the ADA2 domain is tethered by the linker to the C terminus or the N terminus of the RNA-guided DNA endonuclease domain. In a preferred embodiment, the ADA2 domain is tethered by the linker to the N terminus of the RNA-guided DNA endonuclease domain. In another embodiment, the DNA binding domain is tethered by the linker to the C terminus of the RNA-guided DNA endonuclease domain. Hence, in some embodiments, the fusion protein of the first aspect comprises, from the N to the C terminus: ADA2 monomer - flexible linker- ADA2 monomer - linker - RNA-guided DNA endonuclease domain. In some embodiments, the fusion protein comprises, from N to C terminus: ADA2 monomer -flexible linker - ADA2 monomer - linker - Cas domain. In some embodiments, the fusion protein comprises, from N to C terminus: ADA2 monomer - flexible linker - ADA2 monomer - linker - Cas nickase domain.

[0053] In a second aspect, the present invention is directed to a nucleic acid encoding the fusion protein according to the first aspect, including all embodiments as outlined above. The nucleic acid may be DNA or RNA, including mRNA.

[0054] In a third aspect, the present invention is directed to a vector comprising the nucleic acid according to the second aspect.

[0055] In some embodiments, the vector is an expression vector. In such embodiments, the nucleic acid of the second aspect is typically functionally linked to a promoter sequence that induces the expression of the encoding nucleic acid.

[0056] In a fourth aspect, the present invention is directed to a ribonucleoprotein (RNP) complex comprising the fusion protein according to the first aspect, including all embodiments as outlined above, and at least one target-specific guide RNA (gRNA).

[0057] In a fifth aspect, the present invention is directed to a particle comprising the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the vector according to the third aspect, or the RNP complex according to the fourth aspect.

[0058] The particle may be selected according to the cargo that is comprised in the particle. If, for example, the fusion protein is comprised in the particle, the particle may be a polymeric (e.g., PLGA, gelatin, chitosan, hyaluronic acid) or a virus-like particle. In another example, the cargo may be a nucleic acid such as an expression vector or mRNA, the particle may be a lipid nanoparticle (LNP). Yet further, the particle may comprise an RNP complex, and the particle may be an LNP, a gold nanoparticle ora polymeric particle. Accordingly, in some embodiments, the particle is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a herpes simplex viral vector, a baculoviral vector, an Epstein-Barr viral vector, a poxvirus vector, a virosome, a lipid nanoparticle (LNP) and a liposome. The skilled person is well aware of suitable particles that may be used, depending on the cargo that is comprised in the particle. On a general level, any standard delivery technology known to and available for the skilled person, including commercially available delivery technology, may be used.

[0059] In a sixth aspect, the present invention is directed to a cell comprising the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the vector according to the third aspect, the RNP complex according to the fourth aspect, or the particle according to the fifth aspect. The cell may be inside a human or animal body or the cell may be outside the human or animal body. A cell outside the human or animal body may in particular be used to produce the fusion protein according to the first aspect. A cell inside the human or animal body may in particular be a patient’s cell undergoing genome editing. However, such gene editing may also be carried out in a cell outside the human or animal body, wherein the cell has been gained from a patient and wherein the cell is put pack into the patient after the genome editing.

[0060] In a seventh aspect, the present invention is directed to a pharmaceutical composition comprising the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the vector according to the third aspect, the RNP complex according to the fourth aspect, or the particle according to the fifth aspect, including all embodiments of each aspect as outlined above, and at least one pharmaceutically suitable excipient.

[0061] In an eighth aspect, the present invention is directed to the use of the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the vector according to the third aspect, the RNP complex according to the fourth aspect, the particle according to the fifth aspect, or the pharmaceutical composition according to the seventh aspect (including all embodiments of each aspect as outlined above) in gene editing. The gene editing is preferably the conversion from a dA to a dl. In some embodiments, the use of the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the vector according to the third aspect, the RNP complex according to the fourth aspect, the particle according to the fifth aspect, or the pharmaceutical composition according to the seventh aspect in gene editing comprises the site-directed modification of the genome of a cell or a cell population. It is noted that the site-directed modification of the genome may be one or more than one site-directed conversion of a dA to a dl. The skilled person is aware that a dl is recognized as a dG such that the at least one site-directed conversion from a dA to a dl is basically a conversion from a dA to a dG, i.e., the enzymes recognize a dG instead of a dA. The cell may be a cell inside a human or animal body, i.e., a living cell inside an organism, or the cell may be a cell outside the human or animal body, i.e., an isolated cell comprised e.g. in cell culture (which may be referred to as an in vitro situation). The cell population may be a cell population inside a human or animal body, i.e., a living cell population inside an organism, or the cell population may be a cell population outside the human or animal body, i.e., an isolated cell population comprised e.g. in cell culture (which may be referred to as an in vitro situation).

[0062] In a ninth aspect, the invention provides a method of gene editing comprising the steps of

[0063] i) providing a double-stranded DNA; and

[0064] ii) contacting the double stranded DNA with the fusion protein according to the first aspect and at least one target-specific gRNA, or the RNP complex according to the fourth aspect, including all embodiments of each aspect as outlined above.

[0065] In some embodiments, the method comprises the further step of providing a cell in vivo or in vitro, wherein the cell comprises the double-stranded DNA (such that the double-stranded DNA is provided in step i)). Accordingly, in some embodiments, the method may be an in vitro method or an in vivo method.

[0066] In some embodiments, the contacting step ii) results in a conversion of a dA to a dl at one or more targeted position(s) in the double-stranded DNA. In a tenth aspect, the invention provides a method of gene editing comprising administering the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the vector according to the third aspect, the RNP complex according to the fourth aspect, the particle according to the fifth aspect, or the pharmaceutical composition according to the seventh aspect (including all embodiments of each aspect as outlined above) to a subject in need thereof.

[0067] In an embodiment, this may also be referred to as a method of treating a disease by gene editing in a patient in need thereof comprising administering the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the vector according to the third aspect, the RNP complex according to the fourth aspect, the particle according to the fifth aspect, or the pharmaceutical composition according to the seventh aspect (including all embodiments of each aspect as outlined above) to the patient.

[0068] In some embodiments, the gene editing comprises the conversion of a dA to a dl at one or more targeted position(s) in the double-stranded DNA.

[0069] In an eleventh aspect, the invention provides a kit comprising the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the vector according to the third aspect, the RNP complex according to the fourth aspect, the particle according to the fifth aspect, or the pharmaceutical composition according to the seventh aspect; and instructions for use.

[0070] In some embodiments, the kit comprises the fusion protein according to the first aspect, instructions for use and a buffer solution to facilitate cellular / nuclear penetration.

[0071] In some embodiments, the kit comprises the fusion protein according to the first aspect, at least one target sequence-specific gRNA, instructions for use and a buffer solution to facilitate either RNP complex formation or cellular / nuclear penetration.

[0072] DADA2 treatment

[0073] As noted above, the inventors also surprisingly found that ADA2 acts predominantly in the lysosome and not only in the extracellular space. An effective treatment of DADA2 can thus be achieved by an ADA2-replacement therapy, wherein ADA2 is targeted to the lysosome to “do the job” of the lacking / mutant endogenous ADA2 or the endogenous ADA2 present at a much lower level, respectively. In a twelfth aspect, the invention provides a pharmaceutical composition comprising adenosine deaminase 2 (ADA2) or a functional variant thereof and at least one pharmaceutically acceptable excipient, wherein the pharmaceutical composition is targeted to the lysosome.

[0074] An exemplary way to target a protein or a particle to the lysosome resided in the use of a lysosometargeting peptide tat. Lysosome-targeting peptide tags are derived from proteins that traffic to the lysosome, or synthetically designed. In one embodiment, the ADA2 or functional variant thereof is fused to a lysosome-targeting peptide tag corresponding to or derived from IGF-II (including a fragment thereof). Lysosome-targeting peptide tags include glycosylation-independent lysosomal targeting (GILT) tag, described in Maga et al. 2013, JBC 288(3): 1428-1438, hereby incorporated by reference. Synthetic endocytosis-triggering binding proteins called EndoTags described in Huang et al. (Nature 638, 796-804, 2025, doi 10.1038 / s41586-024-07948-2), hereby incorporated by reference.

[0075] In some embodiments, the targeting to the lysosome is achieved by a modification of the ADA2. Such a modification of the ADA2 may in particular be an altered glycosylation pattern of ADA2. In some embodiments, modifying the glycosylation pattern of ADA2 comprises addition of mannose 6-phosphate or mannose. In some embodiments, the ADA2 comprises more mannose 6-phosphate and / or mannose compared to wild-type ADA2, preferably at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 5 times or at least about 10 times more mannose 6-phosphate and / or mannose compared to wild-type ADA2. In some embodiments, the ADA2 is fused to IGF-II (SEQ ID NO: 44) or a fragment thereof, or GILT (SEQ ID NO: 45).

[0076] In some embodiments, the composition comprises a particle comprising ADA2, wherein the particle is targeted to the lysosome. In this setup, the ADA2 may be modified as outlined above, i.e., the ADA2 may also be modified to target the lysosome. In some embodiments, the particle comprises IGF-II (SEQ ID NO: 44) or a fragment thereof, or GILT (SEQ ID NO: 45) on the surface of the particle.

[0077] In some embodiments, the ADA2 is a primate ADA2 or functional variant thereof. The primate ADA2 may be selected from the group consisting of human, chimpanzee, bonobo, gorilla, orangutan, macaque, and new world monkey (Platyrrhini) ADA2. In preferred embodiments thereof, the ADA2 or functional variant thereof is a human ADA2 or a functional variant thereof.

[0078] ADA2 is a homodimer. Homodimers are assembled from two monomers. Each monomer may comprise several active domains. Typically, when expressing monomers of ADA2 in a cell, the monomers will assemble into a homodimer via molecular interactions such as hydrogen bonds, hydrophobic interactions, ionic bonds or van der Waals forces.

[0079] In some embodiments, the ADA2 comprises two ADA2 monomers, optionally wherein at least one monomer is a functional variant of ADA2. Thus, the first ADA2 monomer may be ADA2 and the second ADA2 monomer may be a functional variant of ADA2. Alternatively, the first ADA2 monomer may be a functional variant of ADA2 and the second ADA2 monomer may be ADA2. Yet alternatively, the first and the second ADA2 monomers may be ADA2. Still alternatively, both ADA2 monomers may be functional variants of ADA2. It is preferred in these options that the ADA2 or functional variant thereof is human ADA2 or a functional variant thereof.

[0080] In some embodiments, the ADA2 domain comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0081] In some embodiments, the ADA2 domain comprises two amino acid sequences of SEQ ID NO: 1 or two amino acid sequences having independently at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, the ADA2 domain comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0082] In some embodiments, the ADA2 domain comprises two amino acid sequences of SEQ ID NO: 2 or two amino acid sequences having independently at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0083] In some embodiments, each of the two ADA2 monomers is individually selected from a species of the group consisting of human, chimpanzee, bonobo, gorilla, orangutan, macaque, new word monkey (Platyrrhini).

[0084] In some embodiments, the two monomers or amino acid sequences, respectively, are connected by a linker and / or by a cleavable peptide domain. Suitable linkers are for example disclosed in Chen et al.

[0085] 2012, Adv Drug DelivRev. 65(10):1357-1369, hereby incorporated by reference. In some embodiments, the linker comprises 4-50 consecutive amino acids. In some embodiments, the linker comprises 4-40, 4-30, 4-20, 4-10, 10-40, 20-40, 30-40, 10-30, or 20-30 consecutive amino acids.

[0086] In some embodiments, the DNA binding domain and the ADA2 domain are connected by a linker, preferably a linker as defined above linking the two monomers. In a preferred embodiment, the linker is a flexible linker. In some embodiments, the linker comprises glycine and / or serine residues (“GS” linker). In some embodiments, the linker is selected from the group consisting of (GGGGS)n(SEQ ID NO: 3), (GGS)n(SEQ ID NO: 4), and (SGSG)n(SEQ ID NO: 5), wherein n is from 1 to 20; and Gn, wherein n is 4-20 (SEQ ID NO: 6). In some embodiments, the linker is a flexible “GS-rich” linker, including a linker comprising or consisting of the amino acid sequence GSAGSAAGSGEF (SEQ ID NO: 7), KESGSVSSEQLAQFRSLD (SEQ ID NO: 8), EGKSSGSGSESKST (SEQ ID NO: 9), SGSETPGTSESATPES (SEQ ID NO: 10) or SGGSPKKKRKVGSSGS (SEQ ID NO: 11).

[0087] In some embodiments, the linker is selected from:

[0088] i) rigid alpha-helix forming linkers, such as (EAAAK)n, wherein n is 1-5 (SEQ ID NO: 26);

[0089] A(EAAAK)n, wherein n is 1-5 (SEQ ID NO: 27); A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 28); or AEAAAKEAAAKA (SEQ ID NO: 29);

[0090] ii) rigid Pro-rich linkers, such as (XP)n, wherein X is any amino acid (SEQ ID NO: 30), preferably Ala (SEQ ID NO: 31), Lys (SEQ ID NO: 32) or Gin (SEQ ID NO: 33), and wherein n is 2-20); such as (AP)7(SEQ ID NO: 34); or PAPAP (SEQ ID NO: 35); or

[0091] iii) mixed flexible-rigid linkers comprising GS-rich and alpha-helix forming elements, such as (EAAAK)n(GGGGS)m, wherein n is 1-2 and m is 2-3 (SEQ ID NO: 36).

[0092] In other embodiments, the linker is selected from the group consisting of, (EAAAK)2(GGGGS)3(SEQ ID NO: 37), (EAAAK)1(GGGGS)3(SEQ ID NO: 38), (EAAAK)1(GGGGS)2(SEQ ID NO: 39), and (EAAAK)2(GGGGS)2(SEQ ID NO: 40). In some embodiments, the cleavable peptide domain is a 2A peptide domain, preferably selected from the group consisting of T2A, P2A, E2A and F2A. In this case, once the cleavable peptide has been cleaved, a monomer located previously C-terminally to the cleavage peptide will assemble with the monomer located previously N-terminally to the cleavage peptide to form a homodimer, wherein the monomer located previously N-terminally to the cleavage peptide is fused to the DNA binding domain. In a thirteenth aspect, the invention provides a pharmaceutical composition comprising a nucleic acid encoding lysosome-targeted adenosine deaminase 2 (ADA2) ora functional variant thereof and at least one pharmaceutically acceptable excipient. The nucleic acid may in particular be mRNA. In some embodiments, the nucleic acid encodes ADA2 linked to a lysosome-targeting peptide (e.g., ADA2 of the amino acid sequence of SEQ ID NO: 2). In this setup, the ADA2 is expressed in the cytosol and then, due to the lysosome-targeting peptide, transported to the lysosome.

[0093] In a fourteenth aspect, the invention provides the pharmaceutical composition of the twelfth aspect or the thirteenth aspect for use in the treatment of DAD2. In other words, the invention provides a method for treatment of a subject suffering from DADA2 comprising administering the pharmaceutical composition of the twelfth aspect or the thirteenth aspect including all embodiments thereof to the subject.

[0094] In some embodiments, the treatment of DADA2 is a treatment of a DADA2 symptom, wherein the DADA2 symptom is preferably selected from the group consisting of systemic inflammation, vasculopathy, recurrent fever, livedoid rash, cytopenia, stroke, immunodeficiency and bone marrow failure.

[0095] Treatment of a TLR9-linked disease

[0096] As noted above, the inventors also surprisingly found that dA-to-dl editing of DNA by ADA2 in the lysosome facilitates immune detection of DNA mediated by TLR9, thus increasing TLR9 activity.

[0097] Providing ADA2 to macrophages is particularly useful for the treatment of TLR9-mediated diseases and phenotypes. As shown herein by the inventors, deamination of dA residues of DNA by ADA2 modulates TLR9 activation. Accordingly, ADA2 activity is associated with the significant upregulation of TLR9 signaling. Among the DNA bases, dC is the most frequently deaminated. At the same time, spontaneous deamination of dA is a minor reaction that occurs at 2-3% of the rate of dC deamination. The data presented herein suggest that this phenomenon can happen enzymatically in the lysosomes. Because dl is recognized as dG, converting dA residues in mammalian DNA modulates the TLR9 agonist potency of DNA molecules. TLR9 is generally believed to preferentially recognize pathogen-derived DNA containing a higher frequency of unmethylated CpG dinucleotides. Its stimulation leads to the production of type-1 IFN in human pDCs. The ability of TLR9 to regulate innate immune responses makes it an attractive target for translational medicine, particularly in cancer immunotherapy and inflammatory diseases.

[0098] In a fifteenth aspect, the invention provides a pharmaceutical composition comprising adenosine deaminase 2 (ADA2) or a functional variant thereof and at least one pharmaceutically acceptable excipient, wherein the pharmaceutical composition is targeted to a macrophage. In an embodiment thereof, the pharmaceutical composition is targeted to the lysosome of a macrophage. In some embodiments, the pharmaceutical composition comprises a particle comprising ADA2 or a functional variant thereof, and the particle comprises

[0099] (i) a material that passively targets macrophages, preferably selected from the group consisting of PLGA, chitosan, iron oxide, gold and silica,

[0100] (ii) a ligand on the surface of the particle that is specific for a binding partner or receptor expressed by macrophages, preferably selected from the group consisting of oligosaccharide, preferably mannose; folate, UNO peptide, M2pep, CXCR4 ligand and RGD peptides;

[0101] (iii) an antibody or antigen-binding fragment thereof (such as, e.g., a nanobody) specific for a macrophage surface marker on the surface of the particle, preferably wherein the macrophage surface marker is selected from the group consisting of CD206, CD163, CCL2, and CXCR4; and / or

[0102] (iv) pathogen-mimicking molecular patterns on the surface of the particle that are recognized by macrophages and induce phagocytosis;

[0103] or the particle has a natural tropism for macrophages, preferably wherein the particle is an exosome or a synthetic vesicle.

[0104] In some embodiments, the particle size is about 100 to about 1000 nm, preferably about 200 to about 500 nm.

[0105] In some embodiments, the ADA2 or functional variant thereof comprised in the particle is, in addition to the targeting of the particle to a macrophage, modified to target the lysosome. Such a modification of the ADA2 may in particular be an altered glycosylation pattern of ADA2. In some embodiments, modifying the glycosylation pattern of ADA2 comprises addition of mannose 6-phosphate or mannose. In some embodiments, the ADA2 comprises more mannose 6-phosphate and / or mannose compared to wild-type ADA2, preferably at least about 1.5 times, at least about 2 times, at least about 2.5 times, or at least about 5 times more mannose 6-phosphate and / or mannose compared to wild-type ADA2. In some embodiments, the ADA2 is fused to IGF-II (SEQ ID NO: 44) or a fragment thereof, or GILT (SEQ ID NO: 45).

[0106] In some embodiments, the ADA2 is a primate ADA2 or functional variant thereof. The primate ADA2 may be selected from the group consisting of human, chimpanzee, bonobo, gorilla, orangutan, macaque and new word monkey (Platyrrhini) ADA2. In preferred embodiments thereof, the ADA2 or functional variant thereof is a human ADA2 or a functional variant thereof.

[0107] ADA2 is a homodimer. Homodimers are assembled from two monomer monomers. Each monomer may comprise several active domains. Typically, when expressing monomers of ADA2 in a cell, the monomers will assemble into a homodimer via molecular interactions such as hydrogen bonds, hydrophobic interactions, ionic bonds or van der Waals forces.

[0108] In some embodiments, the ADA2 comprises two ADA2 monomers, optionally wherein at least one monomer is a functional variant of ADA2. Thus, the first ADA2 monomer may be ADA2 and the second ADA2 monomer may be a functional variant of ADA2. Alternatively, the first ADA2 monomer may be a functional variant of ADA2 and the second ADA2 monomer may be ADA2. Yet alternatively, the first and the second ADA2 monomers may be ADA2. Still alternatively, both ADA2 monomers may be functional variants of ADA2. It is preferred in these options that the ADA2 or functional variant thereof is human ADA2 or a functional variant thereof.

[0109] In some embodiments, the ADA2 domain comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0110] In some embodiments, the ADA2 domain comprises two amino acid sequences of SEQ ID NO: 1 or two amino acid sequences having independently at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0111] In some embodiments, the ADA2 domain comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0112] In some embodiments, the ADA2 domain comprises two amino acid sequences of SEQ ID NO: 2 or two amino acid sequences having independently at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0113] In some embodiments, each of the two ADA2 monomers is individually selected from a species of the group consisting of human, chimpanzee, bonobo, gorilla, orangutan, macaque, new word monkey (Platyrrhini).

[0114] In some embodiments, the two monomers or amino acid sequences, respectively, are connected by a linker and / or by a cleavable peptide domain. Suitable linkers are for example disclosed in Chen et al.

[0115] 2012, Adv Drug DelivRev. 65(10):1357-1369, hereby incorporated by reference. In some embodiments, the linker comprises 4-50 consecutive amino acids. In some embodiments, the linker comprises 4-40, 4-30, 4-20, 4-10, 10-40, 20-40, 30-40, 10-30, or 20-30 consecutive amino acids.

[0116] In some embodiments, the DNA binding domain and the ADA2 domain are connected by a linker, preferably a linker as defined above linking the two monomers. In a preferred embodiment, the linker is a flexible linker. In some embodiments, the linker comprises glycine and / or serine residues (“GS” linker). In some embodiments, the linker is selected from the group consisting of (GGGGS)n(SEQ ID NO: 3), (GGS)n(SEQ ID NO: 4), and (SGSG)n(SEQ ID NO: 5), wherein n is from 1 to 20; and Gn, wherein n is 4-20 (SEQ ID NO: 6). In some embodiments, the linker is a flexible “GS-rich” linker, including a linker comprising or consisting of the amino acid sequence GSAGSAAGSGEF (SEQ ID NO: 7), KESGSVSSEQLAQFRSLD (SEQ ID NO: 8), EGKSSGSGSESKST (SEQ ID NO: 9), SGSETPGTSESATPES (SEQ ID NO: 10) or SGGSPKKKRKVGSSGS (SEQ ID NO: 11).

[0117] In some embodiments, the linker is selected from: i) rigid alpha-helix forming linkers, such as (EAAAK)n, wherein n is 1-5 (SEQ ID NO: 26); A(EAAAK)n, wherein n is 1-5 (SEQ ID NO: 27); A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 28); or AEAAAKEAAAKA (SEQ ID NO: 29);

[0118] ii) rigid Pro-rich linkers, such as (XP)n, wherein X is any amino acid (SEQ ID NO: 30), preferably Ala (SEQ ID NO: 31), Lys (SEQ ID NO: 32) or Gin (SEQ ID NO: 33), and wherein n is 2-20); such as (AP)7(SEQ ID NO: 34); or PAPAP (SEQ ID NO: 35); or

[0119] iii) mixed flexible-rigid linkers comprising GS-rich and alpha-helix forming elements, such as (EAAAK)n(GGGGS)m, wherein n is 1-2 and m is 2-3 (SEQ ID NO: 36).

[0120] In other embodiments, the linker is selected from the group consisting of, (EAAAK)2(GGGGS)3(SEQ ID NO: 37), (EAAAK)1(GGGGS)3(SEQ ID NO: 38), (EAAAK)1(GGGGS)2(SEQ ID NO: 39), and (EAAAK)2(GGGGS)2(SEQ ID NO: 40).

[0121] In some embodiments, the cleavable peptide domain is a 2A peptide domain, preferably selected from the group consisting of T2A, P2A, E2A and F2A. In this case, once the cleavable peptide has been cleaved, a monomer located previously C-terminally to the cleavage peptide will assemble with the monomer located previously N-terminally to the cleavage peptide to form a homodimer, wherein the monomer located previously N-terminally to the cleavage peptide is fused to the DNA binding domain. In a sixteenth aspect, the invention provides the pharmaceutical composition of the fifteenth aspect for use in the treatment of a TLR9-linked disease. In other words, the invention provides a method for treatment of a subject suffering from a TLR9-linked diseases comprising administering the pharmaceutical composition of the fifteenth aspect including all embodiments thereof to the subject. In some embodiments, the TLR9-linked disease is selected from the group consisting of (i) cancer, in particular melanoma, breast cancer and lung cancer including NSCLC; (ii) an autoimmune and / or inflammatory disease, systemic lupus erythematosus (SLE), autoimmune thyroiditis, atherosclerosis, multiple sclerosis, inflammatory bowel disease and Crohn’s; (iii) a viral or bacterial infection, in particular an infection caused by HBV, HCV, HPV, EBV, HIV, Mycobacterium tuberculosis, Streptococcus penumoniae and Candida albicans; and (iv) a neurodegenerative disease.

[0122] In some embodiments, ADA2 modulates the activity of TLR9 inside lysosomes, preferably wherein modulating the activity of TLR9 comprises increasing the activity of TLR9 by at least about 10%. In some embodiments, ADA2 increases the activity of TLR9 inside lysosomes by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In some embodiments, the treatment targets TLR9 in macrophages. In some embodiments, the treatment increases the activity of TLR9 compared to an untreated control. Brief Description of the Drawings

[0123] Figure 1: ADA2 is a lysosomal adenosine deaminase acting on DNA

[0124] (A) EndoV assay operating principle. EndoV from Escherichia coli (EcEndoV) cuts dl-containing DNA. The DNA is labeled with y-32P before being treated or not with EcEndoV or EcEndoV and ADA2 and visualized by phosphorimaging. C. P.1, cleaved product 1 generated by EcEndoV in the presence of dis; C. P.2, cleaved product 2 occurs when additional (ADA2-generated) dis are introduced.

[0125] (B) Top: different amounts of hrADA2 were incubated with DNA substrates at 37°C for 45 min before EcEndoV treatment. H. I., heat inactivated at 95°C for 30 min. Bottom: ADA2 (200 fmol) was incubated with DNA substrates, samples were withdrawn at different time points and treated with EcEndoV, and reaction products were analyzed as above. The DNA substrates are shown under the panels.

[0126] (C) Competition assays were performed by adding competitors as indicated and 20 fmol ADA2 to the reaction mixture (dA = deoxyadenosine; Ado = Adenosine, dCof = deoxycoformycin).

[0127] Figure 2: ADA2 dA-to-dl activity depends on the catalytic domain.

[0128] (A) dA-to-dl activity of the catalytic dead ADA2 proteins mutant G358R (0.5-10 fmol) on various DNA substrates.

[0129] (B) dA-to-dl activity of increasing amount of ADA1. rA2 (5 fmoles) has been included as control Figure 3: ADA2 edits terminal dA(s) residues, EndoV assay.

[0130] Different DNA substrates were incubated with different amount of hrADA2 at 37°C for 45 min before EcEndoV treatment. The DNA substrates are detailed under the panels.

[0131] Figure 4: ADA2 edits terminal dA(s) residues, Dot blot assay.

[0132] Sequences of the Oligonucleotides (ODNs) used and tested to detect dis by dot blot using an antiinosine antibody.

[0133] Figure 5: ADA2 edits terminal dA(s) residues, Mass Spectrometry assay.

[0134] ADA2 was incubated with the indicated DNA substrates for 1 h at pH 5.5. Samples were then subjected to mass spectrometry for quantification of nucleobases. The dl levels are shown as percentage of nucleobases (top) and as dl / dA ratio (x100) (bottom).

[0135] Figure 6: ADA2 edits terminal dA(s) residues, Sequencing gel assay.

[0136] Recombinant human ADA2 (rA2) or endogenous porcine ADA2 (pA2) were incubated with ssllTI or dsAATA substrates, and after treatment with EcEndoV, samples were run on DNA sequencing gels. The length of the different fragments (in nt) is shown to the left. Multiple bands are seen in samples with pA2, likely generated by co-purifying DNases. The ADA2+EcEndoV cleavage product migrates slightly slower than 2 nt because EcEndoV produces 3'OH termini with less negative charge than 3'P termini made by DNases.

[0137] Figure 7: ADA2 edits dA(s) residues when they are present in nicks and gaps. Different DNA substrates were incubated with different amount of hrADA2 at 37°C for 45 min before EcEndoV treatment. The DNA substrates are detailed under the panels (left panel, oligo dsNick, middle panel, oligo dsGap).

[0138] Figure 8: ADA2 can edit A(s) residues in RNA.

[0139] ADA2-mediated A-to-l deamination on ssRNA and dsRNAwas tested using human EndoV, as reported in Vik E. S. et al.( Nat Commun 2271;2013), which cleaves RNA in the presence of inosine residues. The sequence of the used oligos is shown under the gels.

[0140] Figure 9: ADA2 is conserved in primates.

[0141] Multiple sequence alignment of mammalian adenosine deaminase 2 (ADA2 / CECR1) orthologs. Protein sequences are listed at the bottom, annotated with species and UniProt / GenBank identifiers. Conserved regions are highlighted with residue-specific colors, while dashes represent alignment gaps. This alignment reveals conserved domains and interspecies variability.

[0142] Figure 10: ADA2 dA-to-dl activity is conserved between different species.

[0143] (A) Percent of identity (number of identical amino acids * 100 / length of the alignment) between Sus scrofa and Homo sapiens ADA2 and comparison between S. scrofa and Homo sapiens ADA2 structure as depicted in the figure.

[0144] (B) ADA2-mediated dA-to-dl deamination on DNA from Sheep ADA2 tested using ecEndoV assay, recombinant human ADA2 is used as positive control. dA-to-dl activity from Sus Scrofa is shown in figure 6.

[0145] Figure 11: TLR9 is expressed on human macrophages and inhibits IP10 production.

[0146] (A) Expression of TLR9 from human monocyte-derived macrophages evaluated by Western blot. (B) CXCL10 production upon IFN stimulation with or wothouth TLR9 stimulation (CpG) evaluated by legendplex.

[0147] Figure 12: TLR9 stimulation inhibits interferon signature in human macrophages, evidence from GM-CSF differentiated macrophages.

[0148] Volcano plot showing differential gene expression results in GM-CSF differentiated human macrophages stimulated or not with CpG. Each point represents a gene, plotted by log2 fold change (x-axis) and -log10 adjusted p-value (y-axis). Genes with significant upregulation are colored red, and significantly downregulated genes are colored blue. The majority of the downregulated genes are interferon-stimulated genes (ISGs), such as MX1, IFIT1 / 2 / 3, RIGI, HERC5, and OAS3. Selected genes of interest are labeled.

[0149] Figure 13: TLR9 stimulation inhibits interferon signature in human macrophages, evidence from M-CSF differentiated macrophages.

[0150] Volcano plot illustrating differential gene expression in M-CSF differentiated human macrophages stimulated or not with CpG. Each dot represents a gene, plotted by shrunken log2 fold change (x-axis) and -log10 adjusted p-value (y-axis). Significantly upregulated genes are shown in red, and significantly downregulated genes in blue. Most of the downregulated genes, including MX1, OAS2, TRIM25, IFIT2, IFIH1, BISPR, OAS1, and IRF7, are interferon-stimulated genes (ISGs), indicating reduced interferon response in the condition analyzed.

[0151] Figure 14: Deoxyinosine can be recognized by human TLR9

[0152] (A) Sequences of the CpG analogs used for stimulation experiments. CpI and CpG indicate ODNs in which the dG residues required for DNA recognition by TLR9 are replaced with dl or dC, respectively. Ctrl represents unstimulated cells.

[0153] (B) TLR9 activation was measured using human HEK-Blue TLR9 reporter cells. Cells were incubated with 5 pM of the indicated ODNs, and the magnitude of SAEP induction was measured after 20 h as an indicator of TLR9 activation.

[0154] (C) Type I IFN production by human PBMCs upon incubation with CpG analogs as in (B).

[0155] Figure 15: Deoxyadenosine deamination of DNA molecules controls lysosomal DNA sensing, facilitating TLR9 activation.

[0156] (A) Sequences of different ODNs with dA residues replaced by dl as highlighted the figure.

[0157] (B) Activation of TLR9 measured using human HEK-Blue TLR9 reporter cells as in Figure 14(C) following stimulation with the ODNs (5 pM) described in (A).

[0158] Figure 16: ADA2 control lysosomal DNA sensing, facilitating TLR9 activation.

[0159] (A) TLR9 activation was measured using human HEK-Blue TLR9 reporter cells transduced with ADA2 or an empty vector. Cells were incubated with the indicated compounds, and the magnitude of SAEP induction was measured after 20 hours as an indicator of TLR9 activation.

[0160] (B) Concentration of IL-8 in the supernatant of HEK293T-hTLR-UNC93B1 reporter cells after overnight stimulation with the indicated compounds.

[0161] (C) TLR7 activation was measured using human HEK-UNC93B1 -human TLR7 reporter cells transduced with ADA2 or an empty vector. Cells were incubated with the indicated compounds, and the magnitude of IL8 production in the supernatants was measured as readout of the TLR7 activation.

[0162] Figure 17: TLR9 response in DADA2 patients.

[0163] (A-B) IFN-a (A) and TNF (B) were measured by ELISA in the supernatants of PBMCs from healthy donors and DADA2 patients after overnight stimulation with the indicated compounds. Three DADA2 patients with the indicated genotype and three age and gender-matched healthy donors have been compared.

[0164] (C) Gating strategy for sorting plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (eDCs).

[0165] (D) pDCs, eDCs, and monocytes were sorted and stimulated overnight with 5 pM CpG2216. The amount oftype-l IFN in the supernatants was measured using HEKtype- 1 IFN reporter cells. (E) Type-I IFN measured by HEK-type-l IFN reporter cells in the supernatants of sorted pDCs from healthy donors or DADA2 patients after overnight stimulation with CpG2216.

[0166] (F) Type I IFN production was measured using HEK-type-l IFN reporter cells by the indicated sorted human immune cell subtypes upon incubation with 69bp dsDNA using the endolysosomal transfection reagent PLA.

[0167] (G) Type-I IFN production by PBMCs from healthy donors or DADA2 patients upon lysosomal sensing of dsDNA.

[0168] Figure 18: Immunohistochemistry evidence on ADA2 expression and localization in lysosomes in monocytes and macrophages

[0169] (A) ADA2 mRNA expression is indicated by a z-score. Box limits indicate the first and third quartiles. Data from Monac G et al

[0030] (GSE107011).

[0170] (B) ADA2 protein expression (LFQ intensity) in different immune cell subtypes. Data from Rieckmann J. et al

[0031] ,

[0171] (C) CD14 and ADA2 staining of a human tonsil as shown in the figure. The panel shows germinal centers (black dotted line) with cytoplasmic ADA2 positivity in tingible body macrophages (TBM) (marked by asterisks in the upper and lower panel), whereas CD14 highlights the follicular dendritic cell meshwork (1 Ox original magnification).

[0172] (D) In higher magnifications, CD14+dendritic cells in the crypt epithelium (upper panel) and surface epithelium (lower panel) show few small ADA2 positive intracytoplasmic granules (highlighted by arrowheads, exemplary; 20x original magnification).

[0173] (E) Higher magnification (40x original magnification) showing TBM (black dotted line), containing ADA2 positive organelles resembling phagolysosomes. Two TBMs are further magnified in the lower panels.

[0174] Figure 19: Immunofluorescence evidence on ADA2 expression and localization in lysosomes in monocytes and macrophages.

[0175] (A) Immunofluorescence of human tonsils showing ADA2 positive cells (asterisks) in the germinal centers (dotted line) (20x original magnification).

[0176] (B) Higher (40X) magnification ofTBMs with LAMP1 positive lysosomes containing ADA2.

[0177] (C) Color channels as in the figure show single staining of ADA2 and LAMP.

[0178] (D) Expression of ADA2and LAMP1 in monocytes.

[0179] (E) Color channels (upper row) and surface channels (lower row) rendered with Imaris show the signal of ADA2 and LAMP1. The right panel shows the co-localization signal rendered by Imaris.

[0180] (F) Quantification of co-localization expressed as co-localizing voxels. Co-localization between nuclear (DAPI) and ADA2 has been measured as negative control (*** = p < 0.0001, Mann-Whitney test) Figure 20: Immunofluorescence evidence on ADA2 expression and localization in dendritic cells lysosomes. Expression of ADA2 and LAMP1 in human conventional dendritic cells (eDCs) (upper row) and plasmacytoid dendritic cells (lower row) evaluated by immunofluorescence. Channels were split to show a single signal for ADA2 and LAMP1, and the two channels merged.

[0181] Figure 21: ADA2 localization in lysosomes, evidence from glycan structures.

[0182] Heatmap comparing the glycan structures of pA2 and other lysosomal and extracellular proteins. The color scale represents the percentage of glycans linked to the indicated amino acid residues.

[0183] Figure 22: ADA2 interactions with DNA and structural preferences (EMSA assay)

[0184] (A) Diagrams showing structures and sequences of the ODN used (O Oligonucleotide, c complementary, PY pseudo-Y, Fl flap).

[0185] (B) ADA2 binding to DNA as measured by EMSA. hrADA2 was incubated with various [y-32P]-labeled DNA substrates at pH 5.5 before separating bound and free DNA on 7% native polyacrylamide gels.

[0186] (C) EMSA was performed also with [y-32P]-labeled DNA substrates of different lengths.

[0187] (D) EMSA was performed also with [y-32P]-labeled DNA substrates (HpTT42) at pH 5.5 and pH 7.5. (E) EMSA was performed also with [y-32P]-labeled DNA substrates (Pseudo-Y) at pH 5.5 and pH 7.5.

[0188] (F) Binding to DNA of hrADA2 mutant proteins tested by EMSA.

[0189] Figure 23: ADA2 interactions with DNA and structural preferences (FRET assay).

[0190] (A) Correlation between FRET signal (y-axis, fluorescence measured at 575 nm) and ADA activity (x-axis, EC50 in pg / ml) for the wild-type hrADA2 and ADA2 mutant proteins.

[0191] (B) Evaluation of the interaction between ADA2 and ADA1 and DNA as a measure of FRET at different pH or NaCI values.

[0192] (C) Evaluation of the ADA2 and ADA1 -induced FRET signals at different protein concentrations and pH5.5.

[0193] (D) FRET signal measurement, expressed as Fluorescence Unit at different concentrations for human recombinant ADA2 (ADA2 wt), seven mutated ADA2 proteins, ADA1, and a protein purified with the control His tag.

[0194] (E) ADA activity was also measured on the same proteins at different concentrations. These measurements were used to calculate the respective EC50 described in the methods. Detailed Description

[0195] 1. Definitions

[0196] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. The herein described and disclosed embodiments, preferred embodiments and very preferred embodiments should apply to all aspects and other embodiments, preferred embodiments and very preferred embodiments irrespective of whether it is specifically again referred to or its repetition is avoided for the sake of conciseness.

[0197] As used in the specification and the claims, the singular forms of “a” and “an” also include the corresponding plurals unless the context clearly dictates otherwise.

[0198] The term “at least one”, as used herein, means one or more, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or more of the referenced species. Similarly, “one or more”, as used herein, relates to at least one and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. In connection with a given species, the term does not relate to the total number of molecules, but rather to the type of species.

[0199] Numeric values specified without decimal places here refer to the full value specified with one decimal place, i.e., for example, 99 % means 99.0 %, unless otherwise defined.

[0200] The term “about” in the context of the present invention denotes an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10% and preferably ±5%.

[0201] It needs to be understood that the term “comprising” is not limiting. For the purposes of the present invention, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also meant to encompass a group which preferably consists of these embodiments only.

[0202] As used herein, the term “fusion protein” refers to a continuous chain of amino acids, wherein at least two domains have been fused to result in the continuous chain of amino acids. In fusion proteins, domains are often separated by a linker. For example, the fusion protein described herein is a continuous chain of amino acids comprising an ADA2 domain and a DNA binding domain, optionally an RNA-guided DNA endonuclease domain, and further optionally comprising linkers between the domains. As used herein, a fusion protein is not a naturally occurring protein.

[0203] The term “linker” as used herein refers to an amino acid sequence that joins the different domains of a fusion protein together. Direct fusion of functional domains without a linker might lead to undesirable outcomes such as misfolding of the fusion protein, low yield in protein production or impaired functionality. Linker sequences may be derived from natural proteins, for example from multi-domain proteins. Natural linkers have been reported to have average lengths of between about 4 amino acids to about 22 amino acids (Chen et al. 2014, Adv Drug Deliv Rev 65(10):1357-1369, hereby incorporated by reference). Linkers have different characteristics that influence their functionality, including rigidity (flexible or rigid) and cleavability.

[0204] As used herein, the term “functional variant” refers to an amino acid sequence that differs from the wild-type amino acid or underlying amino acid sequence, respectively, of an enzyme, in the present case in particular ADA2, but still retains the enzymatic activity of the underlying enzyme. Differences in the amino acid sequence can include at least one amino acid truncation, insertion, deletion, substitution, or other modification of the underlying sequence. The skilled person can identify the activity of a variant, i.e., whether the variant is still functional, in particular by a suitable enzymatic assay. In the present case, a suitable enzymatic assay for ADA2’s activity is, e.g., the conversion of dA to dl (see the examples, wherein also other assays to test ADA2’s function are outlined, which may also be used to determine whether or not a functional variant of ADA is present). A functional variant may have a different level of activity compared to the underlying enzyme from which it was derived. For example, a variant of ADA2 (in particular human ADA2) may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 80% or at least 90% of the dA to dl editing activity of the wildtype ADA2 (in particular human ADA2) from which it was derived, wherein an activity of at least 50% can be preferred. Similarly, the variant of ADA2 may also have the same or a higher dA to dl editing activity compared to the underlying ADA2, such as about 100%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% or at least 150% activity. Hence, a functional variant of ADA2 as described herein may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% or at least 150% of the dA to dl editing activity of the underlying (wild-type) ADA2 from which it was derived.

[0205] As used herein, the term “ADA2” or “Adenosine Deaminase 2” refers to member of a subfamily of the adenosine deaminase protein family. The protein is one of two adenosine deaminases found in humans. As can be derived from the examples, the protein is conserved in mammals. Mutations in the ADA2 gene lead to the disease “Deficiency of Adenosine deaminase 2” (“DADA2”), a monogenic disease associated with systemic inflammation and vasculopathy. The UniProt number of human ADA2 is Q9NZK5 (ADA2_Human).

[0206] As used herein, the term “nuclear localization sequence” or “NLS” refers to a specific sequence of amino acids comprised in a protein that directs and / or facilitates its transport into the nucleus. This sequence is recognized by nuclear transport receptors, allowing the protein to be actively transported through the nuclear pore complex into the nucleus, and appears to be preferred for proteins that function in processes such as transcription regulation, DNA replication, DNA editing and repair, to assist in their localization in order to carry out their action.

[0207] As used herein, the term “RNA-guided DNA endonuclease enzyme or domain, respectively”, also termed RNA-programmable DNA endonuclease enzyme in literature, refers to an endonuclease that can unwind DNA and find sites that are complementary to a guide RNA (gRNA) sequence and cleave the DNA by inducing a single or double strand break. An “endonuclease” is an enzyme that cleaves the phosphodiester bond within a polynucleotide chain. An endonuclease may cut both strands of a double-stranded nucleic acid molecule (typically referred to as “cleaving”) or cut only one strand of a double-stranded nucleic acid molecule (typically referred to as “nicking”). Thus, an endonuclease that cuts only one strand of a double-stranded nucleic acid molecule is also referred to as a “nickase”. The term “double strand break” or “DSB” as used herein refers to a cut in which both strands of the DNA double helix are severed.

[0208] The term “gene editing” as used herein refers to the targeted alteration of genetic material, in particular the genome, of a cell or organism by inserting, replacing, modifying or deleting a nucleic acid sequence. In the present context, the term in particular relates to a replacement of a dA by a dl (which is ultimately understood by the cellular machinery as being a replacement from a dA to a dG). Gene editing systems using RNA-guided DNA endonuclease enzymes include CRISPR systems, and any other suitable systems, such as, e.g., the obligate mobile element-guided activity (OMEGA) systems. Corresponding CRISPR genome editing systems are, e.g., described in Pacesa et al., Cell 187, February 29, 2024 (https: / / doi.org / 10.1016 / j.cell.2024.01.042), which is incorporated herein by reference - the systems referred to therein as “base editors” may in particular be the fusion proteins according to the present invention with the deaminase domain being ADA2 (see, e.g., Figures 2 to 4 therein).

[0209] The term “CRISPR genome editing” or “CRISPR editing” as used herein refers to a system derived from a natural mechanism that bacteria use to protect themselves from harmful viruses, similar to an immune system. In the event of an infection, an RNA-guided DNA endonuclease enzyme such as Cas9 breaks down the DNA of the invading viruses. The fragments are inserted in short, repeating fragments into a special section (CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats) in the bacteria's genome. If the virus attacks again, its DNA is compared with that stored in the CRISPR section. If the DNA matches the stored sequences, the Cas9 protein cuts the viral DNA, which is subsequently degraded, thus defending the cell against the virus.

[0210] Some CRISPR / Cas systems, like CRSPR / Cas9 and CRISPR / Cas12, can be reduced to a (single- or double-molecule) guide RNA and a Cas nuclease. The resulting gene editing system works not only in bacteria, but in all living cells, including those of plants and animals. It can be used to cut a DNA-strand in a sequence-specific manner at a predetermined site that can be targeted specifically via a guideRNA sequence that pairs with a targeted sequence using sequence homology and, during the subsequent repair, individual DNA building blocks can be cut out, replaced or inserted. A preferred Cas-endonuclease of the invention is Cas9, Cas3 or Cas12, or functional variants thereof including in particular nickase (mutants) thereof.

[0211] The OMEGA (Obligate Mobile Element Guided Activity) system” is a new class of programmable gene editing tool, similar to CRISPR, discovered in bacteria. It uses small RNA molecules (coRNAs) to guide DNA-cutting enzymes, like TnpB and Fanzor, to a specific DNA sequence for modification. Because OMEGA system proteins are much smaller than those used in CRISPR, such as Cas9, they offer a significant advantage for delivery into cells, making them a promising candidate for developing new gene editing therapies. For the purpose of targeted CRISPR gene editing, RNA-guided DNA endonuclease enzymes can associate with synthetic exogenous guide RNA (gRNA) sequences comprising CRISPR RNA (crRNA), which binds to the target-specific region, and trans-activation crRNA (tracrRNA), which activates the endonuclease activity. The OMEGA system is based on small endonucleases such as IscB, IsrB and tnpB, that associate with an small guide RNAs called coRNA, which guides the enzyme to a dsDNA target.

[0212] The term “Cas” as used herein when in the specific context of RNA-guided DNA endonucleases, specifically refers to Cas proteins that cut or cleave dsDNA, including Cas9, Cas3, and Cas12 (including Cas12a). It also includes Cas proteins that cut a single strand of dsDNA, so-called nickases. For specific proteins described herein (e.g., Cas9), the named protein includes any of the protein's naturally occurring forms, or engineered variants or homologs that maintain the activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form.

[0213] Thus, a “CRISPR associated protein 9”, “Cas9” or “Cas9 protein” as referred to herein includes any of the recombinant or naturally-occurring forms of the Cas9 endonuclease or engineered variants or homologs thereof that maintain Cas9 endonuclease or Cas9 nickase enzyme activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas9). In case of Cas9, the activity comprises inducing a blunt-ended double strand DNA break. In case of Cas9 nickase, the activity comprises inducing a single strand DNA break. In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Cas9 protein. In embodiments, the Cas9 protein is substantially identical to the protein identified by the UniProt reference number Q99ZW2 (SEQ ID NO: 41) or a variant or homolog having substantial identity thereto. Cas9 may refer for example to the Cas9 protein from Streptococcus pyogenes, Streptococcus thermophilus, Staphylococcus aureus, Neisseria meningitidis, or Campylobacter jejuni. In a preferred embodiment, Cas9 is from Streptococcus pyogenes.

[0214] Likewise, a “CRISPR associated protein 12”, “Cas12” or“Cas12 protein” as referred to herein includes any of the recombinant or naturally-occurring forms of the Cas12 endonuclease or variants or homologs thereof that maintain Cas12 endonuclease or Cas9 nickase enzyme activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas12). In case of Cas12, the activity comprises inducing a double strand DNA break with 5 nucleotide 5’ overhangs. In case of Cas12 nickase, the activity comprises inducing a single strand DNA break. In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Cas12 protein. In embodiments, the Cas12 protein is substantially identical to the protein identified by the UniProt reference number G2FJU2 or a variant or homolog having substantial identity thereto. Cas12 may refer for example to the Cas12 protein from Francisella novicida, Acidaminococcus spec, Lachnospiraceae spec, or Prevotella spec. Cas12 as used herein also includes variants of Cas12 such as Cas12a, Cas12h and Cas12i. Cas12a protein is substantially identical to the protein identified by the UniProt reference number U2UMQ6 or a variant or homolog having substantial identity thereto.

[0215] Likewise, a “CRISPR associated protein 3”, “Cas3” or “Cas3 protein” as referred to herein includes any of the recombinant or naturally-occurring forms of the Cas3 endonuclease or variants or homologs thereof that maintain Cas3 endonuclease or Cas3 nickase enzyme activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas3). In case of Cas3, the activity comprises inducing a single strand DNA cleavage conveyed by the nuclease domain, followed by DNA unwinding and degradation involving helicase and nuclease domains. In case of Cas3 nickase, the activity comprises inducing a single strand DNA cleavage. In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring Cas3 protein. In embodiments, the Cas3 protein is substantially identical to the protein identified by the UniProt reference number 027158 or a variant or homolog having substantial identity thereto. Cas12 may refer for example to the Cas3 protein from Thermobifida fusca, Salmonella spec., or Pseudomonas aeruginosa.

[0216] A “nickase” as used herein in the context of Cas enzymes is a mutant version of Cas wherein one of two catalytic centers is disabled by a mutation. Cas9 possesses two nuclease domains: His-Asn-His (HNH) domain responsible for cis-cleavage of the target strand (TS) of dsDNA and RuvC domain responsible for cis-cleavage of the non-target strand (NTS) of dsDNA. By mutating the catalytic site of either the HNH domain or the RuvC domain, a Cas9 nickase can be generated that introduces singlestrand nicks in dsDNA without causing double-strand breaks. Hence, Cas9 nickases carry either a D10 mutation, such as D10A, which inactivates the RuvC domain, or an H840 mutation, such as H840A which inactivates the HNH domain. The D10A nickase (SEQ ID NO: 42) cleaves only the target strand. Conversely, the H840A nickase (SEQ ID NO: 43) cleaves only the non-target strand.

[0217] Cas3 comprises one nuclease and one helicase domain, hence, a Cas3 nickase can be produced by inactivating the helicase through mutation of at least one of several essential residues (see for example Hao et al. Open Biol. 2022 Jan;12(1):210241. doi: 10.1098 / rsob.210241. Epub 2022 Jan 12. PMID: 35016549; PMCID: PMC8753164, hereby incorporated by reference). The Cas12 family includes naturally occurring nickase enzymes, such as Cas12h, and engineered nickase mutants such as Cas12a RuvC mutant R1226A (see for example Xiang et al. 2025, Cell Reports 44(5):115718, doi 10.1016 / j.celrep.2025.115718, hereby incorporated by reference).

[0218] The terms “guide RNA” or “gRNA” or single guide RNA “sgRNA” refer to a short RNA sequence that functions as a guide for endonucleases, for example as part of the CRISPR / Cas system. Other RNA-guided endonucleases are, for example, Argonaute, which is part of the RNA interference (RNAi) system; and the endonucleases of the OMEGA system. All these systems use an RNA that is complementary to a target nucleic acid sequence and upon binding, the guide RNA transports an endonuclease enzyme to the target nucleic acid sequence, resulting in the enzyme cutting the target. In the case of RNAi, the target is usually an mRNA that is degraded after being cut. In the case of the CRISPR or OMEGA gene editing system, the cut is usually repaired by either NHEJ, HDR or HITI, which can result in a targeted knock-out or knock-in in the presence of a repair template.

[0219] As used herein, the term “protospacer adjacent motif” or “PAM” refers to a DNA sequence adjacent to a target sequence (e.g., a TTR target sequence) to which a complex comprising an RNA guide (e.g., a TTR-targeting RNA guide) and a Cas polypeptide binds. In a double-stranded DNA molecule, the strand containing the PAM motif is called the “PAM-strand” and the complementary strand is called the “non-PAM strand.” The RNA guide binds to a site in the non-PAM strand that is complementary to a target sequence disclosed herein.

[0220] As used herein, the term “target sequence” or “target DNA” refers to a DNA fragment adjacent to a PAM motif (on the PAM strand). The complementary region of the target sequence is on the non-PAM strand. A target sequence may be immediately adjacent to the PAM motif. Alternatively, the target sequence and the PAM may be separated by a small sequence segment (e.g., up to 5 nucleotides, for example, up to 4, 3, 2, or 1 nucleotide). A target sequence may be located at the 3’ end of the PAM motif or at the 5’ end of the PAM motif, depending upon the CRISPR nuclease that recognizes the PAM motif, which is known in the art.

[0221] A “ribonucleoprotein complex,” or “RNP” as provided herein refers to a complex or particle including a nucleoprotein and a ribonucleic acid. A “nucleoprotein” as provided herein in particular refers to a protein capable of binding a nucleic acid (e.g., RNA, DNA). Where the nucleoprotein binds a ribonucleic acid it is referred to as “ribonucleoprotein.” The interaction between the ribonucleoprotein and the ribonucleic acid may be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In embodiments, the ribonucleoprotein includes an RNA-binding motif non-covalently bound to the ribonucleic acid. For example, positively charged aromatic amino acid residues (e.g., lysine residues) in the RNA-binding motif may form electrostatic interactions with the negative nucleic acid phosphate backbones of the RNA, thereby forming a ribonucleoprotein complex. Nonlimiting examples of ribonucleoproteins include ribosomes, telomerase, RNAseP, hnRNP, CRISPR associated proteins 9, 3 and 12, and small nuclear RNPs (snRNPs). The ribonucleoprotein may be an enzyme. In embodiments, the ribonucleoprotein is an endonuclease. Thus, in embodiments, the ribonucleoprotein complex includes an RNA-guided DNA endonuclease and a ribonucleic acid.

[0222] As used herein, the terms “upstream” and “downstream” refer to relative positions within a single nucleic acid (e.g., DNA) sequence in a nucleic acid molecule. “Upstream” and “downstream” relate to the 5’ to 3’ direction, respectively, in which RNA transcription occurs. A first sequence is upstream of a second sequence when the 3’ end of the first sequence occurs before the 5’ end of the second sequence. A first sequence is downstream of a second sequence when the 5’ end of the first sequence occurs after the 3’ end of the second sequence.

[0223] The term "polynucleotide" or “nucleic acid sequence” or “nucleic acid molecule” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A nucleic acid sequence is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the nucleic acid sequence is RNA). Thus, the term polynucleotide sequence or nucleic acid sequence is the alphabetical representation of a polynucleotide molecule. A polynucleotide may comprise modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.

[0224] The term “DNA” as used herein is the usual abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotide monomers. These nucleotides are usually deoxy-adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate and deoxy-cytidine-monophosphate monomers or analogs thereof which are - by themselves - composed of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety, and polymerize by a characteristic backbone structure. The backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide, i.e. deoxyribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar / phosphate-backbone, is called the DNA-sequence. DNA may be single stranded or double stranded. In the double stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g. by A / T-base-pairing and G / C-base-pairing.

[0225] The term “RNA” as used herein relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a p-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs are considered analogs of naturally-occurring RNA. In some embodiments, the RNA may have modified ribonucleotides. Examples of modified ribonucleotides include, without limitation, 5-methylcytidine, pseudouridine and / or 1-methyl-pseudouridine. In some embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine. In some embodiments, the RNA according to the present disclosure comprises a 5'-cap. In some embodiments, the RNA of the present disclosure does not have uncapped 5'-triphosphates. In some embodiments, the RNA may be modified by a 5'-cap analog. The term "5'-cap" refers to a structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via a 5' to 5' triphosphate linkage. In some embodiments, this guanosine is methylated at the 7-position. Providing an RNA with a 5'-cap or 5'-cap analog may be achieved by in vitro transcription, in which the 5'-cap is co-transcriptionally expressed into the RNA strand, or may be attached to RNA post-transcriptionally using capping enzymes. The term “mRNA” as used herein relates to an RNA transcript which encodes a peptide or protein. As established in the field, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide coding region and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In some embodiments, the mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxy ribonucleotides.

[0226] The term “encodes" or “encoding” as used herein refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, mRNA, or miRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. As used herein, “isolated” refers to a nucleic acid molecule or a nucleic acid sequence that has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells, chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins. Isolated proteins or nucleic acids, or cells containing such, in some examples are at least 50% pure, such as at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% pure.

[0227] The term “isolated” may also refer to a cell or sample cells. An isolated cell or sample cells are a single cell type that is substantially free of many of the components which normally accompany the cells when they are in their native state or when they are initially removed from their native state. In certain embodiments, an isolated cell sample retains those components from its natural state that are required to maintain the cell in a desired state. In some embodiments, an isolated (e.g. purified, separated) cell or isolated cells, are cells that are substantially the only cell type in a sample. A purified cell sample may contain at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of one type of cell. An isolated cell sample may be obtained through the use of a cell marker or a combination of cell markers, either of which is unique to one cell type in an unpurified cell sample. In some embodiments, the cells are isolated through the use of a cell sorter. In some embodiments, antibodies against cell proteins are used to isolate cells.

[0228] A "plasmid" or "expression vector" as used herein is an expression construct used for cloning and gene expression. A plasmid is an extra chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently from the chromosomal DNA. A plasmid can be used as a vector, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.

[0229] The terms "polypeptide", “amino acid sequence” and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component. The term “amino acid sequence” as used herein likewise refers to a continuous string of amino acids. This term can be used interchangeably with the terms “polypeptide” or “protein”. The term “amino acid sequence” may refer to a full-length protein, to a domain of a protein, to a functional region of a protein, or to a fragment or portion of a protein, wherein the fragment may be of any length. A recombinant polynucleotide or amino acid sequence as used interchangeably herein can refer to a nucleotide or an amino acid sequence that is the product of various combinations of cloning, restriction and / or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide or amino acid sequence found in nature.

[0230] A “fragment” or “portion” of an amino acid sequence can be understood to mean an amino acid sequence of reduced length relative to a reference amino acid sequence and comprising, consisting essentially of, or consisting of an amino acid sequence of contiguous amino acids identical or almost identical to the reference amino acid sequence. Such an amino acid fragment or portion according to the disclosure can be, where appropriate, included in a larger amino acid sequence of which it is a constituent. In some embodiments, the amino acid sequence fragment may be reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more amino acids in reference to the full-length amino acid sequence. In some embodiments, the amino acid sequence fragment may be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the reference amino acid sequence.

[0231] A “fragment” or “portion” of a nucleotide sequence can be understood to mean a nucleic acid sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, or consisting of a nucleic acid or nucleotide sequence of contiguous nucleotides identical or almost identical to the reference nucleic acid sequence. In some embodiments, the nucleic acid fragment may be reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more nucleotides in reference to the full-length nucleic acid sequence. Such a nucleic acid sequence fragment or portion according to the disclosure can be, where appropriate, included in a larger polynucleotide of which it is a constituent. In an aspect, a fragment or portion of a nucleotide sequence or nucleic acid sequence can comprise the sequence encoding an exon having one or more mutations. In some embodiments, the nucleic acid fragment may be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the reference nucleic acid sequence.

[0232] “Sequence identity” or “percentage identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned. For example, sequence similarity or identity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences can have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more. Such sequences are also referred to as “variants” herein, e.g., other variants of a missing, deficient, and / or mutant protein or enzyme.

[0233] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0234] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0235] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid ora small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0236] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M). The words “complementary” or “complementarity” refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.

[0237] The term “antibody” as used herein refers to a glycoprotein belonging to the immunoglobulin superfamily. The term "full-length antibody" may refer to an immunoglobulin molecule that binds to a target molecule and contains four peptide chains: two heavy chains and two light chains which are connected to each other through disulfide bonds. Antibodies may comprise several regions or domains, respectively. An antibody typically recognises an antigen via the fragment antigen-binding (Fab) variable region. The fragment crystallizable region (Fc region) is the tail region of an antibody that may allow antibodies to activate the immune system. The hinge region is a stretch of heavy chains linking the Fab and Fc regions. The heavy chain and light chain may each comprise a variable domain and one or more constant domains. For example, in IgG antibodies, a heavy chain comprises a variable domain (VH) and three constant domains (CH1, CH2, and CH3) and a light chain comprises a variable domain (VL) and one constant domain (CL). Examples antibodies include a human antibody, a mouse antibody, a camelid antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a monoclonal antibody, and a polyclonal antibody.

[0238] The term “antigen-binding fragment” as used herein refers to a fragment of an antibody, or a genetically engineered product of one of more fragments of an antibody, which fragment is involved in binding with the target molecule. Examples of antigen-binding fragments include a Fragment antigenbinding region (Fab), a Fab', a Fab'-SH, a fragment antibody (F(ab’)2), a variable region (Fv), a single chain antibody, such as a single chain variable fragment (scFv), a single-domain antibody (sdAb), a nanobody (which may also be referred to as VHH). The term “antigen-binding fragment” also refers to a region of an antibody that binds to antigens and is composed of one constant and one variable region of each of the heavy and the light chain. The term “fragment antibody” or “F(ab’)2” refers to a region of an antibody that remains following digestion of the Fc region while leaving intact some of the hinge region. The term “Fab”’ refers to a fragment formed by the reduction of a F(ab')2 fragment. The term “Fab’-SH” refers to a Fab’ fragment with a free sulfhydryl group. The term “Single chain variable fragment” or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region which are connected to each other. The term “nanobody” or “VHH” refers to a single-domain antibody, which is an antigen-binding fragment typically derived from heavy-chain-only antibodies (found in particular in camelids such as llamas and alpacas).

[0239] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. The terms “transfection”, “transduction”, “transfecting” or “transducing” can be used interchangeably and are defined as a process of introducing a nucleic acid molecule and / or a protein to a cell. Nucleic acids may be introduced to a cell using non-viral or viral-based methods. The nucleic acid molecule can be a sequence encoding complete proteins or functional portions thereof. Typically, a nucleic acid vector, comprising the elements necessary for protein expression (e.g., a promoter, transcription start site, etc.). Non-viral methods of transfection include any appropriate method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. For viral-based methods, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some aspects, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art. The terms “transfection” or “transduction” also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0240] The term “particle” as used herein refers to a delivery vehicle to deliver the fusion protein according to the first aspect, the nucleic acid according to the second aspect, the vector according to the third aspect or the RNP complex according to the fourth aspect into a cell, wherein a “particle” may e.g. be a vector or an LNP as defined in the following. Many suitable particles are known to the skilled person from the common general knowledge, which are designed for delivering the different types of cargo as mentioned above, and the skilled person is well aware of selecting a suitable particle depending on the cargo. The term "vector" as used herein refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. A "lentivirus" as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells. They can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo. The term “lipid nanoparticle” or “LNP” as used herein refers to a particle comprising at least one lipid, preferably a cationic lipid, wherein the lipid forms a complex with and / or encapsulates the nucleic acid comprised therein.

[0241] The term “pharmaceutically suitable excipient” or “pharmaceutically acceptable excipient” as used herein includes a carrier, a diluent, a solvent, a dispersion, a coating, a buffer, a preservative, a stabilizer, and the like, which are compatible with pharmaceutical administration. A pharmaceutically acceptable excipient (e.g., a carrier or a diluent) for therapeutic use is well known in the pharmaceutical art, and described, e.g., in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit.

[0242] 1985). Pharmaceutical excipients can be selected with regard to the intended route of administration and standard pharmaceutical practice. A “pharmaceutically acceptable excipient” as used herein is not pharmaceutically active.

[0243] The term “treatment” as used herein refers to clinical intervention in order to cure or ameliorate a disease, prevent recurrence of a disease, alleviate symptoms of a disease, diminish any direct or indirect pathological consequences of a disease, achieve a stabilized (i.e., not worsening) state of disease, prevent metastasis, decrease the rate of disease progression, and / or prolong survival as compared to expected survival if not receiving treatment.

[0244] The term “subject” as used herein in particular relates to a human of either gender. The subject may be of any age. In some embodiments, the subject is female. In another embodiment, the subject is male. The term “DADA2” as used herein refers to the disease “deficiency of adenosine deaminase 2”. This disease is a monogenic disease associated with systemic inflammation, vasculopathy and bone marrow alterations that affects a wide variety of organs in different patients. Manifestations of the disease include but are not limited to recurrent fever, livedoid rash (reticularis or racemosa), various cytopenias, stroke, immunodeficiency, and bone marrow failure. Symptoms often onset during early childhood, but some cases have been discovered as late as 65 years old. DADA2 is caused by mutations in the ADA2 gene, and is inherited in an autosomal recessive manner.

[0245] The term “TLR9” as used herein refers to Toll-like receptor 9 is a protein that in humans is encoded by the TLR9 gene. TLR9 has also been designated as CD289 (cluster of differentiation 289). It is a member of the toll-like receptor (TLR) family. TLR9 is a receptor expressed in immune system cells including dendritic cells, macrophages, natural killer cells, and other antigen presenting cells. TLR9 is expressed on endosomes internalized from the plasma membrane, binds DNA (preferentially DNA containing unmethylated CpGs of bacterial or viral origin), and triggers signaling cascades that lead to a pro-inflammatory cytokine response. Cancer, infection, autoimmune diseases and tissue damage can all modulate TLR9 expression and activation and vice versa. The UniProt entry of human TLR9 is Q9NR96.

[0246] “Wild-type”, “control” or “reference” gene expression, mRNA or protein levels are determined by a control sample, cell or organisms, or by averaging the expression levels from multiple control samples, cells or organisms. In the context of the present invention, the term “wild-type” or "control" refers to a cell or organism that is healthy or a sample from a subject that is healthy or to a cell or organism with a specific disease that is different from the disease to be treated. 2. Sequences

[0247] SEQ ID NO: 1: ADA2 without leader sequence IDETRAHLLLKEKMMRLGGRLVLNTKEELANERLMTLKIAEMKEAMRTLIFPPSMHFFQAKHLIERSQV FNILRMMPKGAALHLHDIGIVTMDWLVRNVTYRPHCHICFTPRGIMQFRFAHPTPRPSEKCSKWILLED YRKRVQNVTEFDDSLLRNFTLVTQHPEVIYTNQNVVWSKFETIFFTISGLIHYAPVFRDYVFRSMQEFY EDNVLYMEIRARLLPVYELSGEHHDEEWSVKTYQEVAQKFVETHPEFIGIKIIYSDHRSKDVAVIAESIR MAMGLRIKFPTVVAGFDLVGHEDTGHSLHDYKEALMIPAKDGVKLPYFFHAGETDWQGTSIDRNILDA LMLNTTRIGHGFALSKHPAVRTYSWKKDIPIEVCPISNQVLKLVSDLRNHPVATLMATGHPMVISSDDP AMFGAKGLSYDFYEVFMGIGGMKADLRTLKQLAMNSIKYSTLLESEKNTFMEIWKKRWDKFIADVATK SEQ ID NO: 2: ADA2 MLVDGPSERPALCFLLLAVAMSFFGSALSIDETRAHLLLKEKMMRLGGRLVLNTKEELANERLMTLKIA EMKEAMRTLIFPPSMHFFQAKHLIERSQVFNILRMMPKGAALHLHDIGIVTMDWLVRNVTYRPHCHICF TPRGIMQFRFAHPTPRPSEKCSKWILLEDYRKRVQNVTEFDDSLLRNFTLVTQHPEVIYTNQNVVWSK FETIFFTISGLIHYAPVFRDYVFRSMQEFYEDNVLYMEIRARLLPVYELSGEHHDEEWSVKTYQEVAQK FVETHPEFIGIKIIYSDHRSKDVAVIAESIRMAMGLRIKFPTVVAGFDLVGHEDTGHSLHDYKEALMIPAK DGVKLPYFFHAGETDWQGTSIDRNILDALMLNTTRIGHGFALSKHPAVRTYSWKKDIPIEVCPISNQVL KLVSDLRNHPVATLMATGHPMVISSDDPAMFGAKGLSYDFYEVFMGIGGMKADLRTLKQLAMNSIKY STLLESEKNTFMEIWKKRWDKFIADVATK SEQ ID NO: 3: (GGGGS)n linker, wherein n is 1-20 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 4: (GGS)nlinker, wherein n is 1-20 GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGS SEQ ID NO: 5: (SGSG)n linker, wherein n is 1-20 SGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSG SGSGSGSGSGSGSGSGSG SEQ ID NO: 6: Gn, wherein n is 4-20

[0248] GGGGGGGGGGGGGGGGGGGG SEQ ID NO: 7 Linker

[0249] GSAGSAAGSGEF SEQ ID NO: 8 Linker

[0250] KESGSVSSEQLAQFRSLD SEQ ID NO: 9 Linker

[0251] EGKSSGSGSESKST SEQ ID NO: 10 Linker

[0252] SGSETPGTSESATPES SEQ ID NO: 11 Linker

[0253] SGGSPKKKRKVGSSGS SEQ ID NO: 12 NLS

[0254] PKKKRKV SEQ ID NO: 13 NLS

[0255] KRPAATKKAGQAKKKK SEQ ID NO: 14 NLS

[0256] GKRKLITSEEERSPAKRGRKS SEQ ID NO: 15 NLS KGKKGRTQKEKKAARARSKGKN SEQ ID NO: 16 NLS

[0257] AVKRPAATKKAGQAKKKKLD SEQ ID NO: 17 NLS MSRRRKANPTKLSENAKKLAKEVEN SEQ ID NO: 18 NLS

[0258] PAAKRVKLD SEQ ID NO: 19 NLS

[0259] KLKIKRPVK SEQ ID NO: 20 NLS RKRCAAGVGGGPAGCPAPGSTPLKKPRR SEQ ID NO: 21 NLS RKPVTAQERQREREEKRRRRQERAKEREKRRQERER SEQ ID NO: 22 NLS FGNYNNQSSNFGPMKGGNFGGRSSGPY SEQ ID NO: 23 NLS TLLLRETMNNLGVSDHAVLSRKTPQPY SEQ ID NO: 24 NLS

[0260] PGKMDKGEHRQERRDRPY SEQ ID NO: 25 NLS GRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFG SEQ ID NO: 26 Linker

[0261] EAAAK SEQ ID NO: 27 Linker

[0262] AEAAAK SEQ ID NO: 28 Linker AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA SEQ ID NO: 29 Linker

[0263] AEAAAKEAAAKA SEQ ID NO: 30 XPnlinker, wherein X is any amino acid wherein n is 2-20 XPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXPXP SEQ ID NO: 31: APn linker, wherein n is 2-20 APAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP SEQ ID NO: 32: KPn linker, wherein n is 2-20 KPKPKPKPKPKPKPKPKPKPKPKPKPKPKPKPKPKPKPKP SEQ ID NO: 33 QPnlinker, wherein n is 2-20 QPQPQPQPQPQPQPQPQPQPQPQPQPQPQPQPQPQPQPQP SEQ ID NO: 34 Linker

[0264] APAPAPAPAPAPAP SEQ ID NO: 35 Linker

[0265] PAPAP SEQ ID NO: 36 EAAAKnGGGGSmlinker wherein n is 1-2 and m is 2-3 EAAAKEAAAKGGGGSGGGGSGGGGS SEQ ID NO: 37 Linker

[0266] EAAAKEAAAKGGGGSGGGGSGGGGS SEQ ID NO: 38 Linker

[0267] EAAAKGGGGSGGGGSGGGGS SEQ ID NO: 39 Linker

[0268] EAAAKGGGGSGGGGS SEQ ID NO: 40 Linker

[0269] EAAAKEAAAKGGGGSGGGGS SEQ ID NO: 41: SpCas9 wildtype MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNL LAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQL IHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SEQ ID NO: 42: SpCas9 nickase mutant D10A MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNL LAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQL IHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SEQ ID NO: 43: SpCas9 nickase mutant H840A MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNL LAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQL IHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSN IMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPID FLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD SEQ ID NO: 44: IGF-II MGIPMGKSMLVLLTFLAFASCCIAAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVE ECCFRSCDLALLETYCATPAKSERDVSTPPTVLPDNFPRYPVGKFFQYDTWKQSTQRLRRGLPALLR ARRGHVLAKELEAFREAKRHRPLIALPTQDPAHGGAPPEMASNRK SEQ ID NO: 45: GILT-taq

[0270] MGIPMGKSMLVLLTFLAFASCCIAAGAPT

[0271] The SEQ ID NOs of the sequences used in the example section are directly indicated in the respective tables (see the next section). 3. Examples

[0272] Materials and Methods

[0273] Cell Culture

[0274] Human TLR9 Reporter HEK293 Cells (NF-KB) and HEK-Blue™ IFN- a / p cells were maintained in DMEM, 4.5 g / l glucose, 10% (v / v) fetal bovine serum (FBS), 100 U / ml penicillin, 100 pg / ml streptomycin, 2 mM L-glutamine, 10 pg / ml of blasticidin and 100 pg / ml of Zeocin®.

[0275] 293XL / hTLR7-HA cell line (Invivogen) and 293XL / hTLR9-HA cell line (Invivogen) both expressing human UNC93B1-mCitrine WT 26 were maintained in DMEM, 4.5 g / l glucose, 10% (v / v) FBS, 100 U / ml penicillin, 100 pg / ml streptomycin and 2 mM L-glutamine.

[0276] Immunohistochemistry of human tonsils

[0277] For histopathological sample analysis, 2 pm sections were taken from buffered formalin fixed paraffin embedded biopsies of human palatine tonsils after positive ethical vote by local authorities (251 / 13_140389, Ethikkommission der Albert-Ludwigs-Universitat Freiburg, Freiburg, Germany) and were put on coated slides (SuperfrostPlus, Langenbrinck, Emmendingen, Germany, cat. # 03-0060). Deparaffinization was done in xylene, followed by 100% ethanol, with a concentration decreasing in aqueous solution until 50% was reached, followed by distilled water. Blocking of endogenous biotin (Biotin blocking reagent, Agilent Dako, Glostrup, Denmark, cat. # X0590) was performed before adding any primary antibodies. Staining was carried out with a primary antibody against CD14 (rabbit monoclonal antibody, clone EP128, BioGenex, Fremont, USA; cat. # AN814GP; dilution 1:100) and no antigen retrieval was necessary. Secondary antibodies were in a ready-to-use (RTU) formulation and incubated afterwards (Dako REAL Detection System, Agilent Dako, cat. # K5005; containing a mixture of biotinylated goat-anti mouse and goat-anti rabbit antibodies) and visualized with an alkaline phosphatase based red chromogen reaction using the same kit, according to the manufacturer's guidelines. After denaturation of the first antibodies as described by von Schoenfeld et al (Int J Mol Sci 23; 2021; 10.3390 / ijms23010223), heat-mediated epitope retrieval was done in a steamer for 30 min in Tris-buffered saline at pH 6.1, followed by staining against ADA2 (anti-CECR1 rabbit polyclonal antibody, Sigma-Aldrich, cat. # HPA007888, dilution 1:80). The secondary antibodies were again taken from the kit, visualized by an alkaline phosphatase based blue chromogen reaction (StayBlue / AP, Abeam, Cambridge, UK, cat. # ab176915). Photos were taken with an Olympus BX 51 microscope (Olympus Hamburg, Germany) with the AxioCam MRc microscope camera (Carl Zeiss, Oberkochen, Germany).

[0278] Immunofluorescence of human tonsils

[0279] For immunofluorescence, 2 pm sections were taken from buffered formalin fixed paraffin embedded biopsies of human palatine tonsils after positive ethical vote by local authorities (251 / 13_140389, Ethikkommission der Albert-Ludwigs-Universitat Freiburg, Freiburg, Germany) and put on coated slides (SuperfrostPlus, Langenbrinck, Emmendingen, Germany, cat. # 03-0060). Subsequent staining was done following the protocol described by von Schoenfeld et al. (Int J Mol Sci 23; 2021; 10.3390 / ijms23010223). The first staining step was done against LAMP1 (mouse monoclonal antibody, clone eBioH4A3, Thermo Fisher Scientific, cat. # 14-1079-80, dilution 1:100) after heat mediated epitope retrieval in a steamer for 30 min in Tris buffered saline at pH 6.1 and visualized in the 488 channel (RTU secondary antibodies from the kit as mentioned above, coupled to Streptavidin AlexaFluor 488 Conjugate, Thermo Fisher Scientific; cat. # S32354, dilution 1:200). The second staining step was done against ADA2 (anti-CECR1 rabbit polyclonal antibody, Sigma-Aldrich, cat. # HPA007888, dilution 1:80) after high-pressure cooking epitope retrieval for two min in citric buffer pH 6 and visualized in the 555 channel (RTU secondary antibodies from the kit as mentioned above, coupled to Streptavidin AlexaFluor 555 Conjugate, Thermo Fisher Scientific; cat. # S32355 dilution 1:200). Nuclei were counterstained with DAPI (Thermo Fisher Scientific; cat. # 62248, dil: 1:1000). Photos were taken with a fluorescence microscope (Axioplan 2, Zeiss) with the AxioCam MRm microscope camera.

[0280] Monocytes isolation from peripheral blood mononuclear cells

[0281] Peripheral blood mononuclear cells (PBMC) were prepared by centrifugation on a Pancoll gradient (PAN Biotech, Pancoll human cat. # P04-60500). Blood CD14+monocytes were isolated from DADA2 patients (ethical vote 322 / 15, Ethikkommission der Albert-Ludwigs-Universitat Freiburg, Freiburg, Germany) or healthy donors’ PBMC by positive selection using magnetic beads (Miltenyi, cat. # 130-050-201) according to the manufacturer's protocol. Monocytes purity was 95%-98% as measured by flow cytometry.

[0282] Immunofluorescence of primary monocytes from peripheral blood of healthy donors and DADA2 patients

[0283] Monocytes were isolated from the blood of healthy donors, as described above. 150.000 cells were seeded in RPMI per well in a chambered coverslip (IBIDI: cat. # 80826) and let settled for 2 hours at 37°C in the incubator. After incubation, RPMI was removed, and cells were washed three times with PBS before fixation with precooled (-20°C) 1:1 acetone / methanol fixative for 30 min at -20°C. Afterward, the fixative was aspirated, and the sample was washed three times in PBS for 5 min. The cells were subsequently permeabilized in PBS with 10% normal goat serum (NGS) (Abeam, cat. # ab7481) and 0,05% Tween (PanReac AppliChem, cat. # A4974) for 10 min. After permeabilization, the cells were blocked in blocking solution (PBS with 10% NGS + 0,05% Tween 20) for > 2 hours at RT. Hereafter, cells were immunostained with a rabbit anti-human CECR1 (Sigma cat. # HPA007888; 1:100 in blocking solution) and anti-LAMP1-AF488 (Cell Signaling, cat. # 58996, diluted 1:200, in blocking solution) overnight at 4°C. After incubation, cells were washed three times in PBS before being incubated with goat anti-rabbit AF546 (Thermo Fisher Scientific, cat. # A-11010, 1:500) and DAPI (Thermo Fisher Scientific; cat. # 62248, dil: 1: 1000) in DPBS with 10% NGS and 0,05% Tween20 for 1 hour at RT. The slides were mounted with Dako mounting medium (Agilent Technologies cat. # S302380-2). The images were taken with the LSM880 (Zeiss) and analyzed with ZEN (Zeiss) and lmageJ / FiJi2. The software Imaris v9.1 (Oxford Instruments) was used for the 3D reconstruction, profile plots, and co-localization. For the quantification of LAMP1 and ADA2 in peripheral monocytes, the Coloc tool from IMARIS was used. The tool allows defining thresholds with FMO or 2nd Ab-only control staining for each experiment. Defining that threshold, IMARIS renders the region of interest for single- or co-localizing staining. The newly created colocalizing channel can then be quantified, i.e., the number of colocalizing voxels. The same steps were performed with ADA2 and DAPI as internal negative control.

[0284] Plasmacytoid and conventional dendritic cells isolation from peripheral blood mononuclear cells for immunofluorescence

[0285] Peripheral blood mononuclear cells (PBMC) were prepared by centrifugation on a Pancoll gradient (PAN Biotech, Pancoll human cat. # P04-60500). Plasmacytoid dendritic cells (pDCs) were sorted as lineage (CD3-FITC, CD14-FITC, CD16-FITC, CD19-FITC)’, HLA-DR-PE-Cy7+, CD11c-BV421dim, CD4-ACP-Cy7hi9h, conventional DC (eDCs) as lineage (CD3-FITC, CD14-FITC, CD16-FITC, CD19-FITC)-, HLA-DR-PE-Cy7+, CD11 c-BV421+, CD4-ACP-Cy7dim.

[0286] Immunofluorescence of dendritic cells from peripheral blood of healthy donors

[0287] Dendritic cell subtypes were isolated from the blood of healthy donors, as described above. During the sorting time, chambered coverslip (IBIDI: cat. # 80826) were coated with 100 pl of Poly-D-Lysine 0.1mg / ml 20’ RT and washed five times with PBS. Subsequently, 25-30.000 cells were seeded in RPMI per well and let settle for two hrs at 37°C in the incubator. After incubation, RPMI was removed, and cells were washed three times with PBS before fixation with precooled (-20°C) 1:1 acetone / methanol fixative for 30 min at -20°C. Afterward, the fixative was aspirated, and the sample was washed three times in PBS for 5 min. The cells were subsequently permeabilized in PBS with 10% normal goat serum (NGS) (Abeam, cat. # ab7481) and 0.05% Tween (PanReac AppliChem, cat. # A4974) for 10 min. After permeabilization, the cells were blocked in a blocking solution (PBS with 10% NGS + 0.05% Tween 20) for > 2 hours at RT. Hereafter, cells were immunostained with a rabbit anti-human CECR1 (Sigma cat. # HPA007888; 1:100 in blocking solution) and anti-LAMP1-AF488 (Cell Signaling, cat. # 58996, diluted 1:200, in blocking solution) overnight at 4°C. After incubation, cells were washed three times in PBS before being incubated with goat anti-rabbit AF546 (Thermo Fisher Scientific, cat. # A-11010, 1:500) in DPBS with 10% NGS and 0.05% Tween20 for 1hour at RT. The slides were mounted with Dako mounting medium (Agilent Technologies cat. # S302380-2). The images were taken with the LSM880 (Zeiss) and analyzed with ZEN (Zeiss) and lmageJ / FiJi2.

[0288] Modeling of dsDNA binding to ADA2 and alignment of ADA2 and ADA1 structures

[0289] The crystal structure of ADA2 (3LGD, PDB DOI: doi 10.2210 / pdb3lgd / pdb) (Zavialo et al, Journal of Biological Chemistry 285, 12367-12377; 2010; 10.1074 / jbc. M109.083527) and double-stranded DNA (1D66) were used for protein-DNA docking using HDOCK server (Yan et al., Nucleic Acids Res 45, W365-W373 (2017). 10.1093 / nar / gkx407). The top-scored complex was subjected to steered molecular dynamic simulation using a TIP3P water model in a solvated box under periodic boundary conditions. Minimization was performed using 100 steepest descent steps at 0.02 A followed by 10 steps of conjugate gradient at 0.02 A. Equilibration was performed for 100000 steps (time step 1fs) using the heater temperature control method. Production was performed for 100000 steps (time step 1fs). Molecular dynamic simulation was performed with UCSF Chimera. For the comparison of ADA2 and ADA1, the crystal structure of ADA2 (3LGD, PDB DOI 10.2210 / pdb3LGD / pdb) and ADA1 (3iar, PDB DOI: 10.2210 / pdb3iar / pdb) were aligned with PyMOL 5 (Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 3.0; 2015).

[0290] Quantification of sequence similarity between ADA2 from Sus scrofa and Homo sapiens Amino acid sequences of ADA2 from Sus scrofa (P58780, uniprotkb / P58780 / entry) and Homo sapiens (Q9NZK5, uniprotkb / Q9NZK5 / entry) were downloaded from Uniprot and the percent of identity expressed as number of conserved amino acids on number of total amino acids.

[0291] Purification of endogenous ADA2 from porcine brain

[0292] Brains were cut into small pieces and homogenized in 0.075 M acetic acid / 0.15 M NaCI (1:2 mass / vol.) using a Waring Blender. The homogenate was centrifuged at 10000g for 10 min. The supernatant was recovered, pH was adjusted by adding 1M Tris base until it reached 7.6, and then heat treated at 60°C for 20 min before being again centrifuged at 10000g for 10 min. The supernatant was added to 20 ml Concanavalin A Sepharose and stirred overnight at 4°C. The slurry was run through a column and washed with PBS. The glycoproteins were eluted using 0.2 M a-methylmannoside in PBS. The protein solution was subsequently loaded onto a 20 ml hydroxyapatite column equilibrated with PBS, and the proteins were eluted at 0.05 M phosphate. After dialysis against 0.02 M Tris, pH 7.6, this fraction was applied to a DEAE anion exchange column equilibrated with the same buffer. The eluted solution was concentrated and applied to a Sephadex S-200 gel filtration column, dialyzed against 0.02 M Tris, pH 7.6, and subjected to CM cation exchange chromatography using a continuous NaCI salt gradient. The fraction eluted at 0.08 M NaCI was dialyzed against 0.02 M Tris, pH 7.6. After dialysis against 0.02 M Tris, pH 7.6, the samples were run in Heparin Sepharose chromatography, and the proteins bound to heparin were eluted at about 0.1 M NaCI. The final eluted preparation was run on SDS / PAGE and subjected to MS / MS analyses. Mass spectrometry data of the porcine brain ADA2 have been deposited to the ProteomeXchange Consortium via the PRIDE 48 (Perez-Riverol, Y. et al, Nucleic Acids Res 47, D442-D450; 2019; 10.1093 / nar / gky1106) partner repository with the dataset identifier PXD019373. Tandem mass spectrometry analysis of porcine brain proteins

[0293] Gel pieces from the heparin sepharose solution prepared as described above were subjected to in gel reduction, alkylation, and tryptic digestion using 6 ng / pl trypsin. OMIX C18 tips (Varian, Inc., Palo Alto, CA, USA) were used for sample clean-up and concentration. Peptide mixtures containing 0.1% formic acid were loaded onto a Thermo Fisher Scientific EASY-nLC1000 system and EASY-Spray column (C18, 2 pm, 100 A, 50 pm, 15 cm). Peptides were fractionated using a 2-100 % acetonitrile gradient in 0.1 % formic acid over 50 min at a 250 nl / min flow rate. The separated peptides were analyzed using a Thermo Fisher Scientific Q-Exactive mass spectrometer. Data was collected in data-dependent mode using a Topi 0 method. The Proteome Discoverer 1.4 software was used to generate mgf peak list files. The mgf files were searched against a mammalian database using an in-house Mascot server (Matrix Sciences, UK). Peptide mass tolerances used in the search were 10 ppm, and fragment mass tolerance was 0.02 Da.

[0294] Site-specific analysis of N-glycan structures linked to porcine brain proteins The N-glycan analysis was carried out essentially as previously described (Yang, W. et al. Anal Chem 86, 6959-6967; 2014; 10.1021 / ac500876p), but without spectral aligning due to the purity of the proteins. Briefly, after separation by SDS / PAGE and staining by Coomassie Blue, bands of interest were cut out and subjected to in-gel reduction, alkylation, and tryptic digestion using 6 ng / pl trypsin. OMIX C18 tips (Varian, Inc., Palo Alto, CA, USA) were used for sample cleanup and concentration. Peptide mixtures containing 0.1% formic acid were loaded onto a Thermo Fisher Scientific EASY-nLC1000 system and EASY-Spray column (C18, 2pm, 100 A, 50pm, 15 cm). Peptides were fractionated using a 2-100% acetonitrile gradient in 0.1 % formic acid over 50 min at a 250 nl / min flow rate. The separated peptides were analyzed using a Thermo Scientific Q-Exactive mass spectrometer. Data was collected in data-dependent mode using a Top10 method. The Proteome Discoverer 1.4 software was used to generate mgf peak list files. The mgf files were searched against a mammalian database using an in-house Mascot server (Matrix Sciences, UK). Peptide mass tolerances used in the search were 10 ppm, and fragment mass tolerance was 0.02 Da. The identification of the ADA2-linked glycopeptides was carried out in five steps:

[0295] 1) The peptide sequences of the glycopeptides were determined by calculating the theoretical masses of the tryptic peptides containing NXS / T glycosylation sequons of the target protein. The calculated masses of these peptides attached to more than 250 different N-glycan structures were determined and compared to the peptide masses obtained from the MS / MS spectra. Those masses that matched were analyzed further.

[0296] 2) The MS / MS-spectra containing glycopeptides were identified by the typical oxonium ions of simple sugars, m / z 163.1 (Hex), m / z 204.1 (HexNAc) and m / z 366.1 (HexHexNAc). Spectra containing mannose-6-phosphorylated glycans were identified by the additional ions, m / z 243.1 (PHex), and its fragmentation ion, m / z 225.1. 3).

[0297] 3) Each glycopeptide spectrum was scanned for fragmentation ions with a mass similar to the deglycosylated peptide and ions with an additional mass of 203.1, corresponding to linkage with a single HexNAc.

[0298] 4) The identity of the peptide part was further confirmed by identifying fragmentation ions corresponding to the calculated y-and b-values.

[0299] 5) The glycan structure linked to the peptide was deduced by comparing its mass from the MS / MS spectrum and theoretical masses of N-glycan structures.

[0300] The glycan structures were divided into the following groups: SN3, sialylated complex structure with three GIcNAc linked to the core; SN2, sialylated complex structure with two GIcNAcs linked to the core; SN1, sialylated complex structure with one GIcNAc linked to the core; NN3, neutral complex structure with three GIcNAcs linked to the core; NN2, neutral complex structure with two GIcNAcs linked to the core; NN1, neutral complex structure with one GIcNAc linked to the core; LMF, core-fucosylated structure with 1-3 mannoses linked to the core chitobiose unit; P2M, bisphosphorylated oligomannosidic structure; P1 M, monophosphorylated oligomannosidic structure; HM, oligomannose with 6-9 mannose; MM, oligomannose with 4-5 mannose; LM, oligomannose with 1-3 mannose. Production and purification of wild-type human ADA2 and mutant ADA2 proteins Protein production was performed in 100 ml (small scale screening), and a selection of the ADA2-mutants in 1 liter (1 L) (large scale), the final expression yield after purification is given in mg (see Table 1 below).

[0301] Table 1: Protein Production and Purification

[0302]

[0303] ADA2 open reading frame DNA (UniProt Q9NZK5: amino acids [aa] 29-511) with a CD33 leader in frame with its N-terminus and a His6-tag on its C-terminus (proprietary vector) was transfected with the PEI method (PEI MAX, Polysciences, cat. # 24765-1) in HKB11 mammalian cells. Freshly grown cells (1.25 x 108 cells) were centrifuged, resuspended in 9 ml fresh M11V3 media (produced at Bioconcept, cat. #V3-k, proprietary formulation) and transferred into 250 ml shake flask. 75 pg DNA and 225 pg PEI were transferred in 1.75 ml media each and incubated for 5 min in separate tubes. After transfer of PEI to DNA and gentle mixing and incubation for another 15 min at RT, the DNA- and PEI-Mix was added to cells and incubated for 4 hours in a shaking incubator (standard cultivation conditions with shaking at 115 rpm at 37°C with 5% CO2and 80% humidity). Subsequently, transfected cells were fed with 87.5 ml of M11 V3-media and incubated in a shaking incubator for seven days.

[0304] Secreted His-tagged proteins from small-scale screening were purified from cell supernatant with affinity chromatography by gravity flow on the bench, with Ni-NTA-Agarose (Qiagen, cat. # 30230), with a column volume of 0.5ml. Washes have been executed with 10CV of IMAC buffer (20 mM sodium phosphate buffer, 500mM NaCI) containing 20mM imidazol, elution has been performed with 6 CV of IMAC buffer containing 500mM imidazol, both at pH7.4. Buffer exchange into PBS pH7.4 has been done with several concentration and dilution steps with Centrifuge Tubes Microsep Advance 10K, 516-0358, VWR. Protein integrity and purity were analyzed by SDS-PAGE and analytical SEC on Superdex 200 Increase 10 / 300 GL column, GE28-9909-44, with PBS, pH7.4 as running buffer, on Agilent 1260 Infinity instrument.

[0305] The transfection protocol was scaled up proportionally for a 1 L large-scale expression of a selection of ADA2-variants. These supernatants were purified on an Akta system (GE Healthcare), with a 5ml HisTrap HP prepacked column with Nickel Sepharose (17-5248-02, GE Healthcare), using the same buffers as described for small scale but with gradient elution. Polishing of these proteins on preparative SEC was done on a Superdex75 column, GE Healthcare, 28-9893-33, with PBS pH7.4.

[0306] Protein integrity and purity were analyzed by SDS-PAGE, analytical SEC on a Superdex 200 Increase 10 / 300 GL column, GE28-9909-44, with PBS, pH7.4 as running buffer, on an Agilent 1260 Infinity instrument, and MS analysis. All proteins showed low aggregation and an identical retention time. For wild-type ADA2 and the ADA2 G47A mutant, inline multi-angle light scattering (MALS) was combined with SEC to confirm the molecular weight of 127kDa of the recombinant protein resembling the mass of an ADA2 dimer. In brief, a Superdex 200 10 / 30 GL column (GE Healthcare) was equilibrated with PBS (Sigma D8537), and samples were run on an Agilent 1200 HPLC system coupled with Wyatt MALS instrumentation at a constant 0.5 ml / min flow. Human Macrophage colony-stimulating Factor! -Receptor (aa 20-512), which comprises nearly the complete extracellular domain of hCSF1-Receptor (aa 20-517; UniprotKB-P07333, full-length aa 1-972) and a His-tag, was expressed and purified as ADA2 using an IMAC column and used as a control.

[0307] Hereafter is reported a summary of the ADA2 mutant proteins described in patients with ADA-deficiency and tested in the manuscript. Brackets provide the PubMed ID (PMID) of the paper where the variant has been originally described.

[0308] E328K (PMID: 27069017): The variant is localized in the catalytic domain. ADA2 activity in the patient’s plasma, measured using HPLC was 1.7 mU / ml, significantly below the level in healthy controls [mean (s.d.) 14 (6.1) mU / ml], A variant affecting the same aminoacid (E328D) has been tested functionally in PMID: 31945408 and has shown no residual ADA activity. G358R (PMID: 28974505): The variant is localized in the catalytic domain. Reported in two different patients who underwent HSCT in the original paper. The variant has also been tested functionally in PMID: 31945408 and has shown no residual ADA activity.

[0309] G47R (PMID: 24552284): The variant is localized in the dimerization domain. This is a frequent variant reported in several papers and individuals. The variant has also been tested functionally in PMID: 31945408 and has been shown to have less than 25% of residual ADA activity.

[0310] P344L (PMID: 28522451): The variant is localized in the catalytic domain. Reported originally in PMID: 28522451 in compound heterozygosity (C. H) in two patients with different clinical phenotypes. The variant has also been tested functionally in PMID: 31945408 and has been shown to have approximately 50% of residual ADA activity.

[0311] P193L (PMID: 25278816): The variant is localized in the catalytic domain. It was reported in a patient in C. H. with the Arg169Gln in a patient with an upregulation of interferon-stimulated gene transcripts in peripheral blood. To our knowledge, the variant has been tested functionally only in our manuscript. Mass spectrometric analysis of recombinant human ADA2

[0312] 1 pg of protein was resuspended in 8M urea, 10 mM HEPES (pH 8), and 10 mM DTT. Alkylation was performed in the dark for 30 min by adding 55 mM iodoacetamide (IAA). A two-step proteolytic digestion was performed. First, samples were digested at room temperature (RT) with LysC (1:50, w / w) for 3 hours. Then, they were diluted to 1:5 with 50 mM ammonium bicarbonate (pH 8) and digested with trypsin (1:50, w / w) at RT overnight. The resulting peptide mixtures were acidified and loaded on C18 StageTips. Peptides were eluted with 80% acetonitrile (ACN), dried using a SpeedVac centrifuge (Savant, Concentrator plus, SC 110 A), and resuspended in 2% ACN, 0.1% trifluoroacetic acid (TFA), and 0.5% acetic acid. Peptides were separated on an EASY-nLC 1200 HPLC system (Thermo Fisher Scientific) coupled online to a Q Exactive mass HF spectrometer via a nanoelectrospray source (Thermo Fisher Scientific). Peptides were loaded in buffer A (0.1% formic acid) on in-house packed columns (75 pm inner diameter, 50 cm length, and 1.9 pm C18 particles from Dr. Maisch, GmbH). Peptides were eluted with a non-linear 120 min gradient of 5%-60% buffer B (80% ACN, 0.1% formic acid) at a 250 nl / min flow rate and a column temperature of 50°C. Raw files were analyzed by MaxQuant software (version 1.5.3.54). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol, Y. et al, Nucleic Acids Res 47, D442-D450; 2019; 10.1093 / nar / gky1106) partner repository with the dataset identifier PXD019382.

[0313] FRET assay of wt and mutant human recombinant ADA2

[0314] ADA2 FRET assay of recombinant human ADA2 was measured by a commercial FRET assay (DNaseAlert, Thermo Fisher Scientific, cat. # AM1970). In brief, 6 pl ADA2 solution at a final concentration of 10 pg / ml was incubated with 3 pl DNA substrate and 40 pl of MIB super buffer 2 (Malonic acid: imidazole: boric acid in the molar ratios 2:3:3) or CHC super buffer 1 (Citric acid: HEPES: CHES in the molar ratios 2:3:4) (Jena Bioscience CS-332- JBScreen Thermofluor Fundament) at the indicated pH and NaCI concentration. ADA2-DNA interaction was measured every 2 min for 30 min with a fluorescent reader (Spectramax, excitation 535 nm and emission 575 nm). FRET activity was measured at 25 pg / ml.

[0315] ADA activity of wt and mutant human recombinant ADA2 proteins

[0316] Adenosine Deaminase (ADA) activity was measured by a commercial assay (Diazyme-DZ117A-K), which is specific for ADA and has no detectable reaction with other nucleosides. 5 pl of ADA2 or control proteins were added to 180 pl of buffer R1 and incubated for 3 min at 37°C, then 90 pl of buffer R2 was added and the plate was incubated at 37°C for 20 min. The enzymatic activity was monitored by measuring the OD at 550 nm with a temperature-controlled fluorescent reader (Synergy H1 from Biotek). The kit was calibrated with the internal ADA calibrator, which was in the expected range of kit specifications. A range of concentrations was measured to describe the activity of the ADA2 recombinant proteins, and the EC50 value was calculated using Prism Software (GraphPad).

[0317] Electrophoretic mobility shift assay (EMSA)

[0318] Oligonucleotide substrates (see Table 2) were 5’-end labeled using T4 polynucleotide kinase (New England Biolabs, cat. # M0201) and [y-32P]ATP (3000 Ci / mmol, Amersham Biosciences). Doublestranded (ds) substrates were generated by annealing (65 °C, 3 min) the labeled single-stranded (ss) oligonucleotide to a complementary strand. All substrates were gel-purified before use. In EMSA, ADA2 (wt or ADA2 mutant proteins; 0.2-1.5 pmol) and labeled substrates (10 fmol) were mixed in 25 mM sodium acetate (NaAc) pH 5.5 in 10 pl reaction volume. Samples were incubated on ice for 15 min, and DNA gel loading dye (6x) was added (Thermo Fisher Scientific cat. # R0611). DNA-ADA2 complexes were separated from unbound substrates on 7% native polyacrylamide gels containing 2% glycerol in 0.5x TBE at 120 V for 45 min on ice. Gels were dried and visualized by phosphorimaging (Amersham Typhoon IP), and ImageQuant IQTL 8.2 was used for quantification (Cytiva). For the test of pH dependence, the buffer was 25 mM T ris-HCI pH 7.5. Table 2: Oligonucleotide substrates.

[0319]

[0320]

[0321]

[0322] DNA deaminase assay

[0323] DNA deaminase activity was measured using the labeled DNA substrates and amounts of recombinant ADA2 (WT or mutants) as indicated in 25 mM NaAc pH 5.5 in 10 pl reaction volume. Incubation was at 37 °C for 40 min or as shown in the figures, and reactions were neutralized by adding 2 pl 0.1 M Tris-HCI pH 9.3. Deoxyinosines were detected using in-house purified Escherichia coli (Ec) EndoV. The EcEndoV open reading frame was cloned in pET28b (Novagen, cat. #69865) in a frame with a c-terminal His-tag, and the protein was purified by Ni-NTA affinity chromatography after expression in the E. coli strain ER2566 (New England Biolabs, cat. # C2566). EcEdnoV (0.8 pmol) and 2.5 pl EcEndoV reaction buffer (final 10 mM Tris-HCI pH 8.5, 2 mM MgCI2, 50 mM KCI, 1 mM DTT, 5% glycerol) were added to the reaction and incubated at 37 °C for 15 min. Samples were exposed to formamide loading dye (95% formamide, 5 mM EDTA, 0,05% bromphenol blue, and 0,05% xylene cyanol), denatured at 60 °C for 3 min, and the reaction products separated on 20% polyacrylamide / urea gels at 200 V for 1 h in 1 x taurine buffer. All experiments were performed at least 2-3 times, and representative experiments are shown. The influence of pH on ADA2-mediated dT-to-dl activity was tested in NaAc buffer (pH 4.0-6.5) or Tris-HCI (pH 7.0). Gels were dried and visualized by phosphorimaging (Amersham Typhoon IP), and ImageQuant IQTL 8.2 was used for quantification (Cytiva).

[0324] DNA dot blot

[0325] Human recombinant ADA2 (25-100 fmol) was incubated with substrates (10 pmol) as indicated for 1 h in 25 mM sodium acetate pH 5.5 at 37 °C in 5 pl reactions. Reactions were neutralized with 0.5 pl 100 mM Tris-HCI pH 9.3 and heat denatured at 60 °C for 3 min. 2.5 pl of the reaction was spotted onto a nitrocellulose membrane (Whatman Protran BA83) that was air dried before crosslinking (120 mJ / cm2) the DNA to the membrane. Inosines in DNA were detected using an anti-inosine antibody (Medical and Biological Laboratories; cat. # PM098, dilution 1:1000 in PBS with 5% nonfat dry milk), HRP-conjugated secondary antibody (Goat anti-rabbit IgG, Vector laboratories cat. # P1-1000-1) and Supersignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific cat. # 34094). As a control of the specificity of the antibody, oligonucleotides with and without inosines were tested in dot blot. Despite being raised against inosines in RNA, the antibody recognized inosines in DNA oligonucleotides (ACTGGACAIITICTCCGAGG, SEQ ID NO: 48, and CCTCGGAGTITTTGTCCAGT, SEQ ID NO: 94), whereas a DNA oligonucleotide without inosine (GCTGGACAAATACTCCGAGG, SEQ ID NO: 47) gave no signal.

[0326] Nucleobases content in DNA by mass spectrometry

[0327] Oligonucleotides (150 fmol) were incubated with human recombinant ADA2 (hrADA2) (100 fmol) in 25 mM ammonium acetate (pH 5.5) in 20 pl reaction volume for 45 min. Samples were heated at 80 °C for 5 min before completely dried under vacuum. The samples' deoxyinosine (dl) content was measured by liquid chromatography-mass spectrometry (LC-MS / MS). DNA was dissolved in 100 pM deaminase inhibitor EHNA (Sigma-Aldrich cat. # E114) and hydrolyzed to nucleosides by 20 U benzonase (Santa Cruz Biotech cat. # sc-391121 B), 0.2 U nuclease P1 (Sigma-Aldrich cat. # N8630), and 0.1 U alkaline phosphatase (Sigma-Aldrich cat. # P5931) in 10 mM ammonium acetate pH 6.0, and 1 mM MgCI2 at 40°C for 40 min, added 3 volumes of acetonitrile and centrifuged (16 000g, 30 min, 4°C). The supernatants were lyophilized at -80°C to minimize spontaneous adenine deamination during drying and dissolved in 50 pl water for LC-MS / MS analyses of dl, inosine, and unmodified nucleosides. Chromatographic separation was performed using an Agilent 1290 Infinity II UHPLC system with a ZORBAX RRHD Eclipse Plus C18 150 x 2.1 mm ID (1.8 pm) column protected with a ZORBAX RRHD Eclipse Plus C185 x 2.1 mm ID (1.8 pm) guard column (Agilent). Fordl and inosine analyses, the mobile phase consisted of water and methanol (both added 10 mM acetic acid) run at 0.25 ml / min, starting with 5% methanol for 0.5 min, followed by a 4 min gradient of 5-90% methanol, and 4 min re-equilibration with 5% methanol. A portion of each sample was diluted to analyze unmodified nucleosides, chromatographed isocratically with water / methanol / formic acid (80 / 20 / 0.1%). Mass spectrometric detection was performed using an Agilent 6495 Triple Quadrupole system with electrospray ionization, for DNA monitoring the mass transitions 251.1 / 135.1 (dl, negative mode), 252.1 / 136.1 (dA, positive mode), 228.1 / 112.1 (dC, positive mode), 268.1 / 152.1 (dG, positive mode) and 243.1 / 127.1 (dT, positive mode), and for RNA monitoring 267.1 / 135.1 (I, negative mode), 268.1 / 136.1 (A, positive mode), 244.1 / 112.1 (C, positive mode), 284.1 / 152.1 (G, positive mode) and 245.1 / 113.1 (U, positive mode).

[0328] DNA sequencing gel

[0329] Sequencing gels were the same 20% polyacrylamide / urea / taurine gels as the ones described in the deamination assay section, but they were run at 35 W for 1 hour. The gels were dried, and radiolabeled fragments were visualized as above.

[0330] DNA deaminase competition assay

[0331] In the competition assays, competitors and ADA2 were preincubated on ice for 5 min in 25 mM NaAc pH 5.5 before the labeled DNA substrate was added, as described in the deamination assay section. Incubation continued at 37 °C for 30 min before neutralization and treatment with EcEndoV as above. The following competitors were used: adenosine, deoxyadenosine, A-DNA (oligo I ITI 24522 of Table 2), non-A-DNA (oligo A1mut 24793 of Table 2), deoxycoformycin (Sigma-Aldrich, cat. # 116860). All experiments were performed at least 2-3 times, and representative experiments are shown.

[0332] RNA deaminase assay

[0333] Oligonucleotide substrates (Table 2) were 5’-end labeled using T4 polynucleotide kinase (New England Biolabs, cat. # M0201) and [y-32P]ATP (3000 Ci / mmol, Amersham Biosciences). Subsequently, RNA deaminase activity was measured using the labeled RNA substrates and amounts of recombinant ADA2 (wt or mutants) as indicated in 25 mM NaAc pH 5.5 in 10 pl reaction volume. Incubation was at 37 °C for 40 min or as shown in the figures, and reactions were neutralized by adding 2 pl 0.1 M Tris-HCI pH 9.3. Detection of inosines was done using in-house purified human EndoV. Protein was expressed and purified as described in Vik E. S. et al. Nat Commun 2271 (2013). hEndoV (0.8 pmol) and 2.5 pl EndoV reaction buffer (final 10 mM Tris-HCI pH 8.5, 2 mM MgCI2, 50 mM KCI, 1 mM DTT, 5% glycerol) were added to the reaction and incubated at 37 °C for 15 min. Samples were exposed to formamide loading dye (95% formamide, 5 mM EDTA, 0,05% bromphenol blue, and 0,05% xylene cyanol), denatured at 60 °C for 3 min, and the reaction products separated on 20% polyacrylamide / urea gels at 200 V for 1 hour in 1 x taurine buffer. TLR9 activation via HEK TLR9-SEAP reporter cells

[0334] Human TLR9 reporter HEK 293 Cells (NF-kB), cat. # hkb-htlr9 were cultured as described above and stimulated according to the recommendations of the manufacturer's protocol. Briefly, cells were detached and resuspended in warm PBS, counted, resuspended (80.000 cells / 180 pl of HEK-Blue Detection medium) and mixed with 20 pl of different stimuli (Oligos) at different concentrations as indicated in the respective figures and incubated overnight. TLR9 activation was measured with HEK-Blue Detection cell culture medium. Secreted embryonic alkaline phosphatase (SEAP) activity was determined by measuring optical density (OD) at 620-655 nm.

[0335] ADA2 Expression and TLR7 and TLR9 activation via HEK 7 / 9-UNCB93B1 reporter cells

[0336] The 293XL / hTLR7-HA and 293XL / hTLR9-HA cell lines (Invivogen) both expressing human UNC93B1-mCitrine (Pelka, K. et al. Immunity 48, 911-922 e917; 2018, 10.1016 / j.immuni.2018.04.011) 1WT were plated at 20.000 cells / 1 OOpL in a 96-well plate. Once adherent, they were transiently transfected with 25ng pLenti-IRES-GFP-Puro or pLenti-ADA2-IRES-GFP-Puro as indicated using Mirus-LT1 according to the manufacturer’s instructions. After 20 hours of plasmid transfection, cells were stimulated with 1 pg / mL R848 or 10pg / mL of E.coli DNA, CpG2006, CpG2216 or 9.2sRNA complexed with 5pL DOTAP for 10 minutes. After 16 hours, cellular supernatants were harvested for ELISA. The day of stimulation cells were detached and resuspended in warm PBS, counted, resuspended (80.000 cells / 180 pl of HEK-Blue Detection medium) and mixed with 20 pl of different stimuli at different concentrations (Oligos) as indicated in the respective figures and incubated overnight. TLR9 activation was measured by quantifying IL8 production by ELISA.

[0337] Lentivirus transduction of HEK-Blue hTLR9 reporter cells with ADA2 or empty vector Lentivirus were produced by transfecting HEK293 T cells with psPAX2, pMD2. G and pLenti-ADA2-IRES-GFP-Puro or pLenti-ADA2-IRES-GFP-ADA2-Puro (Amount per 10 cm dish, respectively 1.3 pmol, 0.72 pmol, 1.64 pmol). Plasmids were transfected by diluting the plasmids in 1 ml Opti-MEM containing a ratio of 1:3 between DNA and PEI (1mg / ml). The transfection mix was added to HEK293T packaging cells and incubated for 18 hours. After incubation the medium was removed and replaced with 15 ml of fresh medium. Supernatants containing virus were harvested at 48 and 72 hours, centrifuged at 500 g for 5 min and filtered through 0.45 pm filter. Subsequently, the supernatants were transferred to HEK Blue hTLR9 reporter cells and incubated for 16 hours in a humidified incubator in an atmosphere of 5-7% CO2. After this the media containing lentiviral particles was removed and substituted with fresh medium. The efficiency of transduction was monitored by measuring GFP expression using flow cytometry. GFP-positive cells were FACS sorted, and the expression of ADA2 was evaluated by Western Blot.

[0338] Measurement of type-l IFN Production with IFN-a / p reporter HEK 293 Cells from PBMCs On the day of measurement, HEK-Blue™ IFN-a / p cells were detached, diluted to a concentration of 280.000 cells / ml, and subsequently seeded in a flat-bottomed 96-well plate at 50.000 cells / well density. To generate a standard curve, a series of 10-fold dilutions of recombinant human IFN-o was prepared with an initial concentration of 2 pg / ml. The cells were then incubated for 24 hours with either 20 pl of the supernatant of the stimulated cells or with the recombinant human IFN-a overnight at 37 °C. Each experimental condition was carried out in triplicate. After incubation, the QUANTI-Blue solution was prepared according to the manufacturer's instructions, and 20 pl of the supernatants from the HEK-Blue™ IFN-a / p mixed with 180 pl of the QUANTI-Blue solution and incubated for 30-40 min at 37°C. The levels of SEAP, which served as an indicator of IFN production, were measured by spectrophotometry at a wavelength of 620 nm.

[0339] Measurement of type-l IFN production in PBMCs

[0340] PBMCs were isolated from healthy donors and resuspended in RPMI medium supplemented with 10% FBS and 100U / ml P / S and subsequently seeded into round-bottomed 96-well plates (90.000 cells / well in a final volume of 100 pl). Cells were stimulated overnight with CpG (final concentration 3 pg / ml) or CpI (final concentration 3 pg / ml) orCpA (final concentration 3 pg / ml) orCpC (final concentration 3 pg / ml) or PolylC (final concentration 1 pg / ml). After stimulation 20 pl of the supernatants were used to measure type-l IFN production with IFN-a / p reporter HEK 293.

[0341] Immune stimulation of PBMCs

[0342] Blood was obtained from DADA2 patients or age and sex-matched healthy donors in EDTA tubes. PBMCs were prepared by centrifugation on a Pancoll gradient (PAN Biotech, Pancoll human cat. # P04-60500). Cells were seeded in 96 well round bottom plate (106 cells / well) in 200 pl of RPMI containing 10% heat inactivated fetal bovine serum, 100 U / ml penicillin, 100 pg / ml streptomycin and stimulated overnight with CpG2216 (10 pg / ml), or CpG2006 (10 pg / ml), or 9.2sRNA (2 pg / ml). Supernatants were harvested after 16h of stimulation for the quantification of TNF or IFN-a by ELISA.

[0343] Measurement of type-l IFN production in dendritic cells (DCs)

[0344] Blood was obtained from DADA2 patients or age and sex-matched healthy donors in EDTA tubes. PBMCs were prepared by centrifugation on a Pancoll gradient (PAN Biotech, Pancoll human cat. # P04-60500). Plasmacytoid dendritic cells (pDCs) were sorted as viability-dy BV 510-, lineage (CD3-FITC, CD14-FITC, CD16-FITC, CD19-FITC)-, HLA-DR-APC+, CD11c-BV421dim, CD4-PEhi9d, conventional DC (eDCs) as lineage (CD3-FITC, CD14-FITC, CD16-FITC, CD19-FITC)-, HLA-DR-APC+, CD11c-BV421+, CD4-PEdim. Cells were seeded in 96 well V bottom plate (500 cells / well) and stimulated overnight with CpG 2216 (5 pM). The day after 20 pl of the supernatant of the stimulated DCs the supernatants were used to measure type-l IFN production with IFN-a / p reporter HEK 293.

[0345] Measurement of type-l IFN production in PBMCs is upon stimulation with PLA conjugated DNA Blood was obtained from DADA2 patients or age and sex-matched healthy donors in EDTA tubes, and PBMCs were prepared by centrifugation on a Pancoll gradient (PAN Biotech, Pancoll human cat. # P04-60500). Cells were seeded in 96 well V bottom plates (90.000 cells / well in a final volume of 10OpI). Oligo 259 and 260 (Table 2) were annealed and complexed with PLA (Sigma cat # P 4663) at a ratio of 1 pg DNA / 1.2 pl PLA in PBS (0.4 pg dsDNA, 0.6pl PLA and 30pl of PBS for each well) and incubated 30 min at RT. After incubation, the DNA was added to the cells and incubated at 37°C for 17 hrs. The day after the production of type-l IFN was measured using HEK-Blue™ IFN-a / p reporter cells as described above. Quantification and statistical analysis

[0346] Statistical analyses were performed with GraphPad Prism 8. The tests performed for statistical analyses are indicated in the respective figure legends.

[0347] Example 1:

[0348] ADA2 is the only known human deoxyribonuclease deaminase that acts naturally on DNA Until now, no naturally occurring human enzyme was known that deaminates deoxyadenosine (dA) to deoxyinosine (dl) (dA-to-dl activity) by directly acting on DNA. The only proteins described, so far, as capable of dA-to-dl activity on DNA have been human ADAR (adenosine deaminase RNA specific) and ADAT2 (adenosine deaminase acting on tRNA) from Branchiostoma japonicum (BjADAT2). ADAR is a cytosolic adenosine deaminase with a primary role in RNA editing but can also deaminate dA in DNA / RNA hybrids (particularly at dA-C mismatches) in the presence of specific mutations such as E-to-Q in its base-flipping domain. BjADAT2, in complex with BjADAT3, primarily performs A-to-l editing on tRNA but additionally acts on DNA converting deoxycytidine to deoxyuridine and dA to dl [1, 2], All other dA deaminases currently employed in synthetic biology and genome editing originate from bacterial TadA (tRNA-specific adenosine deaminase), which naturally acts only on tRNA [3], These enzymes have been engineered through directed evolution and rational mutagenesis to gain the ability to deaminate dA in single-stranded DNA which forms the basis of current adenine base editors (ABEs). No human or mammalian homolog exists that naturally performs this type of DNA-editing function and has been used in synthetic biology and genome editing. Therefore, the determination of human ADA2 as a dA-to-dl deaminase acting naturally on DNA is a breakthrough in the field of genome editing and provides new tools in this field, e.g., the tools disclosed in the present application.

[0349] The ADA2-mediated dA-to-dl activity was first identified using the Endo-V assay. This assay can be used to test whether a protein can deaminate DNA by detecting the formation of dl as a result of deamination of dA bases. Specifically, Escherichia coli Endonuclease V (EcEndoV) recognizes dl, a product of the dA deamination process, and cleaves the DNA strand at a specific location near the dl site. If a test protein (e.g. hrADA2) deaminates dA, subsequent incubation and treatment with EcEndoV results in strand cleavage, which is usually visualized by gel electrophoresis. Therefore, a positive test result indicate that the protein has nucleic acid deaminase activity (Figure 1A). In this case, in-house purified Escherichia coli (Ec) EndoVwas used. The EcEndoV open reading frame was cloned in pET28b (Novagen, cat. # 69865) in a frame with a c-terminal His-tag, and the protein was purified by Ni-NTA affinity chromatography after expression in the E. coli strain ER2566 (New England Biolabs, cat. # C2566). EcEdnoV (0.8 pmol) and 2.5 pl EcEndoV reaction buffer (final 10 mM Tris-HCI pH 8.5, 2 mM MgCI2, 50 mM KCI, 1 mM DTT, 5% glycerol) were added to the reaction and incubated at 37 °C for 15 min. Samples were exposed to formamide loading dye (95% formamide, 5 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol), denatured at 60 °C for 3 min, and the reaction products separated on 20% polyacrylamide / urea gels at 200 V for 1 h in 1 x taurine buffer. ADA2-mediated dA-to-dl activity was measured by incubating DNA at 37 °C for 40 min (Figure 1 B). Incubation of DNA with ADA2 led to the emergence of cleavage products after EcEndoV treatment, indicating ADA2-mediated dA-to-dl conversion.

[0350] Detection of ADA2-mediated dA-to-dl activity with a meagre amount of ADA2 (<1 nM) indicates that ADA2 has a higher affinity towards DNA-dA than free Ado which in turn suggests that DNA is its natural substrate. This is also demonstrated by the inhibition of the dA-to-dl editing activity by equimolar amounts of “cold” DNA but not by a 10,000-fold excess of free Ado nor free dA (Figure 1C).

[0351] It was also tested if the catalytic site of ADA2 mediates its dA-to-dl activity on DNA by comparing mutant ADA2 proteins with different ADA activity. The catalytic dead mutant G358R showed no dA-to-dl activity on DNA demonstrating that the catalytic site of ADA2 indeed mediates this activity (Figure 2A). This was further confirmed by the significant inhibition of dA-to-dl activity by deoxycoformycin which is a potent inhibitor of ADA2's ADA activity and acts by binding to the enzyme's active site (Figure 1C). Further, it has also been demonstrated that ADA1, unlike ADA2, does not perform dA-to-dl deamination of DNA molecules (Figure 2B).

[0352] Example 2:

[0353] ADA2 selectively deaminates terminal deoxyadenosine

[0354] Most of the currently used deaminases in biotechnology, including engineered TadA and TadA-derived adenine base editors, do not discriminate between terminal and internal dA [5], Their activity is generally guided by programmable recruitment mechanisms such as Cas9 targeting, which define an 'editing window' encompassing several contiguous bases within the DNA sequence, regardless of their physical position [6, 7], This lack of specificity for DNA termini increases the risk of off-target and bystander mutations, posing challenges for precise control in gene therapy interventions [8], On the other hand, an enzyme engineered or evolved to recognize and act only on free dA present at DNA ends — whether exposed by nicks or double-strand breaks — would allow for editing, strictly determined by the physical state of the DNA. Such specificity could confine editing activity to damage sites introduced by programmable nucleases, therefore reducing unwanted sequence changes and increasing the accuracy of targeted repair strategies. Discovery of such a class of selective-terminal deaminases would be a representative of significant advance in the safety and precision of next-generation gene editing technologies. The results of the present studies revealed human ADA2 as such selective-terminal deaminase that naturally favors terminal dA residues. It efficiently deaminates dA at DNA ends while absence of any activity for internal dA bases. Accordingly, genome editing tools based on ADA2 correspond to significantly advanced tools in terms of safety and precision.

[0355] ADA2-mediated dA-to-dl deaminase activity was measured by incubating DNA at 37 °C for 40 min with different substrates containing dA residues in various positions. Incubation of DNA with ADA2 led to the emergence of cleavage products after EcEndoV treatment only in the presence of terminal dA(s) residues (Figure 3). Three additional verification experiments were conducted to further confirm these results. Experiment A

[0356] Human recombinant ADA2 (hrADA2) (25-100 fmol) was incubated with substrates (10 pmol) as indicated for 1 h at 37°C in 5 pL reactions and then heat-denatured at 60°C for 3 min. 2.5 pL of the reaction was spotted onto a nitrocellulose membrane (Whatman Protran BA83) that was air-dried before crosslinking (120 mJ / cm2) the DNA to the membrane. Deoxyinosines in DNA were detected using an anti-inosine antibody (Medical and Biological Laboratories, cat. # PM098, dilution 1:1000 in PBS with 5% nonfat dry milk), HRP-conjugated secondary antibody (Goat anti-rabbit IgG, Vector laboratories cat. #P1 -1000-1) and Supersignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific cat. # 34094). As a control of the specificity of the antibody, oligonucleotides with and without inosines were tested in dot blot. Despite being raised against inosines in RNA, the antibody recognized inosines in DNA oligonucleotides (ACTGGACAIITICTCCGAGG, SEQ ID NO: 48 and CCTCGGAGTITTTGTCCAGT, SEQ ID NO: 94), whereas a DNA oligonucleotide without inosine (GCTGGACAAATACTCCGAGG, SEQ ID NO: 47) gave no signal (Figure 4). Results of these experiments confirmed that ADA2 selectively deaminates terminal dA(s).

[0357] Experiment B

[0358] In this experimental verification, quantification of dl residues was carried out through mass spectrometry (see Table 3 below). Oligonucleotides (150 fmol) were incubated with human recombinant ADA2 (hrADA2) (100 fmol) for 45 min at 37 °C. Samples were heated at 80°C for 5 min before being completely dried under vacuum. The deoxyinosine (dl) content of samples were measured using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The signal observed on the Dot blot and measured dis were present only for oligonucleotides (ODNs) with terminal dAs, which again confirms that human ADA2 exclusively edits terminal dA(s) residues (Figure 5). Table 3: Liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0359] I L1

[0360]

[0361] Deoxyinosine in ADA2-treated DNA oligonucleotides

[0362] 1. AATA 24524: ACTGGACAAATACTCCGAGG

[0363] 2. Al nnit 24793: GCTGGACAAATACTCCGAGG

[0364] 3. CpG2216 CpG2216: GGGGGACGATCGTCGGGGGG

[0365] 4. CompC 24978: ATGGGATATACCGGCGACGCATGATGAACA

[0366] 5. CompCCs 24979: TGTTCATCATGCGTCGCCGGTATATCCCA

[0367] fi. Lambda DNA 1DNA (EcoRI-Hindlll) Experiment C

[0368] As third experimental validation, sequencing gel was performed. Sequencing gels were the same 20% polyacrylamide / urea / taurine gels as the ones described in the deamination assay section, but they were run at 35 W for 1 h. The gels were dried and radiolabeled fragments were visualized. Cleavage of an ODN with a 5’-dl by EcEndoV usually generate a 2-nucleotide fragment (Figure 6). A similar two-nucleotide product is obtained when ADA2-EcEndoV sample was separated by DNA sequencing gel electrophoresis which further confirms that ADA2 deaminates preferentially terminal 5’ and 3’ dA residues (Figure 6).

[0369] Finally, in a complementary experimental test, it was investigated whether the protein could perform dA-to-dl activity on nicked DNA terminals as well by comparing different ODNs, as shown in Figure 7. In alignment with the above observations, these experiments also confirmed that the protein can deaminate dA residues on nicked terminals of DNA (Figure 7).

[0370] Example 3

[0371] ADA2 can deaminate adenosine to inosine on RNA molecules

[0372] Modification of DNA bases changes genomic DNA creating stable and heritable changes. This is an important aspect in correcting permanent genetic diseases or in incorporation of the features that need to be inherited during cell division. On the contrary, editing RNA bases changes messenger RNAs after transcription and produces temporary and non-inheritable effects [9, 10], Tools such as ADAR (for A to I conversion) or engineered enzymes for C to U modification act on transcripts rather than on the genome, allowing reversible modulation of protein expression

[0011] , Thus, RNA editing is an ideal tool when a flexible, temporary effect is required, particularly for controlled in vitro experiments or when safety concerns make long-term genomic alterations undesirable. For example, the CRISPR-Cas13 / REPAIR system can correct pathogenic single-nucleotide mutations in cultured human cells, modeling certain diseases or testing drug responses without altering the genome itself. In vitro, RNA editing has also been used to rescue enzyme activity in cell models for disorders such as Hurler syndrome (restoring alpha-L-hyduronidase function), or to modify Alzheimer's disease genes (editing BACE1 cleavage sites)

[0012] ,

[0373] The ability of hADA2 to deaminate A residues into RNA molecules was tested through an RNA deaminase assay. The ADA2-mediated ‘Adenosine (A) to Inosine (I) (A-to-l) activity’ on RNA was observed and confirmed to favor terminal A residues, similar to the preference as seen in the dA-to-dl activity (Figure 8).

[0374] Example 4

[0375] ADA2 is conserved in primates, sheep, pig (sus scrofa)

[0376] To investigate the evolutionary conservation of ADA2, the amino acid sequence of human ADA2 was first analyzed by performing BLASTP search against the NCBI non-redundant protein database. Homologous sequences were obtained from a range of primate and non-primate vertebrates, with redundant or incomplete entries excluded. The resulting dataset, as shown in the alignment, was subjected to multiple sequence alignment using MAFFT, allowing the identification of fully and partially conserved residues as well as species-specific adaptations. The consensus alignment (Figure 9) demonstrated high sequence conservation in key regions, indicative of evolutionary pressure to maintain ADA2 structure and function across diverse primates. This conservation could support the utility of comparative genomics for inferring functionally important motifs and may provide information on therapeutic strategies for DADA2 by highlighting residues or domains that may be suitable for mutagenesis and drug targeting. Additionally, conservation could facilitate the execution of preclinical studies in animal models.

[0377] Although functional tests were not performed on ADA2 derived from the primates, the functional activity of ADA2 obtained from Sus scrofa (Figure 10A, 6 and pA2) and sheep (Figure 10B) were assessed. The figures confirm that dA-to-dl conversion also occurs in these two additional species and therefore indicates that this activity is conserved across species

[0378] Consequently, the conservation across species can guide translational efforts to model ADA2 and develop targeted interventions in experimental settings where in vivo functional validation, pharmacological testing, or genetic manipulation are required, overcoming the limitations presented by the absence of ADA2 in traditional rodent models.

[0379] Example 5

[0380] ADA2 modulates human TLR9 activation

[0381] Lysosomal DNA sensing is mediated by TLR9 [13-15], This receptor is believed to preferentially recognize the pathogen-derived DNA containing a higher frequency of unmethylated CpG dinucleotides. Its stimulation leads to the production of type I IFN in human pDCs

[0016] , However, it was unclear whether it only recognizes bacterial DNA or can also be activated by self-DNA [17, 18], and whether it has a role in cells other than pDCs, particularly in human monocytes and macrophages.

[0382] The ability of TLR9 to regulate innate immune responses makes it an attractive target for translational medicine, particularly in cancer immunotherapy and inflammatory diseases. Based on this potential, multiple clinical trials have already been conducted testing both TLR9 agonists and antagonists in various therapeutic conditions, including cancer, autoimmune disorders, and infectious diseases. Some medicinal agents like cavrotolimod, vidutolimod, and SD-101 have advanced to Phase 1 and 2 clinical studies, where they are often combined with anti-PD-1 therapies. Early data have shown immune activation and durable responses in subsets of patients resistant to checkpoint inhibitors [19-21], However, early-phase trials of other TLR9 agonists, such as SD-101, tilsotolimod, and vidutolimod, have produced mixed results. While preclinical animal studies showed strong anti-tumor effects, human trials often had weaker or highly variable responses, particularly in monotherapy [22, 23], Combination of TLR9 agonists with checkpoint inhibitors, chemotherapy, or radiotherapy are currently appearing as more promising. Notably, local intratumoral delivery of TLR9, as in Phase 1 and 2 trials of cavrotolimod and tilsotolimod, seems to be more effective approach as it concentrates activation within the tumor microenvironment and reduces systemic inflammation [20, 24, 25],

[0383] The interpretation of above mixed results is further complicated by the fact that the function and expression of TLR9 in monocytes and macrophages in humans remain unclear. Some studies suggest that these cells express little or no functional TLR9, while others report context-dependent responses. This uncertainty raises doubts on how TLR9-targeting drugs truly engage the immune system across individuals and tissues. A more precise definition of TLR9 expression and function in the key myeloid populations is therefore essential for designing the next generation of effective TLR9-based therapeutics.

[0384] Experiments were conducted to measure the TLR9 expression in human monocyte-derived macrophages. Cells were differentiated in vitro, starting from CD14-enriched monocytes isolated from healthy donors. Differentiation of cells was carried out for 7 days, respectively, in GM-CSF (30ng / ml, final concentration) or M-CSF (100ng / ml, final concentration). After differentiation, the cell lysate was obtained, and TLR9 expression was measured by Western blot. The results are shown in the Figure 11 A which indicate that TLR9 is significantly expressed in human macrophages.

[0385] More importantly, it was observed that TRL9 stimulation in human macrophages is associated with a significant downregulation of the interferon response. Two experimental approaches were implemented to demonstrate this.

[0386] Experiment A

[0387] Human monocyte-derived macrophages were stimulated with IFN, both with and without CpG costimulation, and cytokine production was measured after 24 hours. The cytokines in the supernatant were quantified using Legendplex (LEGENDplex Multi-analyte Flow Assay Kit).

[0388] Results are reported in Figure 11B where it can be observed that TLR9 stimulation is associated with a significant downregulation of the IP10 production (product of the gene CXCL1O), an interferon-induced protein.

[0389] To gain further insights into this potential immunomodulatory effect of TLR9 in human macrophages, human monocyte-derived macrophages were stimulated with CpG for 6 hours and then RNA was extracted for sequencing. The RNA-seq analysis was performed using a workflow implemented in Nextflow. This pipeline automated quality control, alignment, and quantification of transcript expression data. Raw sequencing reads in FASTQ format were first evaluated for quality with FastQC. Adapter trimming and removal of low-quality bases were conducted as standard preprocessing steps. The cleaned reads were then aligned to the reference genome using STAR, and gene-level read counts were obtained with feature Counts. Finally, MultiQC was used to compile log files, and a comprehensive summary report was generated. The use of Nextflow ensured reproducibility, scalability, and smooth integration of containerized tools (Docker or Singularity) across different computational environments. As shown in Figure 12 and 13 TRL9 stimulation was associated with a significant downregulation of a considerable number of interferon-induced genes. Experiment B

[0390] Having demonstrated that ADA2 is a DNA editing enzyme converting dA-to-dl and it localizes in the lysosomes, further work was carried out to address the impact of ADA2 on TLR9 activation. Whether dl is recognized by TLR9 is currently unknown. However, dl in DNA is generally recognized by cellular proteins as deoxyinosine (dl)

[0026] , Thus, it was investigated whether dl residues in DNA molecules could be “recognized as” dG by TLR9. For this purpose, the human CpG2216 (A-class CpG) TLR9 Iigand26 were modified by replacing the dG residues known to be essential for TLR9 activation with dl (named CpI) or cytosine (CpC) (Figure 14A). These ODNs were tested in human-TLR9 HEK-Blue reporter cells. In these experiments, CpI showed similar activity to CpG, indicating that TLR9 can recognize dl (Figure 14B).

[0391] Analogous results were obtained for type I interferon (IFN) production in human peripheral blood mononuclear cells (PBMCs) (Figure 14C). Additional experiments were carried out using ODNs, where dA residues have been substituted by dl residues (Figure 15A), which confirmed that dA-to-dl editing of DNA plays a role in the potency of potential TLR9 ligands (Figure 15B).

[0392] HEK-human TLR9 reporter cells were transduced with lentiviruses encoding ADA2-GFP or GFP only. Lentiviruses were produced by transfecting HEK293 T cells with psPAX2, pMD2. G, and pLenti-ADA2-IRES-GFP-Puro or pLenti-ADA2-IRES-GFP-ADA2-Puro (Amount per 10 cm dish, respectively 1.3 pmol, 0.72 pmol, 1.64 pmol). Through these experiments, it was observed that ADA2 expression is associated with the significant upregulation of TLR9 signaling upon CpG2216 stimulation (Figure 16A).

[0393] The above finding was further confirmed by using additional HEK293T reporter cell lines with stable expression of TLR7 or TLR9 and UNC93B1, a molecular chaperone. These cells secreted interleukin (IL)-8 upon activation of the respective receptor and were stimulated with CpG2006 (B-class CpG), CpG2216, R848 (TLR7 / 8 small-molecule agonist), and dsDNA from E. coli. ADA2 significantly increases TLR9 activation upon stimulation with CpG2216 and E. coli DNA (Figure 16B), while no differences were observed upon stimulation with CpG2006 and R848 (Figure 16B) and no ADA2-mediated effect was also observed for the activation of hTLR7 (Figure 16C).

[0394] These results establish a basis for therapeutic strategies targeting ADA2 in macrophage lysosomes

[0027] , such as 1) enzyme replacement therapy (ERT) for ADA2-deficient patients and more broadly as 2) strategies to modulate TLR9 activation. This is, e.g., achieved through the production of recombinant ADA2 protein modified to reach the lysosomes, based on existing methods used for lysosomal storage diseases, and further optimized for targeting of human macrophages.

[0395] Example 6

[0396] TLR9 response in DADA2 patients

[0397] In an experiment, PBMCs were taken from DADA2 patients and healthy controls and tested. IFN-a was used as a readout forTLR9 activation in plasmacytoid dendritic cells (pDCs)

[0016] , while TNFa was used as a readout for CpG2006. The TLR7 / 8 stimulus 9.2s RNA

[0029] was included to control the activity of DCs in DADA2 patients since a reduction in their number could have also led to a reduced TLR9 response. A significant decrease is observed in the production of IFN-a in cells from DADA2 patients upon CpG2216 stimulation but not for TNF-a after CpG2006 stimulation (Figure 17A, 17B). These results were further confirmed in pDCs and conventional dendritic cells (cDCs) sorted (Figure 17C,17D) from DADA2 patients and healthy controls (Figure 17E).

[0398] CpG ODN is artificially modified by longer phosphorothioated backbones, which are generally not seen in naturally occurring DNA. To mimic natural TLR9 sensing, a longer (69bp) dsDNA fragment was additionally tested. Different immune populations were separated from PMBCs using fluorescence-activated cell sorting and then stimulated with the 69bp dsDNA using poly-L-arginine (PLA), an endolysosomal transfection reagent

[0028] , In this setting, dsDNA stimulation produces type-l IFN selectively in DCs (Figure 17F). It was found that cells from DADA2 patients are associated with impaired type-l IFN production upon lysosomal dsDNA stimulation (Figure 17G).

[0399] Example 7

[0400] Localization of ADA2 in lysosomes

[0401] ADA2 is expressed preferentially in monocytes (v23.proteinatlas.org / ADA2 / immune_Cells, and Figure 18A, 18B). To obtain data on ADA2 expression in macrophages, human tonsils were analyzed (proteinatlas-CD68-tonsil). It was found that ADA2 is almost exclusively expressed by tingible body macrophages (TBM) in the germinal center (Figure 18C, 18D, 18E), with few dendritic cells in the crypt epithelium and surface epithelium containing ADA2-positive intracytoplasmic granules. Interestingly, at higher magnification, the ADA2-derived signal resembled phagolysosomes (Figure 18E). Whether ADA2 localizes in lysosomes was assessed by immunofluorescence comprising co-staining ADA2 and LAMP1, a glycoprotein expressed in lysosomes and late endosomes. These experiments revealed that ADA2 is comprised in LAMP1 -positive lysosomes (Figure 19A, 19B, 19C). It was also confirmed in these tests that ADA2 is a lysosomal protein found in peripheral monocytes (Figure 19D, 19E, 19F), plasmacytoid and conventional dendritic cells (Figure 20).

[0402] To reach the lysosome, soluble proteins were tagged with mannose-6-phosphate (M6P)

[0029] which can be recognized by the M6P receptor. Except in rodents, ADA2 orthologs are present in all mammals. Furthermore, the protein is highly expressed in microglia. Thus, endogenous ADA2 from porcine brain was purified to access sufficient material for glycan analysis. It was found that porcine ADA2 (pA2) contains a significant amount of M6P-modified glycans linked to three of its glycosylation sites (N127, N174, N378) (Figure 21). Further it was observed that its glycan profile echoes that of known lysosomal proteins, such as lysosomal alpha-mannosidase (LAMAN), phospholipase D3 (PLD3), and ependymin-related protein 1 (EPDR1), and it was distinct from extracellular proteins such as oligodendrocyte myelin glycoprotein (OMgp) and signal regulatory protein alpha (SIRPa) (Figure 8K). These results suggested that ADA2 is a lysosomal protein targeted to endolysosomes via the M6P-dependent pathway. Example 8

[0403] ADA2 interactions with DNA and structural preferences

[0404] Interactions of ADA2 with DNA and its specific structural preferences have been evaluated using electrophoretic mobility shift assay (EMSA). This assay confirmed that ADA2 binds to different DNA substrates (Figures 22A, 22B; Table 2), including single-stranded (ssDNA) and complex / branched DNA molecules (Hairpin [HpTT], Pseudo-Y and 5’Flap-DNA). The strongest shifts were obtained with substrates containing regions of both dsDNA and ssDNA (Hairpin and Pseudo-Y). It was also observed that ADA2 shows a higher binding affinity for longer DNA substrates (Figure 22C; HpTT42 to HpTT20). Further, the effect of pH was evaluated and when it was increased from 5.5 to 7.5 there was an observed reduction in ADA2-DNA binding but not abolished completely (Figure 22D, 22E). These results suggested that the change in DNA conformation might be related to the ADA catalytic activity or catalytic site of ADA2.

[0405] Additionally, several ADA2 mutant proteins (Table 2) were evaluated. The mutant proteins with different ADA activity exhibited different DNA-binding affinities (Figure 22F). The experiments revealed a positive correlation between ADA2 deaminase activity on free Ado and the FRET signal (Figure 23A, 23D, 23E).

[0406] The FRET assay can assess the interaction between DNA and proteins. As shown in Figure 23B and 23C, hrADA2, but not ADA1, induced a significant FRET signal with optimal pH = 5, resembling a lysosomal environment.

[0407] 4. References

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Claims

1. Claims1. A fusion protein comprising (i) an adenosine deaminase 2 (ADA2) domain and (ii) a DNA binding domain, wherein the DNA binding domain is capable of binding to a target DNA sequence.

2. The fusion protein according to claim 1, wherein the ADA2 domain comprises ADA2 or a functional variant thereof, preferably human ADA2 or a functional variant thereof.

3. The fusion protein according to claim 1 or 2, wherein the ADA2 domain comprises two ADA2 monomers, optionally wherein at least one monomer is a functional variant of ADA2.

4. The fusion protein according to any one of claims 1 to 3, wherein the ADA2 domain comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 80% or at least about 95% identity thereto; preferably two amino acid sequences of SEQ ID NO: 1 or amino acid sequences having independently at least about 80% or at least about 95% identity thereto.

5. The fusion protein according to any one of claims 1 to 4, wherein the fusion protein further comprises a nuclear localization sequence (NLS).

6. The fusion protein according to any one of claims 1 to 5, wherein the DNA binding domain is an RNA-guided DNA binding domain, preferably wherein the RNA-guided DNA binding domain is capable of forming a complex with an RNA such as a guide RNA (gRNA).

7. The fusion protein according to any one of claims 1 to 6, wherein the fusion protein further comprises (iii) a DNA endonuclease domain, wherein the DNA endonuclease domain preferably produces a target-specific double strand break or a target-specific single-strand break at a deoxyadenosine.

8. The fusion protein according to claim 6 or 7, wherein the (i) DNA binding domain and (iii) the DNA endonuclease domain are comprised in a single polypeptide, preferably wherein the single polypeptide is selected from a Cas9 endonuclease or a functional variant thereof, including a Cas9 nickase; and a Cas12 endonuclease or a functional variant thereof, including a Cas12 nickase.

9. The fusion protein according to any one of claims 1 to 8, wherein (a) the ADA domain and the DNA binding domain and / or (b) the two ADA2 monomers or two amino acid sequences, respectively, are connected by a linker, preferably a flexible linker, more preferably a linker comprising glycine and / or serine residues (“GS” linker).

10. A nucleic acid encoding the fusion protein according to any one of claims 1 to 9.

11. A vector comprising the nucleic acid according to claim 10, wherein the vector is preferably an expression vector.

12. A ribonucleoprotein (RNP) complex comprising the fusion protein according to any one of claims 1 to 9 and at least one target-specific guide RNA (gRNA).

13. A particle comprising the fusion protein according to any one of claims 1 to 9, the nucleic acid according to claim 10, the vector according to claim 11 or the RNP complex according to claim 12.

14. A cell comprising the fusion protein according to any one of claims 1 to 9, the nucleic acid according to claim 10, the vector according to claim 11, the RNP complex according to claim 12, or the particle according to claim 13.

15. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 9, the nucleic acid according to claim 10, the vector according to claim 11, the RNP complex according to claim 12, or the particle according to claim 13 and at least one pharmaceutically suitable excipient.

16. Use of the fusion protein according to any one of claims 1 to 9, the nucleic acid according to claim 10, the vector according to claim 11, the RNP complex according to claim 12, the particle according to claim 13, or the pharmaceutical composition of claim 15 in gene editing, preferably wherein the gene editing comprises converting a dA to a dl.

17. A method of gene editing comprising the steps of18.i) providing a double-stranded DNA; and19.ii) contacting the double stranded DNA with the fusion protein according to any one of claims 1 to 9 and at least one target-specific gRNA, or the RNP complex according to claim 12.

18. A method of gene editing comprising administering the fusion protein according to any one of claims 1 to 9, the nucleic acid according to claim 10, the vector according to claim 11, the RNP complex according to claim 12, the particle according to claim 13, or the pharmaceutical composition of claim 15 to a subject in need thereof.

19. A kit comprising the fusion protein according to any one of claims 1 to 9, the nucleic acid according to claim 10, the vector according to claim 11, the RNP complex according to claim 12, the particle according to claim 13, or the pharmaceutical composition of claim 15 and instructions for use.

20. A pharmaceutical composition comprising adenosine deaminase 2 (ADA2) or a functional variant thereof and at least one pharmaceutically suitable excipient, wherein the pharmaceutical composition is targeted to the lysosome.

21. The pharmaceutical composition according to claim 20, wherein the targeting to the lysosome is achieved by a modification of the ADA2, preferably an altered glycosylation pattern of ADA2, more preferably wherein the ADA2 comprises more mannose 6-phosphate and / or mannose compared to wild-type ADA2; and / or wherein the ADA2 is fused to IGF-II or a fragment thereof or a GILT tag.

22. The pharmaceutical composition according to claim 20, wherein the composition comprises a particle comprising ADA2, wherein the particle is targeted to the lysosome, preferably wherein the particle comprises IGF-II or a fragment thereof or a GILT tag on the surface of the particle.

23. The pharmaceutical composition according to any one of claims 20 to 22 for use in the treatment of DAD2.

24. A pharmaceutical composition comprising adenosine deaminase 2 (ADA2) or a functional variant thereof and at least one pharmaceutically suitable excipient, wherein the pharmaceutical composition is targeted to a macrophage.

25. The pharmaceutical composition according to claim 24, wherein the composition comprises a particle comprising ADA2 or a functional variant thereof, and the particle comprises27.(i) a material that passively targets macrophages, preferably selected from the group consisting of PLGA, chitosan, iron oxide, gold and silica,28.(ii) a ligand on the surface of the particle that is specific for a binding partner or receptor expressed by macrophages, preferably selected from the group consisting of oligosaccharide, preferably mannose; folate, UNO peptide, M2pep, CXCR4 ligand and RGD peptides;29.(iii) an antibody or antigen-binding fragment thereof specific for a macrophage surface marker on the surface of the particle, preferably wherein the macrophage surface marker is selected from the group consisting of CD206, CD163, CCL2, and CXCR4; and / or30.(iv) pathogen-mimicking molecular patterns on the surface of the particle that are recognized by macrophages and induce phagocytosis;31.or the particle has a natural tropism for macrophages, preferably wherein the particle is an exosome or a synthetic vesicle.

26. The pharmaceutical composition according to claim 24 or 25 for use in the treatment of a TLR9- linked disease.

27. The pharmaceutical composition for use according to claim 26, wherein the disease is selected from the group consisting of (i) cancer, in particular melanoma, breast cancer and lung cancer including NSCLC; (ii) an autoimmune and / or inflammatory disease, systemic lupus erythematosus (SLE), autoimmune thyroiditis, atherosclerosis, multiple sclerosis, inflammatory bowel disease and Crohn’s; (iii) a viral or bacterial infection, in particular an infection caused by HBV, HCV, HPV, EBV, HIV, Mycobacterium tuberculosis, Streptococcus penumoniae and Candida albicans; and (iv) a neurodegenerative disease.

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