The crl5spsb3 ubiquitin ligase targets nuclear cgas for degradation

Abstract

The present invention relates to a variant of cyclic GMP-AMP synthase (cGAS) carrying an amino acid substitution at one or more of (i) asparagine at amino acid position 513, (ii) asparagine at amino acid position 514, (iii) aspartic acid at amino acid position 465, (iv) glutamic acid at amino acid position 509, (v) tyrosine at amino acid position 510, (vi) arginine at amino acid position 512, (vii) lysine at amino acid position 427, and (viii) lysine at amino acid position 428. The present invention also relates to a variant of SplA / Ryanodine Receptor Domain And SOCS Box Containing 3 (SPSB3) carrying an amino acid substitution at one or more of (i) arginine at amino acid position 213, (ii) tyrosine at amino acid position 197, (iii) threonine at amino acid position 162, (iv) threonine at amino acid position 259, (v) arginine at amino acid position 262, (vi) serine at amino acid position 132, (vii) tyrosine at amino acid position 131, and (viii) tyrosine at amino acid position 160.

Description

[0001] The CRL5SPSB3ubiquitin ligase targets nuclear cGAS for degradation

[0002] The present invention relates to a variant of cyclic GMP-AMP synthase (cGAS) carrying an amino acid substitution at one or more of (i) asparagine at amino acid position 513, (ii) asparagine at amino acid position 514, (iii) aspartic acid at amino acid position 465, (iv) glutamic acid at amino acid position 509, (v) tyrosine at amino acid position 510, (vi) arginine at amino acid position 512, (vii) lysine at amino acid position 427, and (viii) lysine at amino acid position 428. The present invention also relates to a variant of SplA / Ryanodine Receptor Domain And SOCS Box Containing 3 (SPSB3) carrying an amino acid substitution at one or more of (i) arginine at amino acid position 213, (ii) tyrosine at amino acid position 197, (iii) threonine at amino acid position 162, (iv) threonine at amino acid position 259, (v) arginine at amino acid position 262, (vi) serine at amino acid position 132, (vii) tyrosine at amino acid position 131 , and (viii) tyrosine at amino acid position 160.

[0003] In this specification, a number of documents including patent applications and manufacturer’s manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0004] Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, tumor (such as cancer), and inflammatory disease and initiates potent innate immune responses via synthesis of 2’3’-cyclic GMP- AMP (cGAMP)1-7. The indiscriminate activity of cGAS towards DNA - a fundamental element of life - demands tight regulatory mechanisms necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA8-15. However, the fate of nuclear cGAS and its role in cell physiology remains unknown. Here it is shown that the ubiquitin-proteasomal system (UPS) degrades nuclear cGAS limiting activation of innate immunity. SPRY domain-containing SOCS box protein 3 (SPSB3) was identified as the cGAS-targeting substrate receptor that associates with the cullin-RING E3 ligase (CRL) 5 complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy (EM) structure of nucleosome-bound cGAS in complex with SPSB3 uncovers a highly conserved NN minimal degron motif in the C terminus of cGAS directing SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon (IFN) signaling and confers heightened activity against DNA virus infection. Our work uncovers titration of nuclear cGAS as a critical determinant in balancing cell intrinsic immunity and provides structural insight into a novel regulatory element on cGAS amenable to therapeutic exploitation. In mitosis, when the nuclear envelope disassembles, cGAS is rapidly recruited onto chromosomes and, through this mechanism, relocated into the nuclear interior16 17. Key early findings reported that the nuclear pool of cGAS is largely immobile and inactive9. This state is achieved through the tight tethering of cGAS to the acidic patch at the nucleosome surface masking essential elements required for DNA binding and enzyme activation8 10-14. Suppression of intranuclear cGAS is further aided by the chromatin architectural protein BAF, which shields dsDNA from cGAS binding15. These insights elucidated principles of cGAS regulation inside the nuclear space. However, how cells coordinate the presence of cGAS on chromatin with general housekeeping genomic processes is unknown.

[0005] The present invention therefore aims at revealing presence of cGAS on chromatin is coordinated in the cellular context and in particular at providing means and methods for controlling the presence of cGAS in the cellular context.

[0006] Accordingly, the present invention relates in a first aspect to a variant of cyclic GMP-AMP synthase (cGAS) carrying an amino acid substitution at one or more of (i) asparagine at amino acid position 513, (ii) asparagine at amino acid position 514, (iii) aspartic acid at amino acid position 465, (iv) glutamic acid at amino acid position 509, (v) tyrosine at amino acid position 510, (vi) arginine at amino acid position 512, (vii) lysine at amino acid position 427, and (viii) lysine at amino acid position 428.

[0007] Cyclic GMP-AMP synthase (cGAS, cGAMP synthase) belongs to the nucleotidyltransferase family. This enzyme is a cytosolic DNA sensor that activates a type-l interferon response. It is part of the cGAS- STING DNA sensing pathway. It binds to microbial DNA as well as self-DNA, and catalyzes cGAMP synthesis. cGAMP then functions as a second messenger that binds to and activates the endoplasmic reticulum protein STING to trigger type-l IFNs production.

[0008] The cyclic GMP-AMP synthase is preferably human cyclic GMP-AMP synthase and the human cyclic GMP-AMP has preferably the amino acid sequence of SEQ ID NO: 33 or is encoded by the nucleotide sequence of SEQ ID NO: 34.

[0009] It is to be understood that the variant of cyclic GMP-AMP synthase maintains or essentially maintains the activity of the cyclic GMP-AMP synthase. The activity of the cyclic GMP-AMP is the production of cGAMP (cyclic guanosine monophosphate-adenosine monophosphate which in turn activates STING, which induces the interferon response, and production of inflammatory cytokines).

[0010] As can be taken from the appended examples, it was unexpectedly revealed herein for the first time that cGAS forms a complex with SPSB3 via a highly conserved NN (2x asparagine) minimal degron motif in the C terminus of cGAS directing the SPSB3 recruitment, ubiquitylation and cGAS protein stability. Hence, under normal cellular conditions cGAS and SPSB3 can form a complex, cGAS is ubiquitylated by CRL5(Cullin-RING Ligase 5)SPSB3as part of an E3 ligase complex, and cGAS becomes degraded by a proteasome. In more detail, it was found that the cGAS residues N513 and N514 form hydrogen bond to SPSB3 loop residues S132, T162, T259, and R262. Additionally, a set of acidic residues on cGAS, including D465, E509, Y510, R512 and E515 bridges to residues Y131 , Y160, Y197, and R213 on SPSB3 through electrostatic interactions reinforcing complex formation. It is of note that if E515 is mutated to A no change of the binding affinity of the SPSB3-cGAS complex was found so that position 515 is not part of the first aspect of the invention. The amino acid and nucleotide sequence of the cGAS variants E515A are SEQ ID NOs 39 and 40. It is furthermore shown in the appended examples that the substitution of the K427 and K428 in cGAS largely abolished cGAS ubiquitylation and nuclear degradation in cells, corroborating their critical role in dictating cGAS protein stability (Fig. 2h, i). K427 and K428 can be found in the catalytic core of cGAS. Hence, it was surprisingly found that amino acid substitution(s) at one or more of (i) asparagine at amino acid position 513, (ii) asparagine at amino acid position 514, (iii) aspartic acid at amino acid position 465, (iv) glutamic acid at amino acid position 509, (v) tyrosine at amino acid position 510, (vi) arginine at amino acid position 512, (vii) lysine at amino acid position 427, and (viii) lysine at amino acid position 428 of cGAS will either interfere with the complex formation of cGAS with SPSB3 (items (i) to (v)) and / or interfere with the ubiquitylation of cGAS. The technical effect of both is that the half-life of nuclear cGAS can be manipulated in a desired fashion. In particular, the nuclear half-life of cGAS can be increased.

[0011] The variant of cGAS of the first aspect advantageously allows (i) the design of cGAS-based genetic adjuvants with altered (in particular increased) protein stability, (ii) to the design cGAS variants with augmented immune-stimulatory capacity, (iii) influencing (in particular decreasing) the CGAS-SPSB3 interaction for targeted cGAS, and (iv) for the opportunity of disrupting CGAS-SPSB3 interaction for enhanced intrinsic GAS activity (which is useful, for example, in a “cGAS-based immuno-therapy”).

[0012] In accordance with a preferred embodiment of the first aspect the variant has increased stability against proteasomal degradation and / or has increased half-life in cells.

[0013] Hence, it is preferred herein that the variant of cGAS of the first aspect is a variant of cGAS being functionally characterized by an increased half-life in cells (in particular increased nuclear half-life), preferably as compared to the wild-type cGAS, preferably the wild-type human cGAS as defined herein above.

[0014] Alternatively or in addition, it is preferred herein that the variant of cGAS of the first aspect is a variant of cGAS that is functionally characterized by increased stability against proteasomal degradation, preferably as compared to the wild-type cGAS, preferably the wild-type human cGAS as defined herein above.

[0015] The above discussed increases are each independently with increasing preference an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, and at least 70%, at least 80%, at least 90% and at least 100%. Means for measuring increased stability against proteasomal degradation and / or increased half-life are known in the art, e.g. the measurement of intact cGAS protein level over time. In accordance with another preferred embodiment of the first aspect the asparagine at amino acid position 513 and / or the asparagine at amino acid position 514 is / are substituted.

[0016] Thus, among the above options (i) to (viii) options (i) and / or (ii) are preferred since N513 and N514 form the primary contact to SPSB3. In addition or alternatively, options (vii) and / or (viii) are preferred since they largely prevent ubiquitylation.

[0017] In accordance with a further preferred embodiment of the first aspect the variant of cGAS (I) comprises or consist of (a) an amino acid sequence selected from any one of SEQ ID NOs 1 to 8, or (b) an amino acid sequence sharing at least 80%, preferably at least 90% identity with SEQ ID NOs 1 to 8, provided that one or more of (i) asparagine at amino acid position 513, (ii) asparagine at amino acid position 514, (iii) aspartic acid at amino acid position 465, (iv) glutamic acid at amino acid position 509, (v) tyrosine at amino acid position 510, (vi) arginine at amino acid position 512, (vii) lysine at amino acid position 427, and (viii) lysine at amino acid position 428 is / are substituted by another amino acid; (c) the amino acid sequence of (a) or (b) further comprising a nuclear localization sequence (NLS), or (d) the amino acid sequence of any one of (a) to (c), wherein a part of or the complete IDR is deleted; or (II) is encoded by a nucleic acid molecule comprising of consisting of (a) a nucleotide sequence selected from any one of SEQ ID NOs 9 to 16, or (b) a nucleotide sequence sharing at least 80%, preferably at least 90% identity with any one of SEQ ID NOs 9 to 16, provided that the base triplet(s) encoding (i) asparagine at amino acid position 513, (ii) asparagine at amino acid position 514, (iii) aspartic acid at amino acid position 465, (iv) glutamic acid at amino acid position 509, (v) tyrosine at amino acid position 510, (vi) arginine at amino acid position 512, (vii) lysine at amino acid position 427, and (viii) lysine at amino acid position 428 is / are substituted by (a) base triplet(s) encoding another amino acid; (c) the nucleotide sequence or (a) or (b) further encoding a nuclear localization sequence (NLS), or (d) the nucleotide sequence of any one of (a) to (c), wherein a part of orthe complete nucleotide sequence encoding the IDR is deleted.

[0018] SEQ ID NOs 1 to 8 are the amino acid sequences of the human cGAS variants N513A, N514A, D465A, E509A, Y510A, R512A, K427R, and K428R and SEQ ID NOs 9 to 16 are the nucleotide sequences encoding the human cGAS variants N513A, N514A, D465A, E509A, Y510A, R512A, K427A, and K428A.

[0019] The sequence sharing at least 80% identity is with increasing preference at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical.

[0020] In accordance with the present invention, the term “percent (%) sequence identity” describes the number of matches (“hits”) of identical nucleotides / amino acids of two or more aligned nucleic acid or amino acid sequences as compared to the number of nucleotides or amino acid residues making up the overall length of the template nucleic acid or amino acid sequences. In other terms, using an alignment, for two or more sequences or subsequences the percentage of amino acid residues or nucleotides that are the same (e.g. 70%, 75% 80%, 85%, 90% or 95% identity) may be determined, when the (sub)sequences are compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. This definition also applies to the complement of any sequence to be aligned.

[0021] Nucleotide and amino acid sequence analysis and alignment in connection with the present invention are preferably carried out using the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), Nucleic Acids Res. 25:3389-3402). BLAST can be used for nucleotide sequences (nucleotide BLAST) and amino acid sequences (protein BLAST). The skilled person is aware of additional suitable programs to align nucleic acid sequences. The NCBI BLAST algorithm is available for protein (Protein BLAST) and nucleotides (Nucleotide BLAST). For Protein BLAST the algorithm parameters are preferably: max target sequences: 100, with automatically adjust parameters for short input sequences, expect threshold 0.05, word size 6, Max matches in a query range 0, matrix BLOSUM62, cap cost existence: 10 extension: 1 , and compositional adjustment. For Nucleotide BLAST the algorithm parameters are preferably: max target sequences: 100, with automatically adjust parameters for short input sequences, Expect threshold 0.05, word size 28, Max matches in a query range 0, match / mismatch scores 1 ,-2, cap costs linear, low complexity regions filter, and ask for look up table only. These are the standard algorithm parameters for protein BLAST and Nucleotide BLAST and they can adjusted, if needed.

[0022] With respect to item (c) it is noted that a variant cGAS with a nuclear localization sequence (NLS) facilitates the transport of the variant into the cell nucleus.

[0023] The nuclear localization sequence (NLS) is an amino acid sequence that 'tags' a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface.

[0024] NLS can be classified as either monopartite or bipartite. The major structural differences between the two are that the two basic amino acid clusters in bipartite NLSs that are separated by a relatively short spacer sequence (hence bipartite - 2 parts), while monopartite NLSs are not separated. The first NLS to be discovered is the sequence PKKKRKV (SEQ ID NO: 35) in the SV40 Large T-antigen (a monopartite NLS). The SV40 Large T-antigen was also used in the appended examples as NLS. The NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 36), is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids. Both signals are recognized by importin a. Importin a contains a bipartite NLS itself, which is specifically recognized by importin p. The latter can be considered the actual import mediator.

[0025] With respect to item (d) it is noted that a variant cGAS may not comprise the intrinsically disordered region (IDR) region that can be found at the N-terminus of cGAS since this region is not necessary for the activity of cGAS, in particular for catalyzing cGAMP synthesis.

[0026] The IDR region can be found at the N-terminus of the human full-length human cGAS and spans with increasing preference the amino acid positions corresponding to amino acid positions 1-157, amino acid positions 1-155 and amino acid positions 1-146 of the full-length human cGAS. As discussed above, the amino acid sequence of the full length wild-type human cGAS is shown in SEQ ID NO: 33 and this amino acid sequence is encoded by SEQ ID NO: 34.

[0027] In accordance with a yet further preferred embodiment of the invention, the nucleic acid molecule encoding said variant cGAS is RNA or DNA.

[0028] The RNA is preferably mRNA. The mRNA may be introduced into a cell, wherein the host cell is preferably an anti-tumor lymphocyte. Means and methods for introducing the mRNA into the genome of a host cell will be discussed herein below, in particular in connection with lipid nanoparticles.

[0029] The DNA is preferably genomic DNA or cDNA comprised in an expression vector. The genomic DNA and expression vector will be further described herein below.

[0030] The nucleic acid molecule encoding said variant cGAS may preferably and generally be formulated as vesicles, such as liposomes or exosomes. Liposomes have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. Liposomal cell-type delivery systems have been used to effectively deliver nucleic acids, such as siRNA in vivo into cells (Zimmermann et al. (2006) Nature, 441 :111-114). Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are phagocytosed by macrophages and other cells in vivo. Exosomes are lipid packages which can carry a variety of different molecules including RNA (Alexander et al. (2015), Nat Commun; 6:7321). The exosomes including the molecules comprised therein can be taken up by recipient cells. Hence, exosomes are important mediators of intercellular communication and regulators of the cellular niche. Exosomes are useful for diagnostic and therapeutic purposes, since they can be used as delivery vehicles, e.g. also for contrast agents or drugs.

[0031] In accordance with a preferred embodiment of the first aspect the one or more amino acids are each individually substituted by a non-conservative amino acid, preferably by an amino acid with a hydrophobic side chain, more preferably by alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan, even more preferably by alanine, valine, isoleucine or leucine, and most preferably by alanine.

[0032] A non-conservative amino acid substitution is preferably the substitution of the respective amino acid with another naturally occurring amino acid that belongs to a different class of amino acids than the amino acid to be substituted. The different class of amino acids are amino acids with a hydrophobic sides, polar / neutral amino acid and acidic amino acids, basic amino acids. The amino acid with a hydrophobic side chain is preferably selected from alanine, valine, methionine, leucine, isoleucine, proline, tryptophan and phenylalanine. The polar / neutral amino acid is preferably selected from tyrosine, threonine, glutamine, glycine, serine and asparagine. The acidic amino acid is preferably selected from glutamic acid and aspartic acid. The basic amino acid is preferably selected from lysine, arginine and histidine.

[0033] It also of note that in amino acid positions 427 and 428 K (lysine) was replaced by R (arginine). Lysine and arginine are both basic amino acids. Hence, for these positions in addition or as alternative to alanine also arginine is most preferred.

[0034] In accordance with a preferred embodiment of the first aspect the variant of cGAS furthermore carries an amino acid substitution of one or both arginines at amino acid positions 255 and 236 and / or an amino acid substitution of one or both lysines at amino acid positions 254 and 258.

[0035] Variants of cGAS carrying an amino acid substitution of one or both arginines at amino acid positions 255 and 236 and / or an amino acid substitution of one or both lysines at amino acid positions 254 and 258 are the subject of the international application PCT / EP2023 / 054168.

[0036] Human variant cyclic GMP-AMP synthase (cGAS) carrying an amino acid substitution of one or both arginines at amino acid positions 255 and 236 and / or one or both lysines at amino acid positions 254 and 258 are untethered and constitutively active.

[0037] Hence, the variant of cGAS of this preferred embodiment is preferably functionally characterized as being untethered and constitutively active as well as displaying increased stability against proteasomal degradation and / or increased half-life in cells, preferably as compared to the wild-type cGAS, preferably the wild-type human cGAS as defined herein above.

[0038] In accordance with an even more preferred embodiment of the first aspect the one or both arginines at amino acid positions 255 and 236 and / or the one or both lysines at amino acid positions 254 and 258 is / are substituted by a non-conservative amino acid, preferably by an amino acid with a hydrophobic side chain and most preferably by alanine or glutamic acid.

[0039] The meaning of a non-conservative amino acid and amino acid with a hydrophobic side chain have been described herein above and these meanings also apply to this even more preferred embodiment. The replacement of the arginines and lysines by alanine or glutamic acid is illustrated by the examples of the international application PCT / EP2023 / 054168.

[0040] The present invention relates in a second aspect to a variant of SplA / Ryanodine Receptor Domain And SOCS Box Containing 3 (SPSB3) carrying an amino acid substitution at one or more of (i) arginine at amino acid position 213, (ii) tyrosine at amino acid position 197, (iii) threonine at amino acid position 162, (iv) threonine at amino acid position 259, (v) arginine at amino acid position 262, (vi) serine at amino acid position 132, (vii) tyrosine at amino acid position 131 , and (viii) tyrosine at amino acid position 160.

[0041] The definitions and preferred embodiments as descried herein above in connection with first aspect apply mutatis mutandis to the second aspect as being amenable for combination with the second aspect.

[0042] The variant of SplA / Ryanodine Receptor Domain And SOCS Box Containing 3 (SPSB3) was predicted in the art to be involved in proteasome-mediated ubiquitin-dependent protein catabolic process, predicted to be located both in the cytosol and nucleus and predicted to be art of the Cullin-RING 5 (CRL4) E3 ligase complex. The appended examples herein below show for the first time that SPSB3 forms a complexwith cGAS and is involved in the ubiquitinylation and proteasomal degradation of cGAS. It is emphasized that none of the other SPSB family members was found to control nuclear cGAS protein stability.

[0043] The SPSB3 is preferably human SPSB3 synthase and the human SPSB3 has preferably the amino acid sequence of SEQ ID NO: 37 or is encoded by the nucleotide sequence of SEQ ID NO: 38.

[0044] As discussed herein above, SPSB3 residues S132, T162, T259, and R262 and in addition Y131 , Y160, Y197, and R213 on SPSB3 are involved in the complex formation with cGAS.

[0045] The technical effect of replacing one or more of residues R213, Y197, T259, R262, S132, Y131 and Y160 in SPSB3 is therefore that the complex formation with cGAS can be regulated, in particular that the complex formation with cGAS can be inhibited. As explained above, the inhibition of the complex formation in turn results in a nuclear half-life increase of cGAS.

[0046] Among options (i) to (viii) one or more of options (iii) and (vi) are preferred since they form the main contact with the above-discussed residues N513 and N514 of cGAS,

[0047] In accordance with a preferred embodiment of the second aspect the variant displays reduced binding to cGAS as compared to SPSB3.

[0048] Hence, it is preferred herein that the variant of SPSB3 of the second aspect is a variant of SPSB3 that is functionally characterized by reduced binding to cGAS as compared to SPSB3, whereby the cGAS is preferably wild-type cGAS, more preferably the wild-type human cGAS as defined herein above.

[0049] The reduced reduced binding to cGAS as compared to SPSB3 is with creasing preference a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, and at least 70%, at least 80%, at least 90% and most preferably no longer (detectable) binding between cGAS and SPSB3.

[0050] Means for measuring decreased stability reduced binding to cGAS are known in the art, e.g. the measurement of the binding strength between variant of SPSB3 and cGAS, whereby the cGAS is preferably wild-type cGAS, more preferably the wild-type human cGAS as defined herein above.

[0051] In accordance with another preferred embodiment of the second aspect the variant of SPSB3 (I) comprises or consist of (a) an amino acid sequence selected from any one of SEQ ID NOs 17 to 24, or (b) an amino acid sequence sharing at least 80%, preferably at least 90% identity with SEQ ID NOs 17 to 24, provided that one or more of (i) arginine at amino acid position 213, (ii) tyrosine at amino acid position 197, (iii) threonine at amino acid position 162, (iv) threonine at amino acid position 259, (v) arginine at amino acid position 262, (vi) serine at amino acid position 132, (vii) tyrosine at amino acid position 131 , and (viii) tyrosine at amino acid position 160; or (c) the amino acid sequence of (a) or (b) further comprising a nuclear localization sequence (NLS); or (II) is encoded by a nucleic acid molecule comprising of consisting of (a) a nucleotide sequence selected from any one of SEQ ID NOs 25 to 32, or (b) a nucleotide sequence sharing at least 80%, preferably at least 90% identity with any one of SEQ ID NO: 25 to 32, provided that the base triplet(s) encoding (i) arginine at amino acid position 213, (ii) tyrosine at amino acid position 197, (iii) threonine at amino acid position 162, (iv) threonine at amino acid position 259, (v) arginine at amino acid position 262, (vi) serine at amino acid position 132, (vii) tyrosine at amino acid position 131 , and (viii) tyrosine at amino acid position 160 is / are substituted by (a) base triplet(s) encoding another amino acid; or (c) the nucleotide sequence or (a) or (b) further encoding a nuclear localization sequence (NLS).

[0052] SEQ ID NOs 17 to 24 are the amino acid sequences of the human SPSB3 variants R213A, Y197A, T259A, R262A, S132A, Y131A and Y160A, and SEQ ID NOs 25 to 32 are the nucleotide sequences encoding the human SPSB3 variants R213A, Y197A, T259A, R262A, S132A, Y131A and Y160A.

[0053] The sequence sharing at least 80% identity is with increasing preference at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical.

[0054] With respect to item (c) it is noted that a variant cGAS with a nuclear localization sequence (NLS) facilitates the transport of the variant into the cell nucleus.

[0055] Further details on the identification of the sequence identity and the NLS are provided herein above and also apply to this preferred embodiment.

[0056] In accordance with another preferred embodiment of the second aspect the one or more amino acids are each individually substituted by a non-conservative amino acid, preferably by an amino acid with a hydrophobic side chain, more preferably by alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan, even more preferably by alanine, valine, isoleucine or leucine, and most preferably by alanine.

[0057] The meaning of a non-conservative amino acid and amino acid with a hydrophobic side chain has been detail herein above and also applies to this preferred embodiment. The present invention relates in a third aspect to a nucleic acid molecule encoding the variant of cGAS of the first aspect or the variant of SPSB3 of the second aspect.

[0058] The definitions and preferred embodiments as descried herein above in connection with first and second aspect apply mutatis mutandis to the third aspect as being amenable for combination with the third aspect.

[0059] The term “nucleic acid molecule” in accordance with the present invention includes DNA, such as cDNA or double or single stranded genomic DNA and RNA. In this regard, "DNA" (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together on a deoxyribose sugar backbone. DNA can have one strand of nucleotide bases, or two complimentary strands which may form a double helix structure. "RNA" (ribonucleic acid) which is another embodiment of the nucleic acid molecule means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and uracil (U), called nucleotide bases, that are linked together on a ribose sugar backbone. RNA typically has one strand of nucleotide bases, such as mRNA. Included are also single- and doublestranded hybrids molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA. The nucleic acid molecule may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analogue, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acid molecules, in the following also referred as polynucleotides, may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Further included are nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers. Such nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2’-0-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA) (see Braasch and Corey, Chem Biol 2001 , 8: 1). LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2’-oxygen and the 4’-carbon. Also included are nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil. A nucleic acid molecule typically carries genetic information, including the information used by cellular machinery to make proteins and / or polypeptides. The nucleic acid molecule of the invention may additionally comprise promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like. In accordance with a preferred embodiment of the third aspect the nucleic acid molecule encoding said variant cGAS or the variant of SPSB3 is RNA or DNA.

[0060] Hence it is preferred that the nucleic acid molecule of the invention is DNA or RNA. The DNA is preferably genomic DNA and the RNA is preferably mRNA.

[0061] The genomic DNA can be added into the genome of a cell, preferably an anti-tumor lymphocyte. In the case of mRNA, the nucleic acid molecule may in addition comprise a poly-A tail. The mRNA may be introduced into a cell, wherein the host cell is preferably an anti-tumor lymphocyte. Means and methods for introducing the mRNA into the genome of a host cell will be discussed herein below, in particular in connection with lipid nanoparticles.

[0062] In accordance with a preferred embodiment of the third aspect the nucleic acid molecule encoding the variant cGAS or the variant of SPSB3 is comprised in lipid nanoparticles (LNP).

[0063] Lipid nanoparticles have been developed as vehicles for small molecule delivery and are now a key component of COVID-19 mRNA vaccines. Lipid nanoparticles are particularly useful as mRNA carriers.

[0064] Lipid nanoparticles are spherical vesicles made of ionizable lipids, which are positively charged at low pH (enabling RNA complexation) and neutral at physiological pH (reducing potential toxic effects, as compared with positively charged lipids, such as liposomes). Owing to their size and properties, lipid nanoparticles are taken up by cells via endocytosis, and the ionizability of the lipids at low pH (likely) enables endosomal escape, which allows release of the cargo into the cytoplasm. In addition, lipid nanoparticles usually contain a helper lipid to promote cell binding, cholesterol to fill the gaps between the lipids, and a polyethylene glycol (PEG) to reduce opsonization by serum proteins and reticuloendothelial clearance. The relative amounts of ionizable lipid, helper lipid, cholesterol and PEG substantially affect the efficacy of lipid nanoparticles, and need to be optimized for a given application and administration route. Moreover, lipid type, size and surface charge impact the behaviour of lipid nanoparticles in vivo.

[0065] Hence, in the case of contacting the LNPs with cells, the LNPs are taken up by the cells via endocytosis and the nucleic acid molecules as comprised in the LNPs are released into the cytoplasm of the cells.

[0066] The LNPs are preferably antibody-functionalized LNPs that can direct LNPs to specific cell types, including T cells for in vivo CAR T cell generation; see, for example, the review Marques et al. (2023), Pharmaceutics; 15(1):216. Here, the antibodies generally bind to a cell surface marker of the cell type of interest.

[0067] The present invention relates in a fourth aspect to a vector, preferably an expression vector comprising a nucleic acid molecule encoding the variant of cGAS of the first aspect or encoding the variant of SPSB3 of the second aspect. The definitions and preferred embodiments as descried herein above in connection with the other aspects apply mutatis mutandis to the fourth aspect as being amenable for combination with the fourth aspect.

[0068] The term “expression vector” in accordance with the invention means preferably a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering which carries the nucleic acid molecule of the invention in an expressible form. The expressible form is preferably a form, wherein the expression can be controlled, such as a Tet-on or Tet-off system. Here, the expression can be turned on and off by tetracycline or doxycycline. A Tet-on system and the use of doxycycline are illustrated in the examples.

[0069] The nucleic acid molecule of the invention may, for example, be inserted into several commercially available expression vectors. Non-limiting examples include lentiviral expression vector and prokaryotic plasmid vectors, such as of the pUC-series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with an expression in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMCI neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1 , pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, plZD35, pLXlN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen) and pCINeo (Promega). Examples for plasmid vectors suitable for Pichia pastoris comprise e.g. the plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen). The vector can also be a viral vector, such as an adeno-associated vector (AAV).

[0070] The nucleic acid molecule inserted into the vector can e.g. be synthesized by standard methods, or isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and / or to other amino acid encoding sequences can also be carried out using established methods. Transcriptional regulatory elements (parts of an expression cassette) ensuring expression in prokaryotes or eukaryotic cells are well known to those skilled in the art. These elements comprise regulatory sequences ensuring the initiation of transcription (e. g., translation initiation codon, promoters, such as naturally-associated or heterologous promoters and / or insulators; see above), internal ribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476) and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Preferably, the polynucleotide encoding the polypeptide / protein or fusion protein of the invention is operatively linked to such expression control sequences allowing expression in prokaryotes or eukaryotic cells. The vector may further comprise nucleic acid sequences encoding secretion signals as further regulatory elements. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used, leader sequences capable of directing the expressed polypeptide to a cellular compartment may be added to the coding sequence of the polynucleotide of the invention. Such leader sequences are well known in the art.

[0071] Furthermore, it is preferred that the vector comprises a selectable marker. Examples of selectable markers include genes encoding resistance to neomycin, ampicillin, hygromycine, and kanamycin. Specifically-designed vectors allow the shuttling of DNA between different hosts, such as bacteria-fungal cells or bacteria-animal cells (e. g. the Gateway system available at Invitrogen). An expression vector according to this invention is capable of directing the replication, and the expression, of the polynucleotide and encoded peptide or fusion protein of this invention. Apart from introduction via vectors such as phage vectors or viral vectors (e.g. adenoviral, retroviral), the nucleic acid molecules as described herein above may be designed for direct introduction or for introduction via liposomes into a cell. Additionally, baculoviral systems or systems based on vaccinia virus or Semliki Forest virus can be used as eukaryotic expression systems for the nucleic acid molecules of the invention.

[0072] The present invention relates in a fifth aspect to a host cell, preferably an anti-tumor lymphocyte comprising the nucleic acid molecule of the third aspect or the vector of the fourth aspect.

[0073] The definitions and preferred embodiments as descried herein above in connection with the other aspects apply mutatis mutandis to the fifth aspect as being amenable for combination with the fifth aspect.

[0074] The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, so that it carries the nucleic acid molecule of the third aspect or the vector of the fourth aspect of the invention.

[0075] The host cell of the invention is typically produced by introducing the nucleic acid molecule or vector(s) of the invention into the host cell which upon its / their presence mediates the expression of the nucleic acid molecule of the invention encoding the variant of the invention. The host from which the host cell is derived or isolated may be any prokaryote or eukaryotic cell or organism, preferably with the exception of human embryonic stem cells that have been derived directly by destruction of a human embryo.

[0076] Suitable prokaryotes (bacteria) useful as hosts for the invention are, for example, those generally used for cloning and / or expression like E. coli (e.g., E coli strains BL21 , HB101 , DH5a, XL1 Blue, Y1090 and JM101), Salmonella typhimurium, Serratia marcescens, Burkholderia glumae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas stutzeri, Streptomyces lividans, Lactococcus lactis, Mycobacterium smegmatis, Streptomyces coelicolor or Bacillus subtilis. Appropriate culture mediums and conditions for the above-described host cells are well known in the art.

[0077] A suitable eukaryotic host cell may be a vertebrate cell, an insect cell, a fungal / yeast cell, a nematode cell or a plant cell. The fungal / yeast cell may a Saccharomyces cerevisiae cell, Pichia pastoris cell or an Aspergillus cell. Preferred examples for host cell to be genetically engineered with the nucleic acid molecule or the vectors) of the invention is a cell of yeast, E. coli and / or a species of the genus Bacillus (e.g., B. subtilis). In one preferred embodiment the host cell is a yeast cell (e.g. S. cerevisiae).

[0078] In a different preferred embodiment the host cell is a mammalian host cell, such as a Chinese Hamster Ovary (CHO) cell, mouse myeloma lymphoblastoid, human embryonic kidney cell (HEK-293), human embryonic retinal cell (Crucell's Per.C6), or human amniocyte cell (Glycotope and CEVEC). The cells are frequently used in the art to produce recombinant proteins. CHO cells are the most commonly used mammalian host cells for industrial production of recombinant protein therapeutics for humans.

[0079] The host cell is preferably a therapeutic cell, A therapeutic is preferably any type of cell being useful for a tumor therapy, in particular a tumor immune cell therapies, such as anti-tumor dendritic cell, anti-tumor macrophage or an anti-tumor lymphocyte. An anti-tumor lymphocyte (or an anti-tumor effector lymphocyte) is a lymphocyte capable of eliciting a cytolytic response that can cause tumor cell death. These lymphocytes are specializing in and equipped for tumor cell elimination. The first category encompasses clonally expanded T lymphocytes expressing a unique T cell receptor (TCR) and recognizing tumor epitopes in the context of the major histocompatibility complex (MHC) molecules. These T cells, optionally together with B cells producing tumor-specific antibodies and dendritic cells (DC) processing and presenting tumor epitopes, can mediate an adaptive immunity against tumors. The second category of effector cells includes natural killer (NK) cells, NK-T cells, and macrophages (M). These cells are not restricted by the MHC molecules in their interactions with tumor targets, and they mediate innate immunity. Each type of effector cells, whether specific or nonspecific, contains subsets of cells at different stages of differentiation and activation. This means that each type of effector cell is potentially able to target tumor cells contains a heterogeneous mix of cells with distinct functional capabilities, depending on their stage of differentiation, maturation, and / or activation (Holland, Frei; Cancer Medicine; 6th edition, chapter “Antitumor Effector Cells in Humans”). All the above-described types of anti-tumor lymphocytes are applicable in accordance with the present invention.

[0080] The anti-tumor lymphocytes as describe herein are preferably T-cells or NK cells.

[0081] A T-cell or T-lymphocyte can be distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell surface. One of the functions of T-cells is mediating immune-mediated cell death, and it is carried out by two major subtypes: CD8+ "killer" and CD4+ "helper" T-cells. CD8+ T cells are cytotoxic which means that they are able to directly kill selected cells. These selected cells are in accordance with the invention tumor cells (such as cancer cells) as well as virus-infected cells. CD4+ cells function as "helper cells". Unlike CD8+ killer T-cells, these CD4+ helper T-cells function by further activating memory B cells and cytotoxic T-cells, which leads to a larger immune response which is in accordance with the invention directed against tumor cells. The specific adaptive immune response regulated by the T-helper cell depends on its subtype, which is distinguished by the types of cytokines they secrete. The T-cells are preferably a CD8+ killer T-cells or mixture of CD8+ killer and CD4+ helper T-cells.

[0082] A natural killer (NK) cell is a type of cytotoxic lymphocyte being critical to the innate immune system that belong to the rapidly expanding family of the innate lymphoid cells (ILC) and represent 5-20% of all circulating lymphocytes in humans. The role of NK cells in innate immune system is analogous to that of cytotoxic T-cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected cells and other intracellular pathogens acting at around 3 days after infection and respond to tumor formation. Typically, immune cells detect the major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release, causing the death of the infected cell by lysis or apoptosis. NK cells are unique, however, as they have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named "natural killers" because they do not require activation to kill cells that are missing "self" markers of MHC class 1 . This role is especially important because harmful cells that are missing MHC I markers cannot be detected and destroyed by other immune cells, such as T-cells.

[0083] The anti-tumor lymphocytes are also preferably chimeric antigen receptor T-cells (CAR T-cells), T-cell- receptor-engineered T-cells (TCR T-cells), chimeric antigen receptor NK-cells (CAR NK-cells), NK cell receptor-engineered NK cells (NCR NK-cells), TCR / CAR hybrid T-cells, NCR / CAR hybrid NK-cells or tumor-infiltrating lymphocytes (TILs).

[0084] Chimeric antigen receptor T-cells (CAR) T-cells are T-cells that have been genetically engineered to produce a chimeric T cell receptor (CAR) for use in immunotherapy. The receptors are chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor. CAR-T cell therapy uses T-cells engineered with CARs for tumor (such as cancer) therapy. The premise of CAR-T immunotherapy is to modify T-cells to recognize tumor cells in order to more effectively target and destroy them. In order to generate CAR T-cells, T-cells are harvested from a subject, genetically altered, and then infused into patients to attack a tumor in the subject. CAR T-cells can be both CD4+ and / or CD8+ cells. A 1 -to-1 ratio of both cell types is preferred since it provides synergistic antitumor effects.

[0085] CAR T-cells are engineered to transfer arbitrary specificity onto an immune effector cell, like a T cell, which specifically eliminates antigen-bearing tumor cells. The CAR may comprise a scFv being derived from an antibody, a CD3 and a transmembrane domain (so-called first-generation CARs). In this way, the engineered CAR is able to recognize specific tumor associated-antigens. Therefore, the CAR has the ability to bind unprocessed tumor surface antigens without MHC processing while TCRs engage with both tumor intracellular and surface antigenic peptides embedded in MHC molecules.

[0086] In contrast, TCRs are a / p heterodimers that bind to the MHC-bound antigens. As discussed above, CARs recognize tumor antigen which lead to T-cell activation with different functions compared with TCRs. CAR-T cell therapy has certain disadvantages like off-tumor toxicities when targeting tumorspecific antigen. Compared with CARs, TCRs have several structural advantages in T cell-based therapy, such as more subunits in their receptor structure (ten subunits vs one subunit), more immunoreceptor tyrosine-based activation motif (ITAMs) (ten vs three), less dependence on antigens (one vs 100), and more co-stimulate receptors (CD3, CD4, CD28, etc.) (Zhao et al. (2021) Front. Immunol., | https: / / doi.org / 10.3389 / fimmu.2021.658753).

[0087] CAR NK-cells are distinguished from CAR T-cells in that the chimeric antigen receptor is introduced into NK cells instead of T-cells. Just as CAR T-cells, CAR-NK cells can be engineered to target diverse antigens, enhance proliferation and persistence in vivo, increase infiltration into solid tumors, overcome resistant tumor microenvironment, and ultimately achieve an effective anti-tumor response.

[0088] Natural cytotoxicity receptors NK cells (NCR NK-cells) are NK-cell that have been genetically engineered to express a NCR. The NCRs have been proposed to bind to many cellular ligands which are implicated in NK cell surveillance of tumor cells. Many of these interactions have been shown to evoke the cytotoxic and cytokine-secreting functions of NK cells. However, it is also possible that the NCRs regulate other anti-tumor pathways. NCRs and their ligands can be successfully targeted for tumor (such as cancer) immunotherapy. NCRs have been classically defined as activating receptors delivering potent signals to NK cells in order to lyse harmful cells and to produce inflammatory cytokines.

[0089] TCR / CAR hybrid T-cells are T-cells that have been genetically engineered to express a TCR and CAR. Similarly, NCR / CAR hybrid NK-cells are T-cells that have been genetically engineered to express a NCR and CAR.

[0090] Tumor-infiltrating lymphocytes (TILs) are white blood cells that have left the bloodstream and migrate towards a tumor. TILs are implicated in killing tumor cells. The presence of lymphocytes in tumors is often associated with better clinical outcomes.

[0091] The tumor-infiltrating lymphocytes are preferably tumor-infiltrating T-cells or tumor-infiltrating NK cells.

[0092] In adoptive T-cell transfer therapy, TILs are expanded ex vivo from surgically resected tumors that have been cut into small fragments or from single cell suspensions isolated from the tumor fragments. Multiple individual cultures are established, grown separately and assayed for specific tumor recognition. TILs are typically expanded over the course of a few weeks with a high dose of IL-2 in 24-well plates. Selected TIL lines that presented best tumor reactivity are then further expanded in a "rapid expansion protocol" (REP), which uses anti-CD3 activation for a typical period of two weeks. The final post-REP TIL is infused back into a patient in order to treat a tumor of the patient. This applies mutatis mutandis to adoptive NK-cell transfer with TILs.

[0093] In addition, the anti-tumor lymphocytes are preferably autologous anti-tumor lymphocytes.

[0094] In an anti-tumor therapy with autologous lymphocytes the lymphocytes are taken from a subject having a tumor and are genetically engineered (e.g. to produce CAR T-cells) and / or selected and / or expanded (e.g. to produce TILs) ex vivo and then transferred back into the same subject. These autologous therapies are subject-specific because the therapeutic cells are created from a subject's own cells.

[0095] In accordance with a preferred embodiment of the fifth aspect the nucleic acid molecule encoding said variant of cGAS and / or said variant of SPSB3 has been inserted into the genome of the host cell by a genome editing technology, such as meganuclease, Zn-finger, TALEN or CRISPR. The nucleic acid molecule encoding the variant cGAS and / or variant SPSB3 may be introduced into the genome of the anti-tumor lymphocytes by homologous recombination. The homologous recombination is preferably Cre-Lox recombination. This is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems.

[0096] Over the past years homologous recombination has been largely replaced by genome editing technologies, such as meganuclease, Zn-finger, TALEN or CRISPR. The genome editing technology is preferably CRISPR.

[0097] Meganucleases are enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large DNA sequences (from 12 to 40 base pairs). The most widespread and best known meganucleases are the proteins in the LAGLIDADG family, which owe their name to a conserved amino acid sequence. Meganucleases, found commonly in microbial species, have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific. In order to find the exact meganuclease required to act on a specific DNA sequence, mutagenesis and high throughput screening methods are available to create a meganuclease variant that recognizes a desired target sequence. It is also possible to fuse meganucleases to each other, thereby creating hybrid enzymes that recognize a new sequence. Moreover, a method named rationally designed meganuclease (US 8,021 ,867) may be used to design sequence specific meganucleases.

[0098] The concept behind ZFNs and TALEN technology is based on a non-specific DNA cutting enzyme, which can then be linked to a specific DNA sequence recognizing peptides such as zinc fingers and transcription activator-like effectors (TALEs). The key to this was to find an endonuclease whose DNA recognition site and cleaving site were separate from each other, a situation that is not common among restriction enzymes. Once this enzyme was found, its cleaving portion could be separated which would be very non-specific as it would have no recognition ability. This portion could then be linked to sequence recognizing peptides that could lead to very high specificity.

[0099] Zinc finger motifs occur in several transcription factors. The zinc ion, found in 8% of all human proteins, plays an important role in the organization of their three-dimensional structure. In transcription factors, it is most often located at the protein-DNA interaction sites, where it stabilizes the motif. The C-terminal part of each finger is responsible for the specific recognition of the DNA sequence. The recognized sequences are short, made up of around 3 base pairs, but by combining 6 to 8 zinc fingers whose recognition sites have been characterized, it is possible to obtain specific proteins for sequences of around 20 base pairs. It is therefore possible to control the expression of a specific gene. The method generally adopted for this involves associating two proteins - each containing 3 to 6 specifically chosen zinc fingers - with the catalytic domain of the Fokl endonuclease. The two proteins recognize two DNA sequences that are a few nucleotides apart. Linking the two zinc finger proteins to their respective sequences brings the two endonucleases associated with them closer together. Fokl requires dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner would recognize a unique DNA sequence. To enhance this effect, Fokl nucleases have been engineered that can only function as heterodimers and have increased catalytic activity.

[0100] Transcription activator-like effector nucleases (TALENs or TAL nucleases) are artificial restriction enzymes generated by fusing a specific DNA-binding domain to a non-specific DNA cleaving domain. The DNA binding domains, which can be designed to bind any desired DNA sequence, comes from TAL effectors, DNA-binding proteins excreted by plant pathogenic Xanthomanos sp. Tai effectors consists of repeated domains, each which contains a highly considered sequence of 34 amino acids, and recognize a single DNA nucleotide. The nuclease can create double strand breaks at the target site that can be repaired by error-prone non-homologous end-joining (NHEJ), resulting in gene disruptions through the introduction of small insertions or deletions. TALEN constructs are used in a similar way to designed zinc finger nucleases, and have at least three advantages in targeted mutagenesis: (1) DNA binding specificity is higher, (2) off-target effects are lower, and (3) construction of DNA-binding domains is easier.

[0101] As is evident from the above, in meganucleases, ZNF nucleases, and TAL nucleases the endonuclease activity and the site-specificity within the target genome are conferred by one compound.

[0102] The CRISPR or CRISPR-Cas genome editing system was adapted from a naturally occurring defense system against foreign DNA (e.g. viruses, plasmid DNA) in prokaryotes. Prokaryotes with CRISPR-Cas system capture fragments of DNA from invading DNA and integrate them into DNA segments known as CRISPR arrays. The CRISPR arrays allow the bacteria / archaea to acquire immunity against the invading DNA (or homologous ones). The bacteria / archaea produce CRISPR-RNAs (crRNAs) from the CRISPR arrays to target the foreign DNA, which in complex with CRISPR nucleases (e.g. Cas9 or a similar enzyme) inactivate the invading DNA by nucleolytic cleavage.

[0103] The CRISPR-Cas system has been harnessed for genome editing in prokaryotes and eukaryotes. A small piece of RNA with a short "guide" sequence that attaches (binds) to a specific target sequence of DNA in a genome is created (the so-called guide RNA (gRNA) or single guide (sgRNA)). The genomic target site of the gRNA can be any ~20 nucleotide DNA sequence, provided it meets two conditions: (i) The sequence is unique compared to the rest of the genome, and (ii) the target is present immediately adjacent to a Protospacer Adjacent Motif (PAM). The PAM sequence is essential for target binding, but the exact sequence depends on which CRISPR endonuclease is used. CRISPR endonuclease and their respective PAM sequences are known in the art (see https: / / www. addgene. org / crispr / guide / #pam- table). Hence, the gRNA also binds to the CRISPR endonuclease (e.g. the Cas9 or Cpf1 enzyme). As in bacteria, the gRNA is used to recognize the DNA sequence, and the CRISPR endonuclease cuts the DNA at the targeted location. Once the DNA is cut, the cell's own DNA repair machinery (NHEJ or HDR) adds or deletes pieces of genetic material, or makes changes to the DNA by replacing an existing segment with a customized DNA sequence. Hence, in the CRISPR-Cas system, the CRISPR nuclease makes a double-stranded break in DNA at a site determined by the short (~20 nucleotide) gRNA which break is then repaired within the cell by NHEJ or HDR. The CRISPR-Cas system can be multiplexed by adding multiple gRNAs. It was demonstrated that, for example, five different simultaneous mutations can be introduced into mouse embryonic stem cells by using five different gRNA molecules and one CRISPR endonuclease.

[0104] As is evident from the above, in the CRISPR technology the endonuclease activity (CRISPR nuclease) and the site-specificity within the target genome (gRNA) are conferred by two separate compounds.

[0105] The present invention relates in a sixth aspect to a pharmaceutical composition comprising the variant of cGAS of the first aspect, the variant of SPSB3 of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, or any combination thereof.

[0106] The present invention relates in a related seventh aspect to the variant of cGAS of the first aspect, the variant of SPSB3 of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, or any combination thereof for use in the treatment or prevention of a disease.

[0107] The definitions and preferred embodiments as descried herein above in connection with the other aspects apply mutatis mutandis to the sixth and seventh aspect as being amenable for combination with the sixth and seventh aspect.

[0108] In accordance with the present invention, the term “pharmaceutical composition” relates to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition of the invention comprises the compounds recited above. It may, optionally, comprise further molecules capable of altering the characteristics of the compounds of the invention thereby, for example, stabilizing, modulating and / or activating their function. The composition may be in solid, liquid or gaseous form and may be, inter alia, in the form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). The pharmaceutical composition of the present invention may, optionally and additionally, comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The therapeutically effective amount for a given situation will readily be determined by routine experimentation and is within the skills and judgement of the ordinary clinician or physician. The length of treatment needed to observe changes and the interval following treatment for responses to occur vary depending on the desired effect. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art. The subject to be treated herein is preferably a mammal, more preferably a primate and most preferably human.

[0109] The modulation of cGAS activity allows to modulate cellular senescence (Yang et al. (2027), PNAS, 114(23):E4612-E4620). The modulation of cellular senescence is a new strategy to treat senescence- associated human diseases that potentially include cancer, neurodegenerative diseases, cardiovascular diseases, and aging. Cellular senescence is important for the maintenance of tissue homeostasis. Dysregulation of senescence is linked to many human diseases, such as cancer, premature aging, and age-related diseases. Thus, the modulation of cGAS activity is a strategy to treat various diseases that potentially include as non-limiting examples tumors, neurodegenerative diseases, cardiovascular diseases, and aging.

[0110] The present invention relates in an eight aspect to the variant of cGAS of the first aspect, the variant of SPSB3 of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the pharmaceutical composition of the sixth aspect, or any combination thereof for use in the treatment of an inflammatory disease, infectious disease or tumor.

[0111] The definitions and preferred embodiments as descried herein above in connection with the other aspects apply mutatis mutandis to the eighth aspect as being amenable for combination with the eighth aspect.

[0112] The type-l interferons (IFN) are cytokines which play essential roles in inflammation and immunoregulation. The cGAS-STING pathway is a therapeutic target in inflammatory diseases (Decout et al. (2021), Nature Reviews Immunology volume 21 , pages548-569 (2021)). For this reason the compounds recited above are suitable to treat inflammatory diseases. The inflammatory disease is preferably an autoinflammatory disease.

[0113] Mice lacking cGAS are more vulnerable to lethal infection by DNA viruses and RNA viruses (Li et al. (2013), Science, 341 (6152): 1390-4). In addition, cGAS has been shown to be an innate immune sensor of retroviruses including HIV. For this reason the compounds recited above are suitable to treat infectious diseases. The infectious disease is preferably a viral infectious disease.

[0114] The tumor will be further defined herein below.

[0115] The present invention relates in a ninth aspect to therapeutic cells, preferably anti-tumor lymphocytes, and the variant of cGAS of the first aspect, the variant of SPSB3 of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the pharmaceutical composition of the sixth aspect, or any combination thereof, provided that the cGAS or the encoded cGAS carries an amino acid substitution of one or both arginines at amino acid positions 255 and 236 and / or an amino acid substitution of one or both lysines at amino acid positions 254 and

[0116] 258 for use in the treatment of a tumor in a subject.

[0117] The definitions and preferred embodiments as descried herein above in connection with the other aspects apply mutatis mutandis to the ninth aspect as being amenable for combination with the ninth aspect.

[0118] The therapeutic cells, anti-tumor lymphocytes and preferred examples thereof have been described herein above.

[0119] As discussed above, a variant cGAS carrying an amino acid substitution of one or both arginines at amino acid positions 255 and 236 and / or an amino acid substitution of one or both lysines at amino acid positions 254 and 258 of the invention designates a constitutively active variant cGAS. In this respect “constitutively active” means that the variant cGAS is capable of permanently producing cGAMP when present within a cell or subject.

[0120] A tumor is an abnormal benign or malignant new growth of tissue that possesses no physiological function and arises from uncontrolled usually rapid cellular proliferation.

[0121] The tumor is preferably cancer. Cancer is an abnormal malignant growth of tissue that possesses no physiological function and arises from uncontrolled usually rapid cellular proliferation. The cancer is preferably selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulva cancer, bladder cancer, salivary gland cancer, pancreatic cancer, thyroid cancer, kidney cancer, lung cancer, cancer concerning the upper gastrointestinal tract, colon cancer, colorectal cancer, prostate cancer, squamous-cell carcinoma of the head and neck, cervical cancer, glioblastomas, malignant ascites, lymphomas and leukemias.

[0122] The tumor or cancer is preferably a solid tumor or cancer. A solid tumor or cancer is an abnormal mass of tissue that usually does not contain cysts or liquid areas by contrast to a liquid tumor.

[0123] The subject may be a subject being at risk of developing a tumor and is preferably a subject wherein a tumor has been diagnosed.

[0124] The subject may receive a further anti-tumor treatment before, along with or after the medical use of the invention. Non-limiting examples of such anti-tumor treatments are surgery, radiation therapy and chemotherapy.

[0125] It is shown in PCT / EP2023 / 054168 that a variant cGAS carrying an amino acid substitution of one or both arginines at amino acid positions 255 and 236 and / or an amino acid substitution of one or both lysines at amino acid positions 254 and 258 leads to tumor regression as well as the activation of immune cells. It is expected that the active cGAS variant not only reduces the tumor load but also enhances and promotes the anti-tumor activity of the anti-tumor lymphocytes by activating them. The combined use of an active cGAS variant and anti-tumor lymphocytes, and in particular of anti-tumor lymphocytes expressing an active cGAS variant is therefore expected to provide a therapeutic effect that exceeds the cumulative effects of the active cGAS variant and the anti-tumor lymphocytes alone. It is of further note that the active cGAS variant as described herein is not only constitutively active but in addition displays increased half-life and / or stability against proteasomal degradation. Hence, it is expected that compounds as recited above are particularly well suitable for the treatment of a tumor in a subject.

[0126] Regarding the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0127] Similarly, and also in those cases where independent and / or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1 . In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

[0128] The figures show.

[0129] Fig. 1 | Discovery of SPSB3 and CRL5 in the regulation of nuclear cGAS. a, Representative image sequence demonstrating cGAS chromosome attachment in mitosis followed by intranuclear redistribution and degradation, respectively, in cGAS-GFP (green) expressing HeLa cells. Scale bar, 10 pm. b, Representative nuclear cGAS-GFP mean fluorescence intensity (MFI) measurement in a postmitotic HeLa cell, c, Relative nuclear cGAS-GFP MFI in post-mitotic HeLa cells treated with Epoxomicin or DMSO (n = 10 cells per condition), d, Relative change of nuclear cGAS-GFP MFI in post-mitotic HeLa cells treated with Epoxomicin (n = 10) or DMSO (n = 9). e, cGAS levels in human cells treated or not with Epoxomicin or Bortezomib by immunoblot (representative of two independent experiments), f, Focused RNAi-based screen of UPS factors regulating nuclear cGAS-GFP abundance. Each dot shows cGAS-GFP mean intensity blotted against integrated intensity. Knockdown of genes yielding increased cGAS-GFP levels (> 3 SD relative to control siRNA) are shown in yellow. Genes encoding for cGAS (dark blue), proteasomal subunits (light blue), CRL5 complex components (dark green) and the SPSB3 gene (light green) are highlighted, g, Schematic model of CRL5-directed substrate ubiquitylation via the ELOBC adaptor complex and SPSB3. h, Relative nuclear cGAS-GFP MFI in post-mitotic HeLa cells treated with siRNA against SPSB3 (n = 18), CUL5 (n =14), control siRNA (n =16) or Epoxomicin (n = 14). i, Nuclear cGAS-GFP measurements by confocal microscopy in HeLa cells treated with Epoxomicin (n = 32), MLN4924 (n = 29), or DMSO (n = 32) (left) or treated with siRNA against SPSB3 (n = 31) or CUL5 (n = 33) or control siRNA (n = 22). j, Relative cGAS protein levels were assessed in HeLa cells pre-treated with MLN4924 or control upon addition of the translation inhibitor cycloheximide (CHX) for 0 to 16 h (n = 2). Data are mean ± SD. Numbers indicate individual cells (c, d, h, i) or numbers of independent biological experiments (j). Two-way ANOVA with Sidak’s multiple comparison test (c, j), Student’s t-test (d), two-way ANOVA with Tukey’s multiple comparison test (h) or one-way ANOVA with Dunnett’s multiple comparison test (i) were used for statistical analysis.

[0130] Fig. 2 | CRL5SPSB3ubiquitylates cGAS. a, Immunoprecipitation with anti-FLAG antibody from lysates of HEK293T cells transfected with constructs for cGAS and FLAG-tagged SPSB3 or FLAG-tagged EGFP. Samples were analysed by immunoblot, b, Immunoprecipitation with anti-FLAG antibody from lysates of HEK293T cells transfected with constructs for FLAG-tagged cGAS, HA-tagged Ubiquitin, and SPSB3 or SPSB3 ASOCS. Samples were analysed by immunoblot, c, Immunoprecipitation with anti- FLAG antibody from lysates of HEK293T cells transfected with constructs for FLAG-tagged cGAS, HA- tagged Ubiquitin and SPSB3 followed by treatment with DMSO or MLN4924. Samples were analysed by immunoblot, d, In vitro Ni-NTA pull-down assaying cGAS complex formation with the SPSB3-ELOBC heterotrimer. His-Halo-tagged cGAS full-length (FL) or catalytic core (CD) cGAS was used, e, Immunoblot showing in vitro ubiquitylation reactions of cGAS by CRL5SPSB3in the presence or absence of the RBR E3 ligase ARIH2. f, Ubiquitylation sites of human cGAS identified by mass spectrometry. Lysines present in the conserved catalytic core were further validated, g, Immunoblot showing in vitro ubiquitylation reactions of wild-type cGAS and cGASK427RK428Rmutant by CRL5SPSB3. h, Immunoprecipitation with anti-FLAG antibody from lysates of HEK293T cells transfected with constructs for FLAG-tagged cGAS or FLAG-tagged cGASK427 / 8R(KK), HA-tagged Ubiquitin, and SPSB3. Samples were analysed by immunoblot, i, Wild-type cGAS or cGASK427 / 8R(KK) was induced in HeLa cGAS KO cells by doxycycline treatment. After 4 days cGAS levels were analysed in whole cell lysates by immunoblot. Vinculin was used as a loading control. One representative of two (a) or three (b-e, g-i) independent experiments is shown.

[0131] Fig. 3 | Structural basis of cGAS targeting by SPSB3. a, A composite cryo-EM density map of the nucleosome-cGAS-SPSB3-ELOBC complex, assembled from two focused-refinement maps (nucleosome-cGAS and cGAS-SPSB3-ELOBC). Different contour levels were used for optimal visualization using UCSF ChimeraX35. b, Ribbon representation of the nucleosome-cGAS-SPSB3- ELOBC complex structure. Arrows indicate B30.2 / SPRY and SOCS box domains of SPSB3. c, Detailed view of the binding interface between cGAS and SPSB3. d, Sequence logo of the cGAS and SPSB3 interface derived from 150 vertebrate species, e, Model of nucleosome-bound cGAS targeted by an activated (neddylated) CRL5SPSB3complex with RBR E3 ligase ARIH2 priming polyubiquitylation by transferring the first ubiquitin onto cGAS. Lysine residues identified as cGAS ubiquitylation sites by mass spectrometry and essential for SPSB3-mediated degradation of cGAS are coloured red; the catalytic Cys310 of ARIH2 is coloured gold. The model is built by docking the nucleosome-cGAS-SPSB3-ELOBC model into a model composed of the ELOBC-CUL5 (PDB: 4JGH)31, CUL5-NEDD8-RBX2-ARIH2 (PDB: 7ONI)29, and ARIH1-UB (PDB: 7B5M)30complexes.

[0132] Fig. 4 | Disrupted nuclear cGAS ubiquitylation elevates the cellular type I IFN tone, a, Bio-layer interferometry binding assays of His-Halo-tagged wild-type (WT) cGAS, N513A or N514A cGAS mutants with SPSB3-ELOBC. Right upper graph shows binding affinity of wild-type cGAS with SPSB3-ELOBC. b, Immunoblot showing in vitro ubiquitylation reactions of wild-type cGAS, cGASN513AN514Amutant and cGASK427RK428Rmutant by CRL5SPSB3. c, Immunoprecipitation with anti-FLAG antibody from lysates of HEK293T cells transfected with constructs for FLAG-tagged cGAS (WT) or FLAG-tagged cGASN513 / 4A(N513 / 4), HA-tagged Ubiquitin, and SPSB3. Samples were analysed by immunoblot, d, Wild-type cGAS or cGASN513 / 4Awas induced in HeLa cGAS KO cells by doxycycline treatment. After 4 days cGAS levels were analysed in whole cell lysates by immunoblot. GAPDH was used as a loading control, e, f, Expression of IFNB1, IFIT1, and ISG15 was assessed by RT-qPCR 4 days after induction of cGAS or cGASN513 / 4A(e) or cGAS or cGASK427 / 8R(f) with doxycycline, g, Expression of IFNB1, IFIT1, and ISG15 was assessed by RT-qPCR 3 days after enforced expression or not of SPSB3 with doxycycline in HeLa cells, h, mRNA levels of IFNB1, IFIT1, and ISG15were measured by RT-qPCR in HeLa and HeLa cGAS KO cells treated with non-targeting control siRNA or SPSB3 siRNAs for 5 days. Ratios of relative IFNB1, IFIT1, and ISG15 mRNA levels normalized to the control are shown, i, Following induction of wild-type cGAS, cGASN513 / 4A, or cGASK427 / 8Rby doxycycline, cells were infected with HSV-1-GFP (left; n = 6) or VACV-GFP (right; n = 3). GFP+cells were analysed by flow cytometry, j, Following enforced expression or not of SPSB3 in wild-type HeLa cells by doxycycline, cells were infected with HSV-1-GFP (left; n = 6) or VACV-GFP (right; n = 4). GFP+cells were analysed by flow cytometry. Data are mean ± SD. Numbers indicate technical replicates (e-h) or independent biological experiments (i-j). P values were obtained by one-way ANOVA (h-i) or two-tailed Student’s t-test (e-g, j). One representative of two (a-d) or three (f-h) independent experiments is shown.

[0133] Fig. 5 | Nuclear cGAS stability is regulated by the ubiquitin-proteasomal system, a, Representative image sequence demonstrating cGAS intranuclear degradation in HeLa cells expressing cGAS-GFP and PCNA. PCNA distribution pattern is used to determine cell cycle phase with PCNA foci formation (arrows) in mid S phase. Scale bar, 10 pm. b, Relative nuclear cGAS-GFP MFI in post-mitotic HeLa cells treated with Leptomycin B (n = 20) or DMSO (n = 12). c, Representative image of a HeLa cell expressing cGAS-Dendra2 before and after photoconversion, d, Relative cGAS-Dendra2 MFI in the nucleus (left; n = 16 cells per condition) or cytoplasm (right; n = 5 cells per condition) in cells that underwent photoconversion or not. e, Relative nuclear cGAS-GFP MFI measurement in post-mitotic HeLa cells treated with Epoxomicin (n = 13), Bortezomib (n = 14) or DMSO (n = 13). f, Relative change of nuclear cGAS-GFP MFI in post-mitotic HeLa cells in G1 phase (left) or S phase (right) treated with Epoxomicin (G1 : n = 10; S: n = 5) or DMSO (G1 : n = 11 ; S: n = 9). g, Relative nuclear photoconverted cGAS-Dendra2 MFI measurement in post-mitotic HeLa cells treated with Epoxomicin (n = 8) or DMSO (n = 6). Data are mean ± SD. Numbers indicate individual cells. Two-way ANOVA with Sidak’s multiple comparison test (b, d, g), Student’s t-test (f) or two-way ANOVA with Tukey’s multiple comparison test (e) were used for statistical analysis.

[0134] Fig. 6 | SPSB3 and CUL5 control nuclear cGAS levels, a, Results from the siRNA screen highlighting cGAS-GFP nuclear abundance by mean fluorescence intensity and integrated fluorescence intensity in cells treated with siRNAs against SPSB family members (n = 3). b, Nuclear cGAS measurements by confocal microscopy in HeLa cells (endogenous cGAS) or U2OS cells (cGAS-GFP expression) transfected with siRNAs against CUL5, SPSB3 and PSMA5 or control siRNA or treated with Epoxomicin or DMSO (n = 2). c, Relative nuclear cGAS-GFP MFI measurement in post-mitotic U2OS cells treated with siRNA against SPSB3 (n = 15) or CUL5 (n =15), control siRNA (n =14) or Epoxomicin (n = 15). d, mRNA levels of cGAS were measured by RT-qPCR in HeLa treated with non-targeting control siRNA or with siRNAs against CUL5 or SPSB3 for 5 days. Ratios of relative cGAS mRNA levels normalized to the control are shown (n = 3). e, mRNA levels of cGAS were measured by RT-qPCR in HeLa cells treated with DMSO or MLN4924. Ratios of relative cGAS mRNA level normalized to the control are shown (n = 6). f, Cytosolic and nuclear fractions were extracted from dox-inducible SPSB3 overexpressing HeLa cells treated or not with doxycycline (4 days) for enforced expression or not of SPSB3 from a doxycycline-inducible promoter. Immunoblots probing cGAS and FLAG are shown. Vinculin and H2B were used as loading control of cytosolic and nuclear fractions, respectively, g-j, Nuclear fractions collected from HeLa cells (n = 3) (g), primary endothelial cells (n = 3) (h), BJ-5ta fibroblast cells (n = 3) (i), or differentiated THP-1 cells (n = 3) (j) treated with non-targeting control siRNA or siRNA against SPSB3 for 5 days were analysed by immunoblot. H2B was used as a loading control. Corresponding mRNA levels of SPSB3 measured by RT-qPCR and shown as ratio normalized to the control are shown below. Data are mean ± SD. Numbers indicate individual cells (c), independent biological experiments (a, b), or technical replicates (d, e, g-j). P values were obtained by two-way ANOVA with Tukey’s multiple comparison test (c), one-way ANOVA (d) or two-tailed Student’s t-test (e, g-j). One representative of two (d, e, h, i) or three (f, g, j) independent experiments is shown.

[0135] Fig. 7 | SPSB3 targets nuclear cGAS. a, b, Airyscan microscopy images of HeLa cells stained for SPSB3 (a) or CUL5 (b) and DAPI (blue). One representative cell is shown of at least n = 6 cells from 3 independent experiments. Scale bars, 10 pm. c, Immunoprecipitation with anti-GFP antibody from lysates of HEK293T cells transfected with constructs for GFP-tagged NLS-cGAS, HA-tagged Ubiquitin with or without SPSB3. Samples were analysed by immunoblot. One representative of two independent experiments is shown.

[0136] Fig. 8 | cGAS is bound and ubiquitylated by CRL5SPSBS3. a, Size-exclusion chromatography and SDS-PAGE of an assembled Halo-cGAS-SPSB3-ELOBC complex, b, Size-exclusion chromatography and SDS-PAGE of an assembled cGAS-SPSB3-ELOBC complex, c, Immunoblot showing in vitro ubiquitylation reactions of human cGAS catalytic domain (CD; aa: 155-522 aa) by CRL5SPSB3. d, Representative spectrum of a peptide comprising ubiquitylated K427 and K428 lysine residues identified by mass spectrometry. The sequence of the peptide, DKKHLDK, is shown at top of the spectrum and ubiquitylation at K427 and K428 is indicated by the 114 increases of mass, as was shown in the fragmentation table below, e, Immunoblot showing in vitro ubiquitylation reactions of wild-type cGAS CD and distinct lysine residue substitutions of cGAS CD by CRL5SPSB3. Representatives of at least n = 2 independent experiments (c, e) are shown.

[0137] Fig. 9 | Structural conservation of ubiquitylated lysine residues on cGAS. a, Sequence alignment of amino acids surrounding K427 and K428 in human cGAS, murine cGAS, bovine cGAS or porcine cGAS. The K427 / K428 lysine pair in human cGAS subject to ubiquitylation by CRL5SPSB3is highlighted in dark red and the structurally conserved lysine pair of the other cGAS homologues as shown in (b) are highlighted in red. b, Comparison of human cGAS (PDB: 4LEV)39, mouse cGAS (PDB: 4K8V)5, porcine cGAS (PDB: 4JLX)34and bovine cGAS (Alphafold2 prediction). The lysine pair on cGAS is rendered by sphere, c, Size-exclusion chromatography and SDS-PAGE of an assembled mouse cGAS-Halo- SPSB3-ELOBC complex, d, Immunoblot showing in vitro ubiquitylation reactions of wild-type mouse cGAS CD (aa: 147-507) and the corresponding K409R / K410R mutant by CRL5SPSB3. Representatives of n = 3 independent experiments are shown (c, d).

[0138] Fig. 10 | Purification and cryo-EM data processing of nucleosome-bound cGAS in complex with SPSB3-ELOBC. a, In vitro Ni-NTA pull-down assaying nucleosome-bound His-Halo-tagged wild-type (WT) cGAS or site C mutant cGAS (K285A / R300A / K427A) complex formation with the SPSB3-ELOBC heterotrimer. b, Size-exclusion chromatography of nucleosome-bound cGAS in complex with SPSB3- ELOBC. c, Left: Coomassie blue staining SDS-PAGE and cryo-EM analysis of the complex peak (red) shown in (b). Middle: Representative micrograph of the nucleosome-cGAS-SPSB3-ELOBC complex in vitrified ice from 9,855 raw images. Scale bar, 100 nm. Right: Zoomed-in cutaways highlighting the complex fiber. Scale bar, 10 nm. d, Selected 2D class averages of nucleosome-cGAS-SPSB3-ELOBC complex particles. Scale bar, 10 nm. e, Cryo-EM data processing flow chart, f, Corrected gold-standard Fourier shell correlation (FSC) curves of the nucleosome-cGAS-SPSB3-ELOBC complex for the 3D EM reconstruction maps, g, Angular distribution of nucleosome-cGAS-SPSB3-ELOBC complex particles included in the final reconstruction, h, Final 3D reconstruction of the nucleosome-cGAS-SPSB3-ELOBC complex; cGAS in blue, SPSB3 in green, nucleosomal DNA in lilac, histones in grey. One representative of at least three independent experiments is shown (a, b, c).

[0139] Fig. 11 | Purification and cryo-EM data processing of nucleosome-bound cGAS C site mutant in complex with SPSB3-ELOBC. a, Left: Size-exclusion chromatography of nucleosome-bound cGAS C site mutant (K285A / R300A / K427A) in complex with SPSB3-ELOBC. Right: Coomassie blue staining SDS-PAGE and cryo-EM analysis of the complex peak (red), b, Representative micrograph and selected 2D class averages of nucleosome-cGASsiteC-SPSB3-ELOBC complex particles in vitrified ice from 15,141 raw images. Scale bar left, 100 nm; scale bar right, 10 nm. c, Cryo-EM data processing flow chart, d, Corrected gold-standard Fourier shell correlation (FSC) curves of the consensus 3D reconstruction of the nucleosome-cGASsiteC-SPSB3-ELOBC complex (top) and the focused 3D reconstruction with a mask on cGASsiteC-SPSB3-ELOBC (bottom), e, Angular distribution of the nucleosome-cGASsiteC-SPSB3-ELOBC complex particles included in the final reconstruction, f, Final 3D reconstruction of the cGASsiteC-SPSB3-ELOBC complex coloured according to the local resolution, g, Cryo-EM density maps (grey mesh) of cGASsiteCand SPSB3 contoured at 5o. The protein structures fitted into the density map are shown by stick models. One representative of at least three independent experiments is shown (a).

[0140] Fig. 12 | A unique interface on SPSB3 for binding cGAS. a, Right: Ribbon diagram and a 3D reconstruction of the nucleosome-cGASsiteC-SPSB3-ELOBC complex. cGAS in blue, SPSB3 in green, nucleosomal DNA in lilac, histones in grey, ELOB in yellow, ELOC in antique white. Left: EM densities (shown as mesh at 5a) for cGAS residues interacting with the SPSB3. Shown are cGAS N513, N514 and R512 / D465 interactions with SPSB3 respectively, b, The CGAS.SPSB3 model is aligned with the VASA-SPSB1 model (PDB: 3F2O)27, individual interacting residues are shown by sticks, c, Sequence alignment of SPSB172 / 3 / 4. Residues used for substrate recognition are highlighted, d, Percent identity matrix of human SPSB1 / 2 / 3 / 4.

[0141] Fig. 13 | Effect of structure-guided mutations on cGAS binding to SPSB3. a, Left panels: Bio-layer interferometry binding assays of His-Halo-tagged cGAS mutants with SPSB3-ELOBC. Right panels: binding affinity of cGAS mutants with SPSB3-ELOBC. b, Bio-layer interferometry binding assays His- Halo-tagged cGAS with an SPSB3 mutant in complex with ELOBC. c, Immunoblot showing in vitro ubiquitylation reactions of wild-type human cGAS catalytic domain (CD; aa: 155-522) or distinct cGAS CD mutants by CRL5SPSB3. d, Immunoprecipitation with anti-FLAG antibody from lysates of HEK293T cells transfected with constructs for GFP-tagged NLS-cGAS (WT) or GFP-tagged cGASN513 / 4A(N513 / 4), HA-tagged Ubiquitin, and SPSB3. Samples were analysed by immunoblot, e, Relative nuclear cGAS- GFP MFI measurement in post-mitotic HeLa cGAS KO cells reconstituted with wild-type (WT) cGAS or cGAS N513 / 4A mutations (n = 15 cells per condition). Data are mean ± SD. Two-way ANOVA with Sidak’s multiple comparison test was used for statistical analysis. One representative of three independent experiments is shown (a-d).

[0142] Fig. 14 | Disrupted nuclear cGAS ubiquitylation primes type I IFN signalling, a, mRNA levels of cGAS were measured by RT-qPCR in HeLa cells cGAS KO cells reconstituted with wild-type cGAS (WT), cGASN513 / 4A(NN), or cGASK427 / 8R(KK). Ratios relative to WT levels are shown (n = 3). b, Expression levels of cGAS, IFNB1, IFIT1, or ISG15 were assessed by RT-qPCR in HeLa cells cGAS KO cells reconstituted with doxycycline-inducible wild-type cGAS (WT) (n = 3) or a cGAS mutant defective in DNA binding and ubiquitylation (KRKKNN (K173E / R176E / K407E / K411A / N513 / 514A)) (n = 3) after 4 days of doxycycline treatment, c, Cells from (b) were lysed and analysed by immunblot for cGAS and ISG levels. Vinculin was used as a loading control, d, mRNA levels of SPSB3 were measured by RT-qPCR after enforced expression or not of SPSB3 with doxycycline in HeLa cells (see Fig. 4g) (n = 3). e-g, mRNA expression levels of SPSB3, IFIT1, IFIT2, ISG15, and IFNB1 were assessed by RT- qPCR in primary endothelial cells (n = 3) (e), BJ-5ta fibroblast cells (n = 3) (f) and differentiated THP-1 cells (n = 3) or THP-1 cGAS KO cells (n = 3) (g) treated with non-targeting control siRNAs or siRNAs against SPSB3 for 5 days. Ratios of the relative expression of each transcript compared to the controls are shown. Data are mean ± SD. Numbers indicate the number of technical replicates (a-b, d-g). P values were obtained by one-way ANOVA (a, e, f-g) or two-tailed Student’s t-test (b, d). One representative of two (a) or three (b-g) independent experiments is shown.

[0143] Fig. 15 | cGAS and ISG15 measurements by immunoblot, a, cGAS and ISG15 measurements by immunoblot in HeLa cGAS KO cells transfected with mRNA of encoding for human cGAS or cGAS N513A / N514A (NN). Cells were harvested on day 0, 1 , 2, or 4 after transfection. Vinculin was used as a loading control, b, cGAS and ISG15 measurements by immunoblot in CT26 eGas KO cells transfected with mRNA of encoding for mouse cGAS or cGAS N498A / N499A (NN). Cells were harvested on day 0, 1 , 2, or 4 after transfection. Vinculin was used as a loading control.

[0144] The examples illustrate the invention.

[0145] Example 1 - Intranuclear degradation of cGAS

[0146] To determine the fate of nuclear cGAS, GFP-tagged cGAS was tracked by live cell imaging in HeLa cells. Consistent with previous findings16 17, nuclear envelope breakdown led to rapid recruitment of cytosolic cGAS onto mitotic chromosomes and prominent relocation of cGAS into the newly forming nuclei of both daughter cells (Fig. 1 a). Notably, over the course of the subsequent cell cycle - most prominently during G1 phase - the abundance of nuclear cGAS progressively decreased (Fig. 1 a, b, ad Fig. 5a). Concomitantly, a steady increase in cGAS presence in the cytosol was observed (Fig. 1 a and Fig. 5a). Chemical inhibition of the major nuclear export machinery by Leptomycin B had no effect on nuclear cGAS levels (Fig. 5b). Selective tracking of cGAS using a photoconvertable fusion construct (cGAS-Dendra2) confirmed prominent decrease of cGAS abundance in the nucleus with no reciprocal augmentation in the cytosol (Fig. 5c, d). To explain nuclear loss of cGAS, protein degradation through the UPS was considered18. Blocking the proteasome with Epoxomicin or Bortezomib stabilised intranuclear cGAS (Fig. 1 c, d, Fig. 5e-g). Critically, proteasomal degradation of nuclear cGAS was similarly observed for endogenous cGAS in distinct human cell lines (Fig. 1 e). Together, these results establish proteolysis via the UPS as an essential principle in the regulation of cGAS.

[0147] Example 2 - Discovery of CRL5SPSB3in cGAS control

[0148] To identify factors involved in nuclear cGAS degradation, an RNA interference (RNAi)-based genetic screen was conducted targeting 980 genes associated with the UPS and monitored nuclear cGAS abundance by confocal microscopy. Validating the experimental approach, siRNA-mediated knockdown of distinct subunits of the proteasome disrupted nuclear cGAS degradation (Fig. 1f). Strikingly, among the top scoring hits, all components of the core multi-subunit cullin-RING E3 ligase (CRL) 5 scaffolding complex (CUL5, RBX2, NEDD8, ELOB, ELOC), the cullin 5 (CUL5)-specific NEDD8 E2 ligase UBE2F, and a poorly characterised substrate receptor SPSB3 (Fig. 1f, g) were identified19-22. SPRY domain- and SOCS box-containing (SPSB) proteins bind - via their SOCS box - the Elongin B-Elongin C (ELOBC) heterodimeric adaptor connecting to the cullin 5-RING-box-protein 2 (CUL5-RBX2) complex and recruit substrates through the SPRY domain specifying protein ubiquitylation2023. In contrast to SPSB3, none of the other SPSB family members scored as hits in the screen, implying a specific role for SPSB3 in controlling nuclear cGAS protein stability (Fig. 6a). It was confirmed that a knockdown of CUL5 and SPSB3 attenuated the nuclear degradation of cGAS, while leaving its transcript levels unaffected (Fig. 1 h, i and Fig. 6b-d). Likewise, pharmacological targeting of cullin-RING E3 ligases by the neddylation inhibitor MLN4924 blocked nuclear cGAS degradation (Fig. 1 i and Fig. 6e). Reciprocally, overexpression of SPSB3 profoundly reduced nuclear cGAS levels (Fig. 6f). Consistent with a function in controlling protein turn-over, MLN4924 treatment increased the half-life of cGAS in cycloheximide chase experiments (Fig. 1j). Regulation of endogenous nuclear cGAS protein levels by SPSB3 was validated in HeLa cells, human monocytic THP-1 cells, BJ fibroblasts, and primary human endothelial cells (Fig. 6g-j). Together, these data identify a role for the cullin 5-RBX2-ELOBC E3 ligase assembly and its substrate receptor SPSB3 - a complex referred to as CLR5SPSB3- in the control of nuclear cGAS stability.

[0149] Example 3 - CLR5SPSB3ubiquitylates cGAS

[0150] Next immunoprecipitation experiments were performed to directly assess cGAS binding and ubiquitylation by CRL5SPSB3. SPSB3 efficiently immunoprecipitated cGAS upon ectopic expression in HEK293T cells (Fig. 2a). Further, expression of SPSB3, but not a SPSB3 mutant bearing a defective SOCS box, promoted cGAS ubiquitylation and this effect was blunted by treatment with MLN4924 (Fig. 2b, c). Both CUL5 and SPSB3 localise inside the nucleus and SPSB3 efficiently modifies cGAS fused to a nuclear localisation signal (NLS), verifying that SPSB3 directs CUL5 E3 ubiquitin ligase activity against the nuclear cGAS pool (Fig. 7).

[0151] To directly probe cGAS recognition by SPSB3, SPSB3 target binding was reconstituted in vitro using a truncated version of SPSB3 comprising the core structured part (amino acids: 83-326) stabilised by the ELOBC heterodimer. Recombinant cGAS robustly interacted with the SPSB3-ELOBC heterotrimeric complex (Fig. 2d and Fig. 8a, b). Deletion of the unstructured N terminus of cGAS (cGAS catalytic core domain (CD); amino acids: 155-522) did not affect its interaction with the substrate receptor complex, suggesting that SPSB3 recognises an element within the cGAS catalytic core (Fig. 2d). In vitro ubiquitylation assays demonstrated that the minimal functional CRL5SPSB3ligase complex promotes ubiquitylation of full-length cGAS and its catalytic domain (Fig. 2e and Fig. 8c). Consistent with accelerating CLR5-dependent ubiquitylation through E3-E3 super-assembly formation, addition of the RING-between-RING (RBR) E3 ligase ARIH2 enhanced cGAS ubiquitylation in vitro (Fig. 2e) (ref2425). To determine which lysine residues are targeted by CRL5SPSB3, ubiquitylated cGAS was analysed by mass spectrometry, which revealed five sites modified by CRL5SPSB3that are situated in the catalytic core region and in the N terminus (Fig. 2f and Fig. 8d). Mutagenesis identified the neighbouring lysine pair KK427 / 8 as the major residues ubiquitylated by CRL5SPSB3with minor contributions emerging from N terminal lysine residues (Fig. 2g and Fig. 8e). Substitution of the KK427 / 8 largely abolished cGAS ubiquitylation and nuclear degradation in cells, corroborating their critical role in dictating cGAS protein stability (Fig. 2h, i). Interestingly, despite the lack of sequence identity for the KK427 / 8 lysine pair at the amino acid level for cGAS orthologues, cGAS from mouse and other non-primate mammals encode for a conserved, surface exposed lysine pair (e.g., mus musculus cGAS KK409 / 410) in a region upstream within the same C terminal alpha helix (Fig. 9a, b). Robust ubiquitylation of the mouse cGAS catalytic domain was verified by CLR5SPSB3in vitro and confirmed the mcGAS KK409 / 410 lysine pair as major target site of ubiquitin modification (Fig. 9c, d). Together, these results demonstrate cGAS ubiquitylation by CLR5SPSB3and establish conserved targeting of a unique lysine pair within the catalytic core as the underlying mechanism of cGAS degradation.

[0152] Example 4 - Structure of the CGAS-SPSB3 complex

[0153] Inside the nucleus, cGAS is attached to chromatin by strong interactions with the histone H2A-H2B formed “acidic patch” on the surface of the nucleosome9-14. Considering a role in nuclear cGAS regulation, it was therefore hypothesised that SPSB3 might recognise nucleosome-bound cGAS. Consistent with this idea, immunoprecipitation assays and size-exclusion chromatography (SEC) indicated that SPSB3-ELOBC forms a complex with nucleosome-bound cGAS (Fig. 10a, b). Human cGAS, but not mouse cGAS, is capable of cross-bridging individual nucleosomes in vitro based on contacts between basic surface patches of the human-specific third DNA binding site, C site, and nucleosomal DNA10 1326. Mutational analysis showed that C site residues are not involved in cGAS binding to SPSB3, an observation that is in line with a shared mechanism of nuclear cGAS detection by the SPSB3 substrate receptor (Fig. 10a and 12a).

[0154] To define the molecular basis of cGAS recognition by SPSB3, Cryo-EM structures of wild-type and C site mutated cGAS catalytic core domains in complex with a nucleosome core particle (NCP) and the SPSB3-ELOBC heterotrimer were determined (Fig. 3a, b, Figs. 11 c-g and 12b-g). The overall topology of the CGAS-SPSB3 subcomplex within the two distinct maps is identical, but the complex incorporating wild-type cGAS is not as well resolved, probably owing to the heterogeneity of the sample with formation of varying higher-order cGAS-bridged nucleosome fibres (Fig. 10c). It was therefore focused on the structural analysis of the SPSB3-cGAS C site complex. A 3.5 A final reconstruction of this complex reveals an intermolecular interface contributed by the C-terminal helix on cGAS (amino acids: 497-515) and five variable loops extending from the bent p-sandwich core of SPSB3 opposite face of the SOCS box domain (Fig. 3a-c). The cGAS residues N513 and N514 form hydrogen bonds to SPSB3 loop residues S132, T162, T259, and R262 (Fig. 3c and Fig. 12a). Additionally, a set of acidic residues on cGAS, including D465, E509, Y510, R512 and E515 bridges to residues Y131 , Y160, Y197, and R213 on SPSB3 through electrostatic interactions reinforcing complex formation (Fig 3c and Fig. 12a). Although the overall composition of the substrate recognition module - comprised of the connecting loops extruding from the central SPRY domain27- is identical across SPSB paralogues, the residues involved in side chain contacts to the substrate are unique to SPSB3, reflecting its specific role in cGAS binding (Fig. 12b-d). Accordingly, the cGAS interface gives rise to a distinctive NN minimal sequence recognition motif differing from the previously described SPSB-recognised consensus motif2728. Notably, contacts comprising these major interactions are shared across all vertebrate DNA-sensing cGAS enzymes and SPSB3 receptors, respectively, defining a conserved mechanism of cGAS protein targeting by E3 ligases (Fig. 3d).

[0155] To model ubiquitylation of cGAS by CRL5SPSB3, an activated (neddylated) ELOBC-CRL5-ARIH2 E3-E3 complex29-31wass docked to the SPSB3-cGAS-nucleosome assembly (Fig. 3e)(ref10). In this model, cGAS and ubiquitin-bound ARIH2 are situated at opposite ends of the bent and elongated CRL5SPSB3core, facilitating ARIH2 to reach across the SPSB3-bound cGAS substrate for ubiquitin ligation. Remarkably, the ARIH2 active site cysteine is juxtaposed with K427 and K428 of cGAS, rationalising preferential ligation of this particular substrate site.

[0156] Next the CGAS-SPSB3 complex structure was combined with biochemical and cellular analysis to establish a molecular mechanism of nuclear cGAS control. Analysing the interaction by Bio-layer Interferometry (BLI), it was observed that SPSB3 detects cGAS with submicromolar affinity (Kd = 352 nM) (Fig. 4a). Strikingly, cGAS single substitutions of either asparagine residue of the predicted interface (N513, N514) disrupted all detectable binding to SPSB3, demonstrating that the conserved NN motif on cGAS is the main contributor to SPSB3 recognition (Fig. 4a). Single point mutation of the supporting cGAS residues (D465, E509, Y510, R512), but not the adjacent position (E515), weakened the interaction with SPSB3 and a combined mutation of all four cGAS residues completely blocked binding, hence confirming their importance in reinforcing complex formation (Fig. 13a). Likewise, a combined substitution of cGAS-interacting residues on SPSB3 (Y160, T162, T259, R262) abrogated cGAS binding (Fig. 13b). As expected from its central role in substrate receptor recruitment, mutation of the NN motif prevented cGAS ubiquitylation by CRL5SPSB3in vitro (Fig. 4b and Fig. 13c). Cellular expression of cGAS or cGAS-NLS together with SPSB3 confirmed that cGAS N513 / 4A (cGAS NN) lost the ability to bind SPSB3 and to undergo ubiquitylation (Fig. 4c and Fig. 13d). Consistently, compared to wild-type cGAS, cGAS NN showed markedly increased protein abundance upon reconstitution in HeLa cGAS KO cells, establishing the NN pair as a bona fide degron motif dictating nuclear cGAS stability (Fig. 4d, and Fig. 13e).

[0157] Example 5 - Nuclear cGAS impacts IFN tone

[0158] To determine the biological consequences of nuclear cGAS control by CRL5SPSB3, cell intrinsic type I IFN responses were monitored, the most prominent cellular outcome of cGAS activity3233. Reconstitution of HeLa cGAS KO cells with SPSB3-binding deficient cGAS (N513 / 4A mutant; NN) or ubiquitylation-resistant cGAS (K427 / 8R mutant; KK) induced heightened levels of IFNB1 and interferon- stimulated gene (ISG) relative to reconstitution with wild-type cGAS (Fig. 4d-f and Fig. 14a). Additional mutation of residues involved in DNA binding834abrogated the type I IFN response induced by expression of the cGAS N513 / 4A variant, demonstrating that cellular activation results from aberrant engagement of nuclear DNA (Fig. 14b, c). Conversely, ectopic expression of SPSB3 from a doxycycline- inducible promoter (Dox) lowered baseline expression of ISGs in HeLa cells (Fig. 4g and Fig. 14d). Using a complementary approach, it was found that silencing of SPSB3 increased type I IFN signalling in various cell types and this response was strictly dependent on cGAS (Fig. 4h and Fig. 14e-g). To probe the relevance of heightened type I IFN signalling, HeLa cGAS KO cells reconstituted with either wild-type cGAS or degradation-defective cGAS variants were infected with the DNA viruses herpes simplex virus-1 (HSV-1) and vaccinia virus (VACV). In both cases, expression of cGAS mutants with disrupted nuclear degradation reduced susceptibility of cells to infection (Fig. 4i). Conversely, enforced degradation of cGAS by SPSB3 overexpression increased the susceptibility of cells to infection with both HSV-1 and VACV (Fig. 4j). Together, these results demonstrate that nuclear cGAS levels impact the cellular IFN tone and reveal a role for CRL5SPSB3in cell intrinsic immunity.

[0159] Example 6 - Methods

[0160] Cell culture

[0161] HeLa (CCL-2) cells were obtained from Sigma-Aldrich. HEK 293T cells were a gift from D. Trono (EPFL), originally purchased from ATCC. U2OS cells were obtained from ATCC. THP-1 cells and BJ-5ta cells were obtained from ATCC. Primary human endothelial cells were obtained from a commercial supplier (Cell Biologies). HeLa and HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, 41965039) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco SKU, 10270106), 100 IU ml-1penicillin / streptomycin (BioConcept, 4-01 F00-H), 2 mM L-glutamine (Thermo Fisher Scientific, 25030024) and 1 mM sodium pyruvate (BioConcept, 5-60F00-H) at 37°C and at atmospheric O2 and 5% CO2. THP-1 cells were cultured in RPMI 1640 Medium (Thermo Fisher Scientific, 21875091) supplemented with 10% FBS, 1x penicillin-streptomycin-L-glutamine (Corning, 30-009-CI), and 1x 2-mercaptoethanol (Gibco) at 37°C and at atmospheric O2 and 5% CO2, and differentiated with 10 pg mL-1phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, P8139) for 2 days prior to experiments. BJ-5ta cells were cultured in a mixed medium consisting of 80% of Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, 41965039) and 20% of Medium 199 (Thermo Fisher Scientific, 41150087) supplemented with 10% FBS, 2 mM L-glutamine, 1x penicillin-streptomycin-L-glutamine. Primary human endothelial cells were cultured in complete human endothelial cell medium (Cell Biologies, H1168), according to the supplier’s instructions. cGAS KO HeLa cells were reported in ref.36. U2OS cGAS KO cells were generated as described in ref.36cGAS KO THP-1 cells were purchased from Invivogen. Cell lines were repeatedly tested for mycoplasma by PCR. No method of cell line authentication was used.

[0162] Plasmids

[0163] For CRISPR / Cas9 plasmids, single guide RNAs (sgRNAs) targeting cGAS were designed using the web tool CRISPOR37. SgRNA targeting cGAS was cloned in pSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid (Addgene, 62988). pBABE-HA-Ubiquitin was generated by inserting amplified HA-Ubiquitin sequences from HA-Ubiqitin (Addgene, 18712) into pBABE vector. The pEFBos-cGAS-FLAG, pEFBos-SPSB3- FLAG, and pEFBos-GFP-FLAG were obtained by inserting cGAS, SPSB3, and GFP sequences flanked by 5’ Xhol and 3’ Notl sites into the pEFBos vector. The pEFBos-based cGAS mutations (N513 / 514A, K427 / 428R and KRKKNN (K173E, R176E, K407E, K411A, N513 / 514A)) and SPSB3 truncations (SPSB3ASOCS) were obtained by site-directed mutagenesis. The pTRIPZ-based cGAS and cGAS mutations (N513 / 514A, K427 / 428R, and KRKKNN (K173E, R176E, K407E, K411A, N513 / 514A)) were generated by inserting the coding sequences of cGAS flanked by 5’ Xhol and 3’ Notl sites into the pTRIPZ vector.

[0164] Generation of cell lines

[0165] To establish HeLa cells or U2OS cells with inducible expression of distinct cGAS constructs, corresponding cGAS KO cells were infected with a pTRIPZ lentiviral vector carrying cGAS-GFP or cGAS-Dendra2 and a puromycin-resistance gene. Cells were selected with puromycin (1 pg ml-1). To generate HeLa cGAS-GFP cells co-expressing PCNA-mCherry, HeLa cGAS-GFP cells were infected with a pRRL lentiviral vector (kind gift from D. Trono, EPFL) carrying PCNA-mCherry and a blasticidin- resistance gene. Cells were selected using blasticidin (5 pg ml-1). HeLa cells expressing doxyxycline- inducible FLAG-cGAS, FLAG-cGAS N513 / 4A, FLAG-cGAS K427 / 8A or FLAG-cGAS KRKKNN (K173E, R176E, K407E, K411A, N513 / 514A), respectively, were generated from HeLa cGAS KO by infection with a pTRIPZ lentiviral vector carrying the corresponding inserts and a puromycin resistance gene. Cells were selected with puromycin (1 pg mL-1).

[0166] Transfection

[0167] For plasmid transfection, cells plated in a 6-well plate were transfected with plasmids and GeneJuice transfection reagent (Millipore, 70967) following the manufacturer’s protocol.

[0168] For siRNA transfection, 3 x 104cells were transfected with Lipofectamine™ RNAiMAX transfection reagent (Invitrogen, 13778075) and 40 pmol siRNA following the manufacturer’s protocol followed by 3- 5 days of incubation. Medium containing transfection reagents was replaced with fresh medium 6 h posttransfection. siRNA targeting SPSB3 (s40519, S40520), CUL5 (s15588) and negative control (4390847) were purchased from Thermo Fisher Scientific.

[0169] RNAi screen targeting factors of the UPS

[0170] To identify regulators of nuclear cGAS stability, HeLa cells expressing doxycycline-inducible cGAS-GFP were treated with siRNAs targeting 980 UPS genes (three independent siRNAs per target) using the A30140 Silencer™ Select Human Ubiquitin siRNA library (Thermo Fisher Scientific) . Each plate (96- well PhenoPlate (PerkinElmer)) comprised 8 control wells, including 2 wells with negative control siRNA, 2 wells with a cGAS-targeting siRNA, and 4 wells containing Epoxomicin (100ng mL-1). For reverse transfection, 5 pL of siRNA (200 nM in Opti-MEM (Thermo Fisher Scientific)) were mixed with 5 pL Lipofectamine™ RNAiMAX (Thermo Fisher Scientific) (pre-diluted 1 :20 in OptiMEM) inside each well. After 15 min incubation at room temperature, 3,500 cells were added to each well in 100 pL complete DMEM (10% FBS, 1 % penicillin-streptomycin-glutamine solution, 1 pg mL-1doxycycline). After 48 hours, Epoxomicin was added to the respective control wells. Following overnight incubation (day 3 after reverse transfection), cells were washed with PBS and fixed by addition of a paraformaldehyde 4% solution in PBS supplemented with Hoechst (1 pg mL-1). Fixed cells were stored in PBS at 4°C prior to imaging. Imaging of cells (entire well; 21 images at 10 x magnification) was performed using the IN Cell Analyzer 2200 microscope (GE Healthcare Life Sciences). Analysis of nuclear cGAS-GFP levels was performed using the CellProfiler software. In brief, each nucleus was identified by its Hoechst signal and intranuclear cGAS-GFP mean fluorescence intensity and integrated fluorescence intensity was quantified. Final results were obtained by calculating relative values for mean fluorescence intensity and integrated fluorescence intensity relative to the controls (cGAS-targeting siRNA = -1 ; negative control siRNA = 0; Epoxomicin = 1) and data were visualized by blotting relative mean fluorescence intensity levels against integrated fluorescence intensity levels. Genes were defined as hits if their relative fluorescence intensity values deviated from the negative control siRNA by more than three times the standard deviation.

[0171] Live cell imaging

[0172] HeLa cells expressing cGAS-GFP were plated in Falcon® 96-well microplates (black; clear flat-bottom; TC-treated) (Corning) at a density of 3,500 to 7.500 cells per well in complete FluoroBrite™ (Thermo Fisher Scientific) DMEM (10% FBS, 2 mM L-Glutamine, 1 % penicillin-streptomycin, 1 pg mL-1doxycycline). At the next day, cells were transfected or not with siRNAs and incubated for two days. Drug treatment, including Epoxomicin (500 nM), Bortezomib (100 nM), Leptomicin B (80 nM), was performed 1 h prior to live cell recording. Imaging was performed using a confocal Leica SP8 Inverted microscope equipped with an HC PL APO 63x / 1 ,40 / oil (magnification / N.A. / immersion) objective and HyD detectors, operated with the Leica LAS X software. Images were acquired every 5 - 10 minutes. Live cell imaging of cGAS-GFP-WT and cGAS-GFP-NN (N513 / 4A) was performed on a Zeiss LSM980 Inverted microscope (Carl Zeiss) equipped with a motorized, heated stage and a full incubation chamber maintaining 37°C and 5% CO2 and operated with the Zeiss ZEN software. Images were acquired every 3-5 minutes. Image analysis was performed by Fiji (v.2.3.0) on a nucleus-by-nucleus basis and data were further quantified by GraphPad PRISM 9 (v.9.3.1). For cell cycle phase analysis, a single cell nucleus was tracked overtime using PCNA-mCherry as a mask for the nucleus, while recording cGAS- GFP nuclear intensity. A change of PCNA-mCherry signal from a diffuse pattern to a dot-like pattern was used to define the transition from G1 to S phase of the cell cycle, whereas the reverse change was used to define the transition from S to G2 phase of the cell cycle.

[0173] In experiments using cGAS-Dendra2, Dendra2 was photoactivated prior to live cell imaging using the 405 nm laser (700 Hz; laser power: 7.5%) to scan the same region of interest four times (frame average).

[0174] Immunofluorescence and confocal imaging

[0175] Cells were plated in CellCarrier-96 Ultra Microplates (Perkin Elmer, 6055302) at a density of 10,000 cells per well with at least 5 h incubation for adherence of the cells. For immunofluorescence, cells were washed once with PBS, then cells were fixed by adding paraformaldehyde 4% in CBS buffer (10 mM MES pH 6.9, 138 mM KCI, 2 mM MgCh, 2 mM EGTA) for 5-10 minutes at room temperature. Cells were washed 3 times at least 5 minutes in PBS before blocking for 1-2 h at room temperature with PBS supplemented with 0.1 % (v / v) Triton X-100 and 5% (v / v) heat-inactivated FBS and incubating overnight at 4°C with the primary antibodies diluted in staining solution (PBS supplemented with 0.1 % (v / v) Triton X-100) and 1 % (w / v) bovine serum albumin (Sigma-Aldrich, A7906)). The cells were washed with PBS 3 times 5 minutes and incubated for 1 h at room temperature in secondary antibodies diluted in staining solution. From then on, the plate was protected from light. Cells were washed twice more (5 min each) in PBS and incubated 30-60 minutes in Hoechst 33342 (Sigma-Aldrich, B2261) 0.2 pg mL-1in PBS. Cells were kept in 100 pL PBS / well and either imaged directly or kept at 4°C until imaging. Imaging was performed using a Zeiss LSM 980 Inverted microscope (Carl Zeiss) using the Plan-Apochromat 63x / 1 ,40 / oil (magnification / N.A. / immersion) objective. Image analysis and quantifications were performed with Fiji (v2.3.0).

[0176] Antibodies

[0177] Primary antibodies used: mouse monoclonal anti-Vinculin (hVIN-1) (Sigma-Aldrich, V9264, immunoblot 1 :5000; Jess 1 :130), rabbit monoclonal anti-GAPDH (14C10) (Cell Signaling Technology, 2118, immunoblot 1 :3000), mouse monoclonal anti-FLAG (M2) (Sigma-Aldrich, F1804, immunoblot 1 :5000), rabbit monoclonal anti-ISG15 (EPR24482-49) (abeam, ab285367, immunoblot 1 :1000), rabbit monoclonal anti-cGAS (D1 D3G) (Cell Signaling Technology, 15102, immunoblot 1 :1000; Jess 1 :75), rabbit monoclonal anti-cGAS (E5V3W) (Cell Signaling Technology, 79978, immunoblot 1 :1000), mouse monoclonal anti-Ubiquitin (P4D1) (Santa Cruz, sc-8017, 1 :500), rabbit polyclonal anti-SPSB3 (Novus Biologicals, NBP2-20480, immunoblot 1 :1000), rabbit polyclonal anti-SPSB3 (Invitrogen, PA5-61257, immunofluorescence 1 :100), rabbit polyclonal anti-Cullin5 (Abeam, ab264284, immunofluorescence 1 :100), mouse monoclonal anti-His-Tag (27E8) (Cell Signaling Technology, 2366, immunoblot 1 :1000), rabbit monoclonal anti-H2B (Abeam, 52484, immunoblot 1 :5000). HRP-conjugated secondary antibodies used: Donkey anti-rabbit IgG (H+L)-HRP (Jackson ImmunoResearch, 711-036-152, immunoblot: 1 :5000) and Donkey anti-mouse IgG (H+L)-HRP (Jackson ImmunoResearch, 715-036- 151 , immunoblot: 1 :5000). Fluorescence-conjugated secondary antibodies used: Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568-conjugated (Invitrogen, A-11011 , immunofluorescence 1 :800).

[0178] Quantitative real-time PCR (RT-qPCR)

[0179] Cells were lysed in the RLT buffer (Qiagen). RNA was extracted following the manufacturer’s protocol (Qiagen RNeasy Mini Kit). RNA was reverse transcribed using PrimeScript™ RT Reagent Kit (Takara) and analysed by RT-qPCR in triplicates using the Maxima SYBR Green / ROX qPCR Master Mix (Thermo Fisher Scientific). The qPCR reactions were run on a Quantstudio 7 Real-Time PCR system (Thermo Fisher Scientific). GAPDH was used as a housekeeping gene for normalization.

[0180] Cell fractionation

[0181] Cells from a 10-cm2dish were lysed with cold 400 pL buffer A (10mM HEPES pH 7.9, 10 mM KCI, 1. 5mM MgCh, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 20% Triton X-100) on ice for 5 min. Cytosolic fractions were collected by centrifugation at 3500 rpm, 4°C for 5 min. Pellets were washed twice with buffer A before being resuspended with 400 pL buffer B (3 mM EDTA, 0.2 mM EGTA) on a rotator at 4°C for 30 min. The soluble nuclear fractions were collected by centrifugation at 4000 rpm, 4°C for 5 min. The chromatin pellets were then washed with buffer B twice until they became almost invisible before being lysed with salt buffer (50 mM Tris pH 7.5, 300 mM NaCI, 1 % Triton X-100, 1 mM DTT) and boiled with 1 x loading buffer for 20 min.

[0182] Immunoblotting and immunoprecipitation Cells were harvested, quickly rinsed with 1x PBS, and lysed in lysis buffer (20 mM Tris pH 7.4, 0.5% Triton X-100, 150 mM NaCI, 1.5 mM MgCh, 2 mM EGTA, 2 mM DTT and 1x complete™ Protease Inhibitor Cocktail (Roche)) on ice for 30 min and centrifuged at 12,000 rpm, 4°C for 10 min. Supernatants were boiled with 4x loading buffer (200 mM Tris pH 6.8, 8% SDS, 40% glycerol, 0.4 M DTT, 0.4% Bromophenol blue) for 10 min. Proteins were resolved by SDS-PAGE using SurePAGE™ precast gels (GenScript) and transferred to nitrocellulose membranes using the Trans-Blot Turbo RTA Midi Nitrocellulose Transfer Kit (Bio-Rad) following the manufacturer’s instructions. Membranes were blocked with 3% skim milk in PBST (PBS + 0.05% Tween-20) at room temperature for 1 h and then incubated with the primary antibody (diluted in PBST) at 4°C overnight. After washing in PBST, membranes were incubated with the secondary antibody at room temperature for 1 h. Membranes were washed with PBST, visualized with Western Blotting Detection Reagent (Bio-Rad), and imaged with using the ChemiDoc XRS Biorad Imager and Image Lab Software.

[0183] For immunoprecipitation, cells were seeded into 6-well plates and were transfected with indicated plasmids. 16 h post-transfection, cells were lysed in IP lysis buffer (PBS supplemented with 1 % TEGITOL solution, 0.5% sodium deoxycholate, 1x complete™ Protease Inhibitor Cocktail (Roche) and 1 pL Benzonase on ice for 30 min and centrifuged at 12,000 rpm 4°C for 10 min. Supernatants were transferred into new tubes and mixed with anti-FLAG M2 Magnetic beads (Sig ma- Aid rich, M8823) or ChromoTek GFP-Trap agarose (Proteintech, gta) at 4°C overnight on a rotator. After 3-6 washes with IP wash buffer (PBS supplemented with 300 mM NaCI), beads or agaroses were boiled with 1x loading buffer for 10 min. Gels were loaded with 20 pL samples, followed by SDS-PAGE and immunoblot analysis.

[0184] Cycloheximide chase experiments

[0185] HeLa cells were treated with 300 nM Pevonedistat (MLN4924) for 8 h followed by treatment with cycloheximide (20 pg ml-1) for different time points. Total protein was extracted from cell pellets using cell lysis buffer (50 mM tris HCI, 0.14 M NaCI, 1 mM EDTA, 1 % NP40, 10% glycerol, 25 pM complete protease inhibitor (Roche)). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Samples were diluted in Laemmli buffer and boiled for 10 min at 95° C. The expression of the indicated proteins was determined using the Jess automated Western blotting system (ProteinSimple, San Jose, CA, USA). Jess reagents (biotinylated molecular weight marker, streptavidin-HRP fluorescent standards, sample buffer, DTT, stacking matrix, separation matrix, running buffer, wash buffer, matrix removal buffer, fluorescent labeled secondary antibodies, antibody diluent, and capillaries) were purchased from the manufacturer and used according to the manufacturer’s standard protocol. Antibodies were diluted with ProteinSimple antibody diluent at the following dilutions: anti-cGAS (1 :75), anti-Vinculin (1 :130). Target protein concentration is quantitated using Compass for SW 6.1 software. The expression of each target protein is normalized to the expression of vinculin. Total protein expression was analyzed using area under the curve (AUC) measurements generated using Compass software for Simple Western (Protein Simple).

[0186] Protein expression and purification Halo-tagged human cGAS WT, Halo-tagged cGAS catalytic domain (aa: 155-522) and the corresponding substitutions in the pET-28 vector were expressed in BL21 (DE3) bacteria (Sigma- Aldrich, CMC0014). A single colony was inoculated in a culture flask with 100 mL LB with kanamycin (50 pg mL-1) and incubated with shaking (200 rpm, Infors-HT Multitron) at 37 °C overnight as preculture. Large-scale expression of the protein was started the next day by pouring 100 mL of the preculture in a 5 L Erlenmeyer flask containing 2 L LB with kanamycin (50 pg mL-1). The cells were grown until the optical density (OD) at 600 nm reached 0.7. Expression was then induced by adding isopropyl p-D-1- thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM while transferring the culture to an 18 °C shaking incubator overnight. The bacteria were then collected by centrifugation, solubilized in HisTrap buffer A (20 mM HEPES, 500 mM NaCI, 20 M imidazole, 1 mM DTT and 5% glycerol, pH 7.5) supplemented with 4-(2-aminoethyl)-benzolsulfonylfluoride-hydrochloride (AEBSF) and complete protease inhibitor (Roche), lysed by sonication, cleared by centrifugation at 20,000 g and then passed through a 5 mL nickel immobilized metal-affinity chromatography column (Cytiva, HisTrap HP, 17524802) on an fast protein liquid chromatography (FPLC) system. The protein of interst was eluted with buffer B (20 mM HEPES, 500 mM NaCI, 500 mM imidazole, 1 mM DTT and 5% glycerol, pH 7.5). Halo-tagged cGASo was then purified by size-exclusion chromatography through a Superdex 75 Hiload 16 / 600 column (Cytiva 28-9893-33). For untagged cGAS, TEV enzyme was mixed with Halo-tagged cGAS overnight, and untagged cGAS was then purified by size-exclusion chromatography through a Superdex 75 Hiload 16 / 600 column (Cytiva 28-9893-33). Halo-tagged mouse cGAS was prepared using the same procedure. All mutants were generated using a PCR-based technique with appropriate primers and confirmed by DNA sequencing.

[0187] The cDNA of human SPSB3 (aa: 83-326) or its corresponding mutant was cloned into a pET-28 vector with an N-terminal His6-SUMO tag. SPSB3 was expressed in BL21 (DE3) (Sig ma- Aid rich, CMC0014). A single colony was inoculated in a culture flask with 400 mL LB with kanamycin (50 pg mL-1) and incubated with shaking (200 rpm, Infors-HT Multitron) at 37 °C overnight as pre-culture. Large-scale expression of the complex (8 L in total: 2 L in four 5-L Erlenmeyer flasks) was started the next day by pouring 100 mL of preculture into 2 L of Auto Induction Media Terrific Broth (Formedium, AIMTB0210) with kanamycin (50 pg mL-1). Flasks were incubated with shaking at 37 °C for 6 h, then incubated at 18 °C overnight. The cells were then collected by centrifugation (4,000 g, 15 min). The cell pellet of a 2 L expression culture was transferred into a Falcon 50-ml tube. The 2-L expression cells were solubilized in PBS with 1 mM DTT, 1 mM EDTA and 2% glycerol at pH 7.5, supplemented with AEBSF and complete protease inhibitors (Roche), then lysed by sonication. The cell lysate was clarified by centrifugation followed by 0.45-pm filtration. The supernatant was first purified on a 5 mL nickel immobilized metal-affinity chromatography column (Cytiva, HisTrap HP, 17524802) on a FPLC system (Cytiva Akta Pure). After ULP1 cleavage at 4 °C overnight in a 3,500 molecular weight cut-off dialysis tubing against PBS with 5% glycerol at pH 7.5. To remove the ULP1 protease as well as free SUMO, the sample was passed through a HisT rap and washed with PBS with 20 mM Imidazole. The flowthrough was collected and concentrated using a Superose 6 HiLoad 16 / 600 size-exclusion chromatography column (Cytiva 29323952) equilibrated in PBS. All mutants were generated using a PCR-based technique with appropriate primers and confirmed by DNA sequencing. Recombinant proteins required for in vitro ubiquitylation assay were expressed in BL21 (DE3) cells, except UBE1 and the neddylated CUL5-RBX2 complex (purchased from Bio-Techne, E-305-025 and E3-451-025). Briefly, the human UBE2R1 (UniProt: P49427) coding sequence was cloned into pET-28- His-thrombin vector to enable expression as an N-terminal His-thrombin fusion protein; the human UBE2L3 (UniProt: P68036) coding sequence was cloned into pGEX-4T-1 vector to to enable expression as an N-terminal GST-fusion protein; the human ARIH2 (UniProt: 095376) coding sequence was cloned to pGEX-4T-1 vector to enable expression as an N-terminal GST-fusion protein. Following transformation of BL21 (DE3) competent cells, protein expression (UBE2R1 , UBE2L3, ARIH2) was induced either by addition 1 mM IPTG when the OD at 600 nm reached 0.6, and bacteria were cultured at 18°C overnight. For His-tagged proteins, cell pellets were collected and suspended in lysis buffer (20 mM imidazole, 50 mM Tris pH 8.0, 300 mM NaCI, 5% glycerol, 5 mM BME) supplemented complete protease inhibitors (Roche). After sonication, cell lysates were clarified by centrifugation at 20,000 g for 30 min. The supernatants were collected and incubated with Ni-NTA agarose at 4°C for 2 h. Resins were washed and bound protein was eluted by 250 mM imidazole in buffer (50 mM Tris pH 8.0, 300 or 400 mM NaCI, 5% glycerol, 5 mM BME). To remove tags, TEV protease or thrombin was added to the eluates and untagged proteins were further purified by gel filtration with superdex 75 increase 10 / 300 column (Cytiva, lot. 29148721) equilibrated with buffer (25 mM Tris pH 8.0, 200 mM NaCI, 5% glycerol, 1 mM DTT). Protein purify was checked by SDS-PAGE electrophoresis and pure fractions were pooled, flash-frozen in liquid nitrogen and stored at -80°C until further use.

[0188] For GST-tagged proteins, cell pellets were collected and suspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCI, 5% glycerol, 5 mM BME) supplemented with complete protease inhibitors (Roche). After sonication, cell lysates were clarified by centrifugation at 20,000 g for 30 min. The supernatants were collected and incubated with Glutathione Sepharose Resins followed by washing and the bound protein was eluted using 15 mM GSH in lysis buffer. Removal of GST tag was facilitated by adding thrombin to the eluates and the untagged protein was further purified by a second Glutathione sepharose column followed by gel filtration with superdex 75 increase 10 / 300 column equilibrated with 25 mM Tris pH 8.0, 200 mM NaCI, 5% glycerol, 1 mM DTT. Fractions containing pure protein were pooled, flash-frozen in liquid nitrogen and stored at -80°C until further use.

[0189] Bio-layer interferometry

[0190] Bio-layer interferometry analyses were performed at 25 °C using a GatorPrime biosensor system (GatorBio) with anti-His probes (GatorBio, 160009). cGAS or cGAS mutants (50 pg ml-1) were immobilized onto the anti-His biosensor for 1 min. The tips were washed with PBS buffer for 2 min to obtain a baseline reading, then the biosensors were dipped into wells containing the various concentrations of SPSB3 or its mutant for 5 min, which was followed by a 10-min buffer wash to allow the dissociation of molecules from the sensor. Data analysis was performed with GraphPad PRISM 9 using a standard 1 :1 binding model. Three independent experiments were performed for each sample.

[0191] Cryo-EM data acquisition

[0192] 1 mg cGAS (WT)-Halo-SPSB3-ELOBC or 1 mg cGAS (SiteC)-Halo-SPSB3-ELOBC complex was incubated with 1 mg recombinant mononucleosome core particle (Active motif, 81770) for 30 min on ice in PBS. Fractionations corresponding to the complex were collected on a Supedex 200 increase 10 / 300 GL column (Cytiva) in PBS and concentrated to 1 mg mL“1. Aliquots of 3 pL of the cGAS-Halo-SPSB3- ELOBC-nucleosome complexes were loaded onto glow-discharged holey carbon grids (Electron Microscopy Sciences, CF-2 / 2-3Cu-50, C-Flat, Cu, R 2 / 2, 300 mesh). Grids were blotted for 4 s and plunge-frozen in liquid ethane using a Vitrobot at 4 °C and with 100% humidity. Grids were screened for particle presence and ice quality on a TFS Glacios microscope (200 kV), and the grids with the best quality were transferred to a TFS Titan Krios G4. Cryo-EM data were collected using a TFS Titan Krios G4 transmission electron microscope (TEM), equipped with a Cold-FEG on a Falcon IV detector in electron counting mode. Falcon IV gain references were collected just before data collection. Data were collected with TFS EPU v.2.12.1 using aberration-free image shift protocol (AFIS), recording eight micrographs per ice hole. Movies were recorded at a 96,000 x magnification, corresponding to the 0.83 A pixel size at the specimen level, with defocus values ranging from -0.8 to -1 .8 pm. Exposures were adjusted automatically to 60 e" A-2total dose, resulting in an exposure time of approximately 5 s per movie. In total, 9,855 micrographs in EER format were collected for the cGAS WT complex and 15,141 micrographs in EER format were collected for the cGAS SiteC sample.

[0193] Cryo-EM data processing

[0194] Motion correction was performed on raw stacks without binning using the cryoSPARC implementation of motion correction. For the cGAS (WT)-Halo-SPSB3-ELOBC-nucleosome complex, a total of 972,457 particles were template-based automatically picked and particles were binned by a factor of 4. Two rounds of two-dimensional (2D) classification were performed, resulting in a particle set of 378,280 particles. Selected particles resulting from the 2D classification were used for ab initio reconstruction with 2 classes, and the one containing the nucleosome shape was selected as the initial model. A heterorefinement was done on three repeating initial models on the resulting 2D particles. A class with clear cGAS density was selected, including 69,435 particles. To deal with the sample heterogeneity, a mask on a single nucleosome and cGAS-SPSB3-ELOBC was generated for masked 3D-Variability analysis with 3 modes. A mode showing variability on SPSB3 was selected for clustering with 10 classes, among which, 4 classes containing SPSB3-ELOBC density were selected and re-centered and re-extracted. The particles were subjected to iterative CTF refinement and non-uniform refinement in cryoSPARC to 4.30 A. The reported resolutions are based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion. Forthe cGAS (SiteC) Halo-SPSB3-ELOBC-nucleosome complex, a total of 5,257,474 particles were template-based automatically picked and particles were binned by a factor of 4. Two rounds of two-dimensional (2D) classification were performed, resulting in a particle set of 1 ,400,892 particles. Selected particles resulting from the 2D classification were used for ab initio reconstruction with 2 classes, and the one containing the nucleosome shape was selected as the initial model, including 1 ,183,961 particles. A non-uniform refinement refines the map to 7.03 A. To deal with the sample heterogeneity, a mask on the single nucleosome and cGAS-SPSB3-ELOBC was generated for masked 3D-Variability analysis with 3 modes. A mode showing variability on SPSB3 was selected for clustering with 20 classes, among which, 9 classes containing SPSB3 density were selected. Then a mask on cGAS-SPSB3-ELOBC was generated and used for local refinement, followed by a masked 3D- Variability analysis with 3 modes with the same mask. A mode showing variability on ELOBC was selected for clustering with 20 classes, among which, 7 classes containing ELOBC density were selected, re-centered and re-extracted. The particles were subjected to iterative CTF refinement and non-uniform refinement in cryoSPARC to a resolution of 2.75- A. A local refinement on CGAS-SPSB3- ELOBC refined the map to 3.51 A. The reported resolutions are based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion. Local-resolution variations were estimated using cryoSPARC38.

[0195] Model building and refinement

[0196] The nucleosome-cGAS-SPSB3-ELOBC model was generated using a published nucleosome cGAS structure (PDB: 6X59 (ref11), 6Y5E (ref10) and 4LEV (ref39)). Direct prediction of the CGAS-SPSB3- ELOBC complex failed to find SPSB3 interacting with cGAS by Alphafold2. The SPSB3-ELOBC model from Alphafold2 was docked into the cryo-EM map in ChimeraX35and tuned by ISOLDE40and Coot41. The whole model was then refined in PHENIX42. Several loop regions of cGAS and SPSB3 were manually adjusted to fit into the map using Coot. The model was refined in real space again in PHENIX. All structure figures were made using UCSF Chimera43and UCSF ChimeraX35.

[0197] Sequence alignments

[0198] 150 human CGAS / SPSB3 orthologues in vertebrates were individually aligned and downloaded in ClustalW format from the Ensembl database. The sequence logo was derived from WebLogo (webloqo.berkeley.edu) on the indicated alignment regions. Human SPSB172 / 3 / 4 sequence as well as their Percent Identity Matrix analysis were downloaded from Uniprot. Geneious Prime software was used to generate the sequence alignments shown in Fig. 9a and Fig. 12c.

[0199] In vitro ubiquitylation assays

[0200] Human cGAS ubiquitylation assay was performed at 37°C for 40 min. Reactions (20 pL) consisted of 50 pM ubiquitin, 100 nM UBE1 , 1 pM UBE2R1 , 0.35 pM CUL5-RBX2-NEDD8 complex, 0.7 pM SPSB3- ELOBC complex, and 1 pM cGAS or cGAS mutants in buffer (50 mM Tris-HCI pH 7.5, 50 mM NaCI, 2.5 mM MgCL, 1 mM ATP, 2 mM DTT). If needed, 1 pM UBE2L3 and 300 nM ARIH2 were also added to the reaction. Reactions were initiated by ATP and quenched with 5x SDS-PAGE loading buffer after 40 min. Ubiquitylated cGAS products were separated on 4%-20% SurePAGE (GenScript) and probed with cGAS monoclonal antibody (Cell Signaling, E5V3W). Ubiquitylation assay for mouse cGAS was slightly modified: 200 nM UBE1 , 2 pM UBE2L3 and 600 nM ARIH2 were added to the reaction and incubation time was extended to 3h at 37°C. Ubiquitylated mcGAS products were probed with His-tag monoclonal antibody (Cell signaling, 27E8).

[0201] Identification of ubiquitylation sites on cGAS

[0202] To map lysine residues on cGAS modified by ubiquitin, in vitro ubiquitylation reactions were carried out in the presence of UBE2L3 and ARIH2. After SDS-PAGE electrophoresis, the gel was stained by Coomassie Blue and putative ubiquitylated cGAS bands were excised for mass spectrometry (MS) analysis. MS-based proteomic experiments were performed at the Proteomics Core Facility of the School of Life Sciences at EPFL. The selected gel pieces were excised and washed twice with 50% ethanol in 50 mM ammonium bicarbonate (Sigma-Aldrich) for 20 min and dried by vacuum centrifugation. Proteins were reduced with 10 mM dithioerythritol (Merck-Millipore) for 1 h at 56°C followed by a washing-drying step as described above. Reduced proteins were alkylated with 55 mM lodoacetamide (Sigma-Aldrich) for 45 min at 37°C in the dark followed by a washing-drying step as described above. Proteins were digested overnight at 37°C using mass spectrometry grade Trypsin Gold (Promega) at a concentration of 12.5 ng pl-1in 50 mM AB supplemented with 10 mM CaCh. Resulting peptides were extracted in 70% ethanol, 5% formic acid (Merck-Millipore) twice for 20 min, dried by vacuum centrifugation and stored at -20 °C until further analysis.

[0203] LC-MS / MS analysis

[0204] Peptides were desalted on C18 StageTips and dried by vacuum centrifugation prior to LC-MS / MS injections44. Samples were resuspended in 2% acetonitrile (Biosolve), 0.1 % formic acid and nano-flow separations were performed on a Dionex Ultimate 3000 RSLC nano UPLC system (Thermo Fischer Scientific) online connected with an Q Exactive Orbitrap Mass Spectrometer (Thermo Fischer Scientific). A capillary precolumn (Acclaim Pepmap C18, 3 pm-100A, 2 cm x 75 pm ID) was used for sample trapping and cleaning. A 50 cm long capillary column (75 pm ID; in-house packed using ReproSil-Pur C18-AQ 1.9 pm silica beads) was then used for analytical separations at 250 nl min-1over 90 min biphasic gradients. The mobile phases were as follows: A, 2% acetonitrile, 0.1 % formic acid in water and B, 90:10 (v / v) acetonitrile:water, 0.1 % formic acid. The mass spectrometer was operated in positive data-dependent acquisition mode, and the full MS range was from 300 to 2,000 m / z. The 12 most intense ions were isolated in the quadrupole and fragmented under high-energy collisional dissociation with a normalized collision energy of 27% with a 30s exclusion list. Precursor and fragment ions were measured at a resolution of 70,000 and 17,500 (at 200 m / z) respectively. Only ions with charge states of 2 and higher were fragmented with an isolation window of 1 .2 m / z.

[0205] Raw data were processed using SEQUEST, MS Amanda45and MS Fragger46in Proteome Discoverer 2.5 against the provided sequences on a Homo sapiens proteome background. Enzyme specificity was set to Trypsin and a minimum of six amino acids was required for peptide identification. Up to two missed cleavages were allowed and a 1 % FDR cut-off was applied both at peptide and protein identification levels. For the database search, carbamidomethylation was set as a fixed modification, whereas oxidation (M), acetylation (protein N-term), PyroGlu (N-term Q), and Phosphorylation (S, T, Y) and K- GG were considered as variable modifications. Data was further processed and inspected in Scaffold 5.1.0 (Proteome Software, Portland, USA).

[0206] Virus infection experiments

[0207] GFP-expressing virus strains were a kind gift from F. Schmidt, University of Bonn, GER, and described previously (VACV-GFP; ref47) or generated as described48. HeLa cGAS KO cells were treated with doxycycline for 4 days to induce cGAS, cGASN513 / 4A, or cGASK427 / 8Rexpression or HeLa cells were treated with doxycycline for 2 days to trigger enforced expression of SPSB3. Infections were performed by incubating virus inoculum (Moi: 0.1) with cells for 3 h, before the cells were washed and cultured in complete DMEM (10% FBS, 1 % penicillin-streptomycin-glutamine solution). After overnight incubation, cells were fixed and GFP+cells were analysed by flow cytometry.

[0208] Reporting summary

[0209] Further information on research design, reagents and software is available in the Nature Research Reporting Summary Checklist linked to this paper.

[0210] Data availability

[0211] Full scans for all immunoblots are provided in Fig. 5. The 3D cryo-EM density maps are deposited in the Electron Microscopy Data Bank under the accession numbers EMD-16933 (Focused refinement with cGAS site C-SPSB3-ELOBC mask), EMD-16936 (composite map of cGAS site C-SPSB3-ELOBC- nucleosome complex), EMD-16937 (Consensus refinement of cGAS site C-SPSB3-ELOBC- nucleosome complex), EMD-16938 (cGAS WT-SPSB3-ELOBC-nucleosome complex at 2:2 ratio). The coordinates are deposited in the PDB with accession numbers 8OKX (cGAS site C-SPSB3-ELOBC) and 8OL1 (cGAS site C-SPSB3-ELOBC-nucleosome).

[0212] Example 7 - Discussion

[0213] Along with previously defined interactions with nucleosomes, our results provide a complete structural model of nuclear regulation of cGAS. Cytosolic exposure of chromatin in mitosis recruits cGAS onto nucleosomes establishing the tightly chromatin-tethered nuclear cGAS pool. Following mitotic exit, most prominently at the onset of cell division, cGAS levels are tuned by SPSB3-directed ubiquitylation achieving almost complete elimination of the protein shortly before the next mitosis. Coordination between nuclear cGAS levels and cell cycle activity might be necessary for balancing of a yet-to-be clarified nuclear function to minimise autoreactivity, specifically prior to genome (DNA) duplication in the replicative phase of the cell cycle. The identified SPSB3-binding site on cGAS featuring the NN degron motif is distinct from all previously described DNA- and protein-binding surfaces. Therefore, in addition to the removal of nucleosome-bound cGAS, SPSB3 might target DNA-bound cGAS assemblies in addition to free cGAS. This broad binding capacity along with the irreversible nature of the degradation process engenders a highly potent mechanism of cGAS inactivation. The conservation of the NN degron, the key to the compatibility for SPSB3-directed protein removal, together with the conservation of the interfaces on SPSB3, points to a common mechanism in limiting the abundance of cGAS to tune cell intrinsic innate immunity.

[0214] Our functional analysis demonstrates the importance of levelling cGAS to maintain immune homeostasis. Significantly, disruption of the NN degron motif or depletion of SPSB3 promoted cGAS activity from within the nucleus resulting in cell autonomous stimulation of innate immunity. Conversely, enhanced SPSB3 activity suppressed tonic cGAS-dependent type I IFN responses. The CRL5SPSB3- mediated checkpoint on cGAS, as defined in this work, may provide opportunities for the development of new immune-targeted therapies that respectively enhance or counteract cGAS-STING immunity in a given disease context. The structure of the CGAS-SPSB3 interface affords obvious insights for the rational design of this new type of cGAS-targeting pharmacology.

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Claims

CLAIMS1 . A variant of cyclic GMP-AMP synthase (cGAS) carrying an amino acid substitution at one or more of(i) asparagine at amino acid position 513,(ii) asparagine at amino acid position 514,(iii) aspartic acid at amino acid position 465,(iv) glutamic acid at amino acid position 509,(v) tyrosine at amino acid position 510,(vi) arginine at amino acid position 512,(vii) lysine at amino acid position 427, and(viii) lysine at amino acid position 428.

2. The variant of cGAS claim 1 , wherein the variant has increased stability against proteasomal degradation and / or has increased half-life in cells.

3. The variant of cGAS of claim 1 or 2, wherein the variant of cGAS(I) comprises or consist of(a) an amino acid sequence selected from any one of SEQ ID NOs 1 to 8, or(b) an amino acid sequence sharing at least 80%, preferably at least 90% identity with SEQ ID NOs 1 to 8, provided that one or more of(i) asparagine at amino acid position 513,(ii) asparagine at amino acid position 514,(iii) aspartic acid at amino acid position 465,(iv) glutamic acid at amino acid position 509,(v) tyrosine at amino acid position 510,(vi) arginine at amino acid position 512,(vii) lysine at amino acid position 427, and(viii) lysine at amino acid position 428 is / are substituted by another amino acid;(c) the amino acid sequence of (a) or (b) further comprising a nuclear localization sequence (NLS), or(d) the amino acid sequence of any one of (a) to (c), wherein a part of or the complete IDR is deleted; or(II) is encoded by a nucleic acid molecule comprising of consisting of(a) a nucleotide sequence selected from any one of SEQ ID NOs 9 to 16, or(b) a nucleotide sequence sharing at least 80%, preferably at least 90% identity with any one of SEQ ID NOs 9 to 16, provided that the base triplet(s) encoding(i) asparagine at amino acid position 513,(ii) asparagine at amino acid position 514,(iii) aspartic acid at amino acid position 465,(iv) glutamic acid at amino acid position 509,(v) tyrosine at amino acid position 510,(vi) arginine at amino acid position 512,(vii) lysine at amino acid position 427, and(viii) lysine at amino acid position 428 is / are substituted by (a) base triplet(s) encoding another amino acid;(c) the nucleotide sequence or (a) or (b) further encoding a nuclear localization sequence (NLS), or(d) the nucleotide sequence of any one of (a) to (c), wherein a part of or the complete nucleotide sequence encoding the IDR is deleted.

4. The variant of cGAS of any one of claims 1 to 3, wherein the one or more amino acids are each individually substituted by a non-conservative amino acid, preferably by an amino acid with a hydrophobic side chain, more preferably by alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan, even more preferably by alanine, valine, isoleucine or leucine, and most preferably by alanine.

5. The variant of cGAS of any one of claims 1 to 4, wherein the variant of cGAS furthermore carries an amino acid substitution of one or both arginines at amino acid positions 255 and 236 and / or an amino acid substitution of one or both lysines at amino acid positions 254 and 258.

6. A variant of SplA / Ryanodine Receptor Domain And SOCS Box Containing 3 (SPSB3) carrying an amino acid substitution at one or more of(i) arginine at amino acid position 213,(ii) tyrosine at amino acid position 197,(iii) threonine at amino acid position 162,(iv) threonine at amino acid position 259,(v) arginine at amino acid position 262,(vi) serine at amino acid position 132,(vii) tyrosine at amino acid position 131 , and(viii) tyrosine at amino acid position 160.

7. The variant of SPSB3 claim 6, wherein the variant displays reduced binding to cGAS as compared to SPSB3.

8. The variant of SPSB3 of claims 6 or 7, wherein the variant of SPSB3(I) comprises or consist of(a) an amino acid sequence selected from any one of SEQ ID NOs 17 to 24, or(b) an amino acid sequence sharing at least 80%, preferably at least 90% identity with SEQ ID NOs 17 to 24, provided that one or more of(i) arginine at amino acid position 213,(ii) tyrosine at amino acid position 197,(iii) threonine at amino acid position 162,(iv) threonine at amino acid position 259,(v) arginine at amino acid position 262,(vi) serine at amino acid position 132,(vii) tyrosine at amino acid position 131 , and(viil) tyrosine at amino acid position 160; or(c) the amino acid sequence of (a) or (b) further comprising a nuclear localization sequence (NLS); or(II) is encoded by a nucleic acid molecule comprising of consisting of(a) a nucleotide sequence selected from any one of SEQ ID NOs 25 to 32, or(b) a nucleotide sequence sharing at least 80%, preferably at least 90% identity with any one of SEQ ID NO: 25 to 32, provided that the base triplet(s) encoding(i) arginine at amino acid position 213,(ii) tyrosine at amino acid position 197,(iii) threonine at amino acid position 162,(iv) threonine at amino acid position 259,(v) arginine at amino acid position 262,(vi) serine at amino acid position 132,(vii) tyrosine at amino acid position 131 , and(viii) tyrosine at amino acid position 160 is / are substituted by (a) base triplet(s) encoding another amino acid; or(c) the nucleotide sequence or (a) or (b) further encoding a nuclear localization sequence (NLS).

9. The variant of SPSB3 of any one of claims 6 to 8, wherein the one or more amino acids are each individually substituted by a non-conservative amino acid, preferably by an amino acid with a hydrophobic side chain, more preferably by alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan, even more preferably by alanine, valine, isoleucine or leucine, and most preferably by alanine.

10. A nucleic acid molecule encoding the variant of cGAS of any one of claims 1 to 5 or the variant of SPSB3 of any one of claims 6 to 9.

11. A vector, preferably an expression vector comprising a nucleic acid molecule encoding the variant of cGAS of any one of claims 1 to 5 or encoding the variant of SPSB3 of any one of claims 6 to 9.

12. A host cell, preferably an anti-tumor lymphocyte comprising the nucleic acid molecule of claim 10 or the vector of claim 11 .

13. A pharmaceutical composition comprising the variant of cGAS of any one of clams 1 to 5, the variant of SPSB3 of any one of claims 6 to 9, the nucleic acid molecule of claim 10, the vector of claim 11 , the host cell of claim 12, or any combination thereof.

14. The variant of cGAS of any one of clams 1 to 5, the variant of SPSB3 of any one of claims 6 to 9, the nucleic acid molecule of claim 10, the vector of claim 11 , the host cell of claim 12, the pharmaceutical composition of claim 13 or any combination thereof for use in the treatment of an inflammatory disease, infectious disease or tumor.

15. Therapeutic cells, preferably anti-tumor lymphocytes, and the variant of cGAS of any one of claims 1 to 5, the nucleic acid molecule of claim 10, the vector of claim 11 , the host cell of claim 12, the pharmaceutical composition of claim 13 or any combination thereof, provided that the cGAS or the encoded cGAS carries an amino acid substitution of one or both arginines at amino acid positions 255 and 236 and / or an amino acid substitution of one or both lysines at amino acid positions 254 and 258 for use in the treatment of a tumor in a subject.

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