Methods and constructs for raav production

Abstract

The present invention relates to a nucleic acid construct encoding for an adeno- associated virus (AAV) Rep and optionally an AAV Cap protein for improved production of recombinant adeno-associated virus. The present invention further provides vectors and cells comprising the nucleic acids construct and a method for producing recombinant adeno- associated virus particles utilizing the nucleic acid construct, the vectors and / or the cells comprising the nucleic acid construct.

Description

[0001] PCT application

[0002] Rei Thera SRL

[0003] Our Ref.: 163-7 PCT

[0004] METHODS AND CONSTRUCTS FOR rAAV PRODUCTION

[0005] FIELD OF THE INVENTION

[0006] The present invention relates to a nucleic acid construct encoding for an adeno- associated virus (AAV) Rep and optionally an AAV Cap protein for improved production of recombinant adeno-associated virus. The present invention further provides vectors and cells comprising the nucleic acids construct and a method for producing recombinant adeno- associated virus particles utilizing the nucleic acid construct, the vectors and / or the cells comprising the nucleic acid construct.

[0007] BACKGROUND OF THE INVENTION

[0008] Adeno-associated viruses (AAVs) form the genus Dependoviruses within the family of parvoviruses Parvoviridae), a family of single-stranded DNA viruses with a non-enveloped, icosahedral capsid. AAV infects a wide range of animals, including humans, and is found worldwide with a seroprevalence in the human population ranging from around 15% to over 90%, depending on the AAV serotype as well as the cohort studied. Infection with AAV is asymptomatic and can remain lifelong.

[0009] The genome of the best characterized AAV serotype, AAV2, has a length of about 4.7 kb and contains as essential elements two open reading frames (ORFs) for the four regulatory (Rep) proteins Rep78, Rep68, Rep52 and Rep40 and the three capsid (Cap) proteins VP1, VP2 and VP3 flanked by two inverted terminal repeats (ITRs), typically about 145 nt in length, which are necessary for viral replication. For example, AAV2 has a capsid comprising 60 capsid proteins arranged into an icosahedral structure, with VP1, VP2 and VP3 present in approximately a 1 : 1 :8 molar ratio.

[0010] As the genus name suggests, AAV can only replicate and assembly new infectious virus particles in the presence of helper factors, which are provided by coinfections by helper viruses from the herpesvirus family (e.g., HSV-1 and human cytomegalovirus, HCMV), adenoviruses (e.g., AdV5), and papillomaviruses (e.g., human papillomavirus type 16, HPV- 16), as well as other viruses such as baculovirus and human bocavirus 1. In the absence of helper viruses, AAV is able to establish a latent infection. In vitro, this often results in integration of the viral DNA into the genome of the cultured cells, whereas, in vivo, the AAV DNA is thought to exist predominantly in an extrachromosomal state as a stable episome.

[0011] AAV has emerged as a key delivery tool in clinical gene therapy due to its low pathogenicity and ability to establish long-term gene expression in various tissues. Recombinant AAV (rAAV) has been engineered for enhanced specificity and has been developed as a tool for the treatment of various diseases.

[0012] However, current vector production methods do not meet the demand for AAV vectors in clinical trials, particularly for certain genetic diseases that require large quantities of high- quality vectors.

[0013] Two main methodological strategies are widely used to generate recombinant adeno- associated virus (rAAV) particles. One method is based on adenovirus-free transient transfection of one or more plasmids containing the rAAV genome with the therapeutic gene of interest, AAV rep / cap coding sequences and AdV2 or AdV5 or a combination of AdV2 and AdV5 helper genes encoding VA RNA, E2a and E4orf6 into suitable host cells encoding the AdV5 helper genes E1A / E1B. Although the transient transfection method produces high-titer AAV vectors that are free of helper virus, it is labour intensive and expensive to scale up for clinical trials and commercialization phases.

[0014] The second strategy comprises transfection-free production methods based on infection of a production cell line with a helper virus, and in particular with a baculovirus, a herpesvirus or an adenovirus. The Sf9 / baculovirus system is used for approved rAAV-based gene therapies and for clinical studies. The Sf9 / baculovirus system shows a better scalability and productivity than the transfection-based method but less vector potency than a mammalian production system, since post-transcriptional modifications as well as vector methylation are not identical in mammalian and insect cells.

[0015] The method relying on recombinant adenovirus (rAd) infection of mammalian cell lines is a two-step process and requires the generation of rAd particles in a production cell line, followed by infection of a packaging cell line with the produced rAd particles. This method can be scaled up to produce high-titer AAV vectors, however, has also some drawbacks depending on the mammalian cell used. For example, the systems using HeLa cells have the problem of producing wild-type helper adenovirus along with the rAAV particles. Contamination with wild-type adenovirus is highly undesirable in terms of vector safety. Therefore, this type of rAAV production requires efficient purification steps to remove the helper viruses from the rAAV vector stocks. Differently, the methods based on rAd infection of HEK293 cells present the problem that these cells constitutively express the Ad El A gene activating AAV Rep gene promoters p5 and pl 9, which induce expression of AAV Rep proteins. Studies have shown that the AAV Rep proteins are cytostatic and cytotoxic, and thus prevent formation of stable cell lines. The tetracycline-enabled self-silencing adenovirus (‘TESSA’, Su, W, etal., 2022, Nature Comms 13, 1182) allows to control rAd replication during rAAV production but presents the disadvantage of requiring continuous presence of doxycycline, and careful quality controls have to be performed to assess complete removal upon rAAV production.

[0016] WO 2024 / 081756 A2 describes an adeno-associated virus (AAV) production system based on a polynucleic acid molecule comprising sequences that encode one or more AAV proteins. These sequences include an excisable element comprising two introns that flank an exon containing a stop codon; each intron contains a recombination site. The AAV proteinencoding sequences, introns, and recombination sites are included in the polynucleic acid molecule with the same sense orientation. The system also includes a recombinase that can recognise and act on these sites. Upon recombinase expression, the DNA sequence flanked by the recombination sites — including the exon with the stop codon — is excised. This restores the intron structure in order to enable splicing and expression of the full-length coding sequence.

[0017] A recent transfection-based method for producing rAAV particles (WO 2003 / 084977 Al; Qiao C. et al., 2002, J Virology, 76(4): 13015-13027) aims to solve the problem of the Rep cytotoxicity by using a rep nucleic acid sequence containing a terminating intron comprising loxP sites and a polyA stop signal that prevents rep expression in a cell. Rep expression is considered to be possible only when a recombinase is introduced into the cell to remove the intron from the rep sequence. However, cells comprising this construct show some leakiness of rep expression in the absence of recombinase. In addition, the rep nucleic acid sequence comprises a wild-type Ris-Ad, and therefore cannot be used for rAd-based methods for rAAV production.

[0018] Therefore, new methods for AAV production are needed to meet the demand for high quality and high quantity rAAV vectors in both preclinical and clinical studies.

[0019] SUMMARY OF THE INVENTION

[0020] In a first aspect, the present invention relates to a nucleic acid construct comprising: (i) an adeno-associated Virus (AAV) rep open reading frame (ORF) comprising a heterologous intron (int), wherein the rep ORF and the int are split within the intron into a 5' portion (rep5 / fw int5 / fw) and an inverted 3' portion (rep3 / rc int3 / rc), (ii) optionally an inverted AAV cap partial ORF (cap / rc) located between int5 / fw and rep3 / rc, and

[0021] (iii) a pair A of recombination sites (recA / fw, recA / rc), and a different pair B of recombination sites (recB / fw, recB / rc), wherein each pair is specific for a recombinase and allows for inversion of rep3 / rc int3 / rc and, if present, also cap / rc, wherein the nucleic acid construct has the 5' to 3' structure: rep5 / fw int5 / fw recA / fw recB / fw (cap / rc -) rep3 / rc int3 / rc recA / rc recB / rc, wherein — is a direct or an indirect link, and - is a direct link.

[0022] In a second aspect, the present invention relates to a vector comprising the nucleic acid construct according to the first aspect.

[0023] In a third aspect, the present invention relates to a collection of vectors, comprising:

[0024] (i) a first vector according to the second aspect comprising cap / rc and a second vector comprising a transgene flanked by AAV ITRs (inverted terminal repeats),

[0025] (ii) a first vector according to the second aspect comprising cap / rc and a transgene flanked by AAV ITRs and a second vector comprising a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct comprised in the vector according to the second aspect, or

[0026] (iii) a first vector according to the second aspect comprising cap / rc, a second vector comprising a transgene flanked by AAV ITRs, and a third vector comprising a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct comprised in the vector according to the second aspect.

[0027] In a fourth aspect, the present invention relates to a cell comprising:

[0028] (a) extra-chromosomally the nucleic acid construct of the first aspect comprising cap / rc, or the vector of the second aspect comprising cap / rc; or

[0029] (b) chromosomally the nucleic acid construct of the first aspect.

[0030] In a fifth aspect, the present invention relates to a kit comprising:

[0031] (A) the collection of vectors of the third aspect, or the vector of the second aspect comprising cap / rc and a transgene flanked by AAV ITRs, and a cell; or

[0032] (B) the cell of the fourth aspect (b), and one or more vectors comprising:

[0033] (i) a transgene flanked by AAV ITRs,

[0034] (ii) a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct of the first aspect, and

[0035] (iii) a nucleic acid comprising a cap ORF, if a cap ORF is not present chromosomally. In a sixth aspect, the present invention relates to a method for producing recombinant adeno-associated virus (rAAV) particles comprising a transgene, the method comprising the steps of:

[0036] (a) - providing the collection of vectors of the third aspect, or the vector of the second aspect comprising cap / rc and a transgene flanked by AAV ITRs, and a cell,

[0037] - transfecting the cell with the vector(s),

[0038] - culturing the cell to produce the rAAV particles; or

[0039] (b) - providing the cell of the fourth aspect (b), and one or more vectors comprising:

[0040] (i) a transgene flanked by AAV ITRs,

[0041] (ii) a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct of the first aspect, and

[0042] (iii) a nucleic acid comprising a cap ORF, if a cap ORF is not present chromosomally;

[0043] - transfecting the cell with the one or more vectors, and

[0044] - culturing the cell to produce the rAAV particles.

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0047] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (TUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland) and as described in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; the "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", edited by Susan Budavari et al., CRC Press, 1996, and the United States Pharmacopeia-25 / National Formulary-20, published by the United States Pharmcopeial Convention, Inc., Rockville Md., 2001. In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they might be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments, which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0048] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. In preferred embodiments, "comprise" can mean "consist of. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise. Whenever the conjunction "and / or" is used herein, the preferred embodiment is "and", unless indicated otherwise.

[0049] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) are hereby incorporated by reference in their entirety.

[0050] Brief description of the drawings

[0051] Figure 1: Schematic structure of repFLEXcap and repFLEX constructs. A) In the repFLEXcap construct, the rep ORF is split into the two fragments rep5 / fw and rep3 / rc, where rep3 / rc is directly linked to the cap partial ORF (cap / rc) and both rep3 / rc and cap / rc are inverted relative to rep5 / fw. Rep5 / fw and rep3 / rc-cap / rc are separated by the 5' fragment (int5 / fw) of a heterologous intron and by two recombination sites (recA / fw and recB / fw). Downstream of the end of the rep3 / rc-cap / rc portion, is present an inverted 3' fragment of the heterologous intron (int3 / rc) followed by a pair of inverted recombination sites (recA / rc and recB / rc). Upon exposure to the recombinase (e.g., a Cre), the portion comprising rep3 / rc-cap / rc and int3 / rc is inverted and the intron can then be spliced out by the cellular splicing machinery so that a nucleic acid comprising a rep ORF and a cap ORF is generated. B) The repFLEX construct has the same structure as repFLEXcap except for the absence of the cap ORF (cap / rc). C) Steps performed by the recombinase to invert the segment comprising rep3 / rc-cap / rc and int3 / rc in repFLEXcap. The same steps are performed to invert the segment comprising rep3 / rc and int3 / rc in repFLEX. D) Localization of rep5, rep3, and cap pORF in the wt AAV2 genome in relation to the rep78, rep68, rep52, rep40, and cap VP1 ORFs. Rep5 / fw corresponds to rep5 in sense direction, rep3 / rc corresponds to the reverse complement of rep3 and cap / rc corresponds to the reverse complement of cap pORF. The 3' end of rep5 (position 1022) and the 5' start of rep3 (position 1023) as indicated represent exemplary examples.

[0052] Figure 2: rAAV genome. In recombinant AAV (rAAV) (bottom of the Figure), the part of the genome encoding for Rep and Cap and the polyadenylation signal pA is replaced by a transgene (also called gene-of-interest, GOI) while the ITR segments are maintained.

[0053] Figure 3: The repLSLcap construct. In the repLSLcap (LSL = lox-stop-lox) construct (WO 2003 / 084977 Al; Qiao C. et al., 2002, J Virology, 76(4): 13015-13027), the rep coding nucleic acid sequence is split by an intron insertion. The intron itself is also split by a multiple transcription stop signal (TS) flanked by two recombination sites LoxP. Upon exposure to a Cre recombinase, the TS segment is eliminated, and a rep-cap coding sequence is generated after splicing of the intron by the cellular splicing machinery. Splicing of the intron is however also possible in the absence of Cre if transcriptional stop readthrough occurs.

[0054] Figure 4: Rep expression by repLSLcap and by repFLEXcap. Infection with lysates prepared from cells transfected with the constructs pRepLSL-pCre (1), pRepFlex-pCre (2) pRepLSL+pCre (3), and pRepFlex+pCre (4) in addition to pAAV-GFP and pHelper. When transfection is performed in the presence of Cre (panels 3 and 4), both repFLEXcap and repLSLcap express Rep and generate rAAVs as evidenced by the fluorescence (GOI = Green Fluorescent Protein GFP) of cells supplemented with such lysates. In the absence of Cre, only repLSLcap shows leakiness of Rep expression as evidenced by the GFP signal (panel 1). No signal is instead evident for repFLEXcap in the absence of Cre (panel 2).

[0055] Figure 5: Yield of rAAV production. repFLEXcap generates rAAV particles with cap either from AAV2 or AAV8, at high levels comparable to the yield obtained by the corresponding three plasmids transfection method. The transgene (gene of interest = GOI) is GFP. vp = viral particles. A) Results of digital droplet PCR performed on lysates of HEK-iCre cells B), D) Fluorescent microscopy images of HEK293T cells infected with rAAV produced through infection of HEK-iCre cells with rAd-GFP and pRepFLEXcap comprising AAV2 cap (B) or AAV8 cap (D); C), E) Fluorescent microscopy images of HEK293T cells infected with rAAV produced through transfection of HEK-iCre cells with 3 plasmids (pAAV-GFP, SEQ ID NO: 166; GFP of SEQ ID NO: 27), prepFLEXcap (SEQ ID NO: 169) comprising AAV2 cap (C) or AAV8 cap (SEQ ID NO: 169 with AAV8 cap of SEQ ID NO: 21) (E), and pHelper (SEQ ID NO: 167).

[0056] Figure 6: Exemplary production method. A) Example of rAAV production using three different rAds. After infection of the packaging cell line with the three rAds, the recombinase (Cre in this example) is produced, the segment comprising rep3 / rc-cap / rc in repFLEXcap is inverted, the intron is spliced generating a functional rep-cap coding sequence, Rep and Cap proteins are produced and the rAAV particles with the desired transgene are produced. B) Example of rAAV production using two different rAds (one containing the transgene / GOI GFP, the other repFLEXcap) and a packaging cell line with Cre integrated into its genome, vp = viral particles.

[0057] Figure 7: Cap protein sequence alignment. Pairwise protein sequence identity of aligned Cap proteins from different AAV serotypes (SEQ ID NOs: 20, 22, 68-89). Rhesus AAVs are labeled AAVrhx or AAVrh.x, human AAVs AAVx and AAVGo is an AAV isolated from goat. Sequences were first aligned using ClustalX and then the alignment was manually optimized. Grey boxes indicate groups of AAV Cap proteins with > 80% intra-group amino acid sequence identity (big block containing AAV2 and AAV8), > 77% intra-group amino acid sequence identity (AAVrh32.33, AAV4, AAV11, AAV12) and > 93% intra-group amino acid sequence identity (AAV5, AAVGo).

[0058] Figure 8: Rep nucleic acid sequence alignment. Pairwise nucleic acid sequence identity of aligned rep sequences from human AAV serotypes (SEQ ID NOs: 12, 13, 90-100). AAV2_S503C encodes a rep having Ser 503 substituted by Cys.

[0059] Figure 9: Rep protein sequence alignment. Pairwise protein sequence identity of aligned Rep protein sequences from human AAV serotypes (SEQ ID NOs: 14, 15, 101-111). AAV2_S503C has Ser 503 substituted by Cys.

[0060] Figure 10: Cap protein sequence alignment. Alignment of AAV Cap proteins (SEQ ID NOs: 20, 22, 68-89). Sequences were initially aligned using ClustalX and the alignment was subsequently optimized manually. Rhesus AAVs are labeled AAVrhx or AAVrh.x, human AAVs AAVx, and AAVGo is an AAV isolated from goat.

[0061] Figure 11: Alignment of Rep proteins from human AAV serotypes. Sequences were initially aligned using ClustalX and the alignment was subsequently optimized manually. AAV2_S503C is the AAV2 Rep protein with Ser 503 substituted by Cys (SEQ ID NOs: 14, 15, 101-111). Figure 12: Analysis of CPE induction by parental rAd. HEK293 cells were infected with the rAd-GOI (SEQ ID NO: 171; GOI=GFP) or rAd-repFLEXCap2 (SEQ ID NO: 174; without expression of a fluorescent marker) at the indicated multiplicity of infection (MOI), and images taken two weeks post infection. It is possible to note that the morphology of cells worsens by increasing the infecting MOI of either rAd-GOI or rAd-repFLEXCap.

[0062] Figure 13: Analysis of CPE induction upon infection with rAAV produced with the rAd / AAV method. HEK293 cells were infected with a volume of post-AAVX sample (see Table 5) containing 1 MOI of contaminating rAd and approximately 14,000 MOI of rAAV (quantified based on the ddPCR results of Table 5). Two weeks after infection, the brightfield image (A) shows that the HEK293 cells had not exhibited cytopathic effect (in contrast with the positive controls of Figure 12), showing that the cells are not infected by residual adenovirus. Accordingly, the cells undergone multiple divisions so that they required splitting to maintain viability and were not 100% GFP positive (B).

[0063] Nucleotide and amino acid sequences

[0064] The present application refers to SEQ ID NOs 1-178. An overview and explanation of these SEQ IDs is given in the following Table 1. Source refers to the NCBI accession number.

[0065] Table 1: SEQ ID NOs referred to in the application

[0066]

[0067]

[0068] Aspects of the invention and particular embodiments thereof

[0069] The invention relates to several aspects as set out above in the summary of the invention. These aspects comprise alternative embodiments and preferred embodiments, which are described below.

[0070] The inventors have found that a nucleic acid construct (repFLEX, repFLEXcap, Figure 1) comprising a rep open reading frame (ORF) split into two fragments, wherein one of the two fragments is inverted in relation to the other and is optionally linked to a cap pORF, allows obtaining high titers of rAAV particles, since during rAd production, cell growth is not inhibited by the cytotoxic Rep protein. The same is true for cell lines chromosomally comprising the repFLEXcap or the repFLEX nucleic acid construct. Restoration of the rep ORF - and optionally the cap ORF - is afforded by two pairs of recombination sites (Figure 1C) and rAAV packaging cells expressing the respective recombinase(s) for the pairs of recombination sites in order to produce rAAV particles. The productivity of a rAd comprising this nucleic acid construct is very high and surprisingly comparable to that of a rAd not comprising an AAV rep (Example 4). Importantly, this nucleic acid construct allows generation of rAAV particles at high levels comparable to the yield obtained by the corresponding three plasmids transfection method (Example 4, Figure 5). Advantageously, the nucleic acid construct with rep from AAV2 can be used to produce rAAV particles with a capsid composition (cap) different from AAV2 (Example 4, Figure 5). Notably, this nucleic acid construct allows not only for a high level rAAV production in large and small volumes (Examples 5-7) but provides also high purity rAAV preparations with low rAd particle content (Examples 5-7, Tables 3-5).

[0071] Accordingly, in a first aspect, the invention provides a nucleic acid construct comprising:

[0072] (i) an adeno-associated Virus (AAV) rep open reading frame (ORF) comprising a heterologous intron (int), wherein the rep ORF and the int are split within the intron into a 5' portion (rep5 / fw int5 / fw) and an inverted 3' portion (rep3 / rc int3 / rc), (ii) optionally, an inverted AAV cap partial ORF (cap / rc) located between int5 / fw and rep3 / rc, and

[0073] (iii) a pair A of recombination sites (recA / fw, recA / rc), and a different pair B of recombination sites (recB / fw, recB / rc), wherein each pair is specific for a recombinase and allows for inversion of rep3 / rc int3 / rc and, if present, also cap / rc, wherein the nucleic acid construct has the 5' to 3' structure: rep5 / fw int5 / fw recA / fw recB / fw (cap / rc -) rep3 / rc int3 / rc recA / rc recB / rc, wherein — is a direct or an indirect link, and - is a direct link.

[0074] Rep5 / fw, and int5 / fw indicate the sense sequences of rep5 and int5 respectively, and rep3 / rc, int3 / rc, and cap / rc indicate the reverse complementary sequences of rep3, int3, and cap pORF, respectively.

[0075] The present nucleic acid construct differs from that of WO 2024 / 081756 A2. In the present nucleic acid construct, rep3 / rc, int3 / rc, the optional cap / rc, recA / rc, and recB / rc are arranged in an inverse direction with respect to rep5 / fw. By contrast, all sequences in the polynucleic acid molecule of WO 2024 / 081756 A2 are oriented in the same sense direction. Importantly, the arrangement of the two recombination sites of a given pair in the same sense direction in WO 2024 / 081756 A2 results in the excision of the fragment with the stop codon comprised between them upon recombinase activity.

[0076] In the present nucleic acid construct, the arrangement of the two recombination sites of a given pair in opposite orientations leads to inversion of the fragment comprised between them upon recombinase activity. This allows a better control of rep and cap expression and prevents leakiness in the absence of a recombinase.

[0077] Moreover, the nucleic acid construct of WO 2024 / 081756 A2 comprises two pairs of splice donor (SD, here referred to as SD1 and SD2) and splice acceptor sequences (SA, here referred to as SAI and SA2), both in the same sense direction. In this arrangement, SD1 can interact with the SA2 located after the stop codon, resulting in the excision of the fragment containing the stop codon, even in the absence of the recombinase. This would enable expression of the full- length coding sequence in the absence of the recombinase, rendering the construct leaky. In contrast, the present nucleic acid construct comprises a splice acceptor sequence in inverse direction with respect to the splice donor sequence. This also allows a better control of rep and cap expression, as well as preventing leakiness in the absence of a recombinase.

[0078] As shown by the results of Example 3, the present nucleic acid construct outperforms the repLSLcap of the application WO 2003 / 084977 Al : When transfection is performed in the presence of Cre (panels 3 and 4 of Figure 4), both repFLEXcap and repLSLcap express Rep and generate rAAVs; in the absence of Cre, no signal is evident for repFLEXcap (panel 2 of Figure 4), whereas repLSLcap shows leakiness of Rep expression (panel 1 of Figure 4).

[0079] Herein, the rAAV particles of the invention are preferably based on the AAV2 serotype. Accordingly, AAV2 is used as the reference serotype throughout the specification. Unless explicitly stated otherwise, all references to AAV herein, including the AAV genome, individual AAV nucleic acid and amino acid sequences, and AAV proteins preferably relate to AAV2, and in particular to SEQ ID NO: 29 (GenBank accession no.: AF043303; RefSeq: NC 001401). The person skilled in the art can easily transfer all the information given in relation to AAV2 to other AAV serotypes.

[0080] One embodiment of the first aspect relates to a nucleic acid construct comprising:

[0081] (i) an adeno-associated Virus (AAV) rep open reading frame (ORF) comprising a heterologous intron (int), wherein the rep ORF and the int are split within the intron into a 5' portion (rep5 / fw int5 / fw) and an inverted 3' portion (rep3 / rc int3 / rc),

[0082] (ii) an inverted AAV cap partial ORF (cap / rc) located between int5 / fw and rep3 / rc, and

[0083] (iii) a pair A of recombination sites (recA / fw, recA / rc), and a different pair B of recombination sites (recB / fw, recB / rc), wherein each pair is specific for a recombinase and allows for inversion of rep3 / rc int3 / rc and cap / rc, wherein the nucleic acid construct has the 5' to 3' structure: rep5 / fw int5 / fw recA / fw recB / fw cap / rc -rep3 / rc int3 / rc recA / rc recB / rc, wherein — is a direct or an indirect link, and - is a direct link.

[0084] Another embodiment of the first aspect relates to a nucleic acid construct comprising:

[0085] (i) an adeno-associated Virus (AAV) rep open reading frame (ORF) comprising a heterologous intron (int), wherein the rep ORF and the int are split within the intron into a 5' portion (rep5 / fw int5 / fw) and an inverted 3' portion (rep3 / rc int3 / rc), and

[0086] (ii) a pair A of recombination sites (recA / fw, recA / rc), and a different pair B of recombination sites (recB / fw, recB / rc), wherein each pair is specific for a recombinase and allows for inversion of rep3 / rc int3 / rc and, wherein the nucleic acid construct has the 5' to 3' structure: rep5 / fw — int5 / fw — recA / fw — recB / fw — rep3 / rc — int3 / rc — recA / rc — recB / rc, wherein — is a direct or an indirect link. Components of the nucleic acid construct

[0087] The term "adeno-associated Virus (AAV) rep open reading frame (ORF)" or "rep ORF" refers to a nucleic acid sequence that is derived from an adeno-associated virus, and which encodes one or more Rep proteins, and preferably for Rep78, Rep68, Rep52 and Rep40 (Figure ID). In other words, a rep ORF comprises a rep78 ORF, rep68 ORF, rep52 ORF and / or rep40 ORF. A rep68 ORF comprises a first and a second rep68 exon (respectively rep68-ExI and rep68-ExII). A rep40 ORF comprises a first and a second rep40 exon (respectively rep40-ExI and rep40-ExII). Preferably, a rep ORF as used herein consists of a rep78 ORF and of rep68- ExII, wherein rep68-ExII is directly linked to rep78 ORF.

[0088] Rep78 and Rep68 are produced from unspliced and spliced transcripts from the p5 promoter, respectively. Rep78 and Rep68 are multifunctional proteins with overlapping functions in almost every stage of the AAV life cycle, such as site-specific DNA binding to the ribosome binding site (RBS), helicase activity, and / or site-specific endonuclease activity, which is required for the separation of replicated viral genomes. Rep78 and / or Rep68 are required in trans for AAV replication and / or excision from the host genome. Rep52 and Rep40 are produced from unspliced and spliced transcripts, respectively, from the pl9 promoter. Rep52 and Rep40 possess helicase and ATPase activity.

[0089] In a preferred embodiment, the rep ORF is of an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1, AAV12, and AAV13. In a more preferred embodiment, the rep ORF is of AAV2.

[0090] Preferably, a rep78 ORF comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12, 13, 90-100, 129, and variants thereof. In a more preferred embodiment, a rep ORF comprises a nucleic acid sequence of SEQ ID NO: 12 or 13, more preferably 13.

[0091] Alternatively, or in addition, a rep78 ORF preferably encodes for an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 15, 101-111, 130, and variants thereof.

[0092] The phrase "the rep ORF and the int are split within the intron into a 5' portion (rep5 / fw — int5 / fw) and an inverted 3' portion (rep3 / rc int3 / rc)" means that a heterologous intron is inserted into, and the rep ORF and the intron are then divided into two fragments each by a division within the intron, so that the first 5' portion comprises the 5' fragment of the rep, i.e., rep5 (rep5 / fw is rep5 in sense direction), and a 5' fragment of the intron, int5 (int5 / fw is int5 in sense direction) , and the second 3' portion comprises a 3' fragment of the rep, i.e., rep3, and a 3' fragment of the intron, i.e., int3. Additionally, the rep3 and int3 are inserted in the nucleic acid construct as inverted (i.e., reverse complementary) sequences in relation to the sense of the 5' fragment, i.e., as rep3 / rc and int3 / rc. Thus, rep5 and rep3 form a rep ORF, when present in the nucleic acid construct in sense direction and directly linked, e.g. after action of the recombinase and splicing.

[0093] The term "rep5" or "rep5 / fw" refers to a fragment of the rep ORF comprising a 5' fragment of the first rep40 exon but not comprising the Ris-Ad, so that the 5' fragment does not comprise a complete coding sequence for any of Rep78, Rep68, Rep52 and Rep40, and it comprises the pl9 promoter but not the p40 promoter (Figure ID). In one embodiment, rep5 / fw is about 700 to about 1460 nt (nucleotide) in length, about 700 to about 1400 nt, about 700 to about 1350 nt, about 700 to about 1300 nt, about 700 to about 1250 nt, about 700 to about 1200 nt, about 700 to about 1150 nt, about 700 to about 1100 nt, about 700 to about 1050 nt, about 700 to about 1000 nt, about 700 to about 950 nt, about 700 to about 900 nt, about 700 to about 850 nt, or about 700 to about 800 nt in length. In a preferred embodiment, rep5 / fw is about 702 nt in length. In a preferred embodiment, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof.

[0094] Consequently, the heterologous intron is inserted within the rep ORF within the first exon of the transcript encoding for Rep40 (Figure ID), preferably within nt 993 and nt 1906 of SEQ ID NO: 29.

[0095] The term "rep3" refers herein to a fragment of the rep ORF comprising a 3' fragment of the first rep40 exon and comprising the Ris-Ad (nucleotides 1781..2060 of the wt AAV2 genome SEQ ID NO: 29), so that it does not comprise a complete coding sequence for any of Rep78, Rep68, Rep52 and Rep40, and it comprises the p40 promoter and cap 5' UTR, which are included in the RIS-Ad, but not the pl9 promoter (Figure ID). In one embodiment, rep3 is about 450 to about 1250 nt in length, about 500 to about 1250 nt, about 550 to about 1250 nt, about 600 to about 1300 nt, about 650 to about 1250 nt, about 700 to about 1250 nt, about 750 to about 1250 nt, about 800 to about 1250 nt, about 850 to about 1250 nt, about 900 to about 1250 nt, about 950 to about 1250 nt, or about 1000 to about 1250 nt. In a preferred embodiment, rep3 is about 1160 nt in length. The term "rep3 / rc" herein refers to the inverse sequence of rep3 as present in the nucleic acid construct of the first aspect. In a preferred embodiment, rep3 / rc comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7 and 8.

[0096] The term "intron" refers herein to a non-coding nucleic acid sequence of varying length, normally present within a gene, which can be removed from a newly transcribed mRNA precursor by the process of splicing between the donor and acceptor splice sites. An intron contains normally three highly conserved sequence elements: a 5' splice site, a branch point, and a 3' splice site.

[0097] The "branch point" is usually located approximately between 10 and 60 nucleotides upstream of the 3' splice site and forms during the splicing process via its conserved adenosine residue a lariat structure with the 5' splice site. Mammalian branch point sequences are well known in the art (e.g. Zhuang et al., 1989. PNAS 86, 2752-2756) and any may be used for the nucleic acid construct of the first aspect.

[0098] The term "splice site" refers herein to a specific nucleic acid sequence that is present at either the 5' end or the 3' end of an intron. Recognition of these sites by the splicing machinery is followed by the excision of an intron or a polynucleotide sequence flanked by these sites. The term splice sites includes naturally occurring, engineered or synthetic, consensus or cryptic splice sites.

[0099] The terms "splice donor site" and "5' splice site" refer herein to a conserved nucleic acid sequence at the exon-intron boundary at the 5' end of an intron that defines the start of the intron. The terms "splice acceptor site" and "3' splice site" refer herein to a conserved nucleic acid sequence at the intron-exon boundary at the 3 ' end of an intron that defines the end of the intron. Many splice donor and acceptors sites have been characterized and described (e.g. in Lodish et al., Molecular Cell Biology, 5th ed., W.H. Freeman & Co, 2004, Chapter 12), are known to the skilled artisan, and any may be used for the nucleic acid construct of the first aspect. For a review see Ohshima et al., 1987. J. Mol. Biol. 195, 247-259. The term "heterologous intron" herein refers to an intron of origin different from AAV, and preferably to an intron of a mammalian cell genome, so that the intron can be spliced out by a mammalian cellular splicing machinery. Preferably, a heterologous intron as used herein comprises at least one splice donor, at least one branch point, and / or at least one splice acceptor site. In particular, a heterologous intron preferably contains appropriate splice donor and splice acceptor sites for the proper splicing of the nucleic acid construct according to the first aspect to restore a rep ORF after the action of the recombinase. Preferably, a heterologous intron as used herein is an excisable heterologous intron sequence. Preferably, a heterologous intron as used here does not comprise a transcriptional stop signal (TS). Preferably, a heterologous intron as used herein is between about 80 nt and about 700 nt in length, about 80 nt and about 650 nt in length, about 80 nt and about 600 nt in length, about 80 nt and about 550 nt in length, about 80 nt and about 500 nt in length, about 80 nt and about 450 nt in length, about 80 nt and about 400 nt in length, more preferably about 352 nt. The term "int5" refers herein to a fragment of a heterologous intron as defined herein comprising the "splice donor site". The term "int3" refers herein to a fragment of a heterologous intron as defined herein comprising the "branch point" and the "splice acceptor site". Int3 is present in the nucleic acid construct of the first aspect as inverse sequence (int3 / rc). Int5 and int3 form a heterologous intron when they are present in the nucleic acid construct according to the first aspect in sense direction and directly linked, e.g. after action of the recombinase and before splicing.

[0100] Preferably, a "int5" or "int5 / fw" as used herein is between about 5 nt and about 350 nt in length, about 5 nt and about 300 nt in length, about 5 nt and about 250 nt in length, about 5 nt and about 200 nt in length, about 5 nt and about 150 nt in length, about 5 nt and about 100 nt in length, about 5 nt and about 50 nt in length, more preferably about 10 nt in length.

[0101] Preferably, a "int3" as used herein is between about 5 nt and about 500 nt in length, about 5 nt and about 450 nt in length, about 5 nt and about 400 nt in length, about 5 nt and about 350 nt in length, about 10 nt and about 350 nt in length, about 50 nt and about 350 nt in length, about 100 nt and about 350 nt in length, about 150 nt and about 350 nt in length, about 200 nt and about 350 nt in length, about 250 nt and about 350 nt in length, more preferably about 342 nt in length.

[0102] The term "cap ORF" refers herein to a nucleic acid sequence that is derived from an AAV, encodes for one or more Cap proteins VP1, VP2 and VP3, and does not comprise the AAV cap promoter.

[0103] The term "AAV cap promoter" as used herein refers to a cap 5' UTR and p40 promoter naturally associated with an AAV cap ORF, wherein the "AAV cap promoter" can be of the same or different serotype of the AAV cap ORF, preferably of the same serotype.

[0104] The term "cap partial ORF (pORF)" refers herein to a nucleic acid sequence that comprises a 3' fragment of a cap full-length ORF and does not comprise a 5' fragment of the cap full-length ORF, which is comprised in the 3' fragment of rep rep3, to which is directly linked in an embodiment of the nucleic acid construct according to the first aspect.

[0105] The term "cap / rc" as used herein refers to a reverse complementary sequence (i.e., inverse) of a cap partial ORF (pORF). Therefore, cap / rc when directly linked to a rep3 / rc in a nucleic acid construct according to the first aspect forms a cap ORF, which is present in the nucleic acid construct of the first aspect as inverse sequence. Preferably, a cap / rc or cap pORF is about 2100-2158 nt in length. Preferably, cap / rc comprises the nucleic acid sequence of SEQ ID NO: 6 or a variant thereof. The phrase "inverted AAV cap partial ORF (cap / rc) located between int5 / fw and rep3 / rc" herein refers to a reverse complementary sequence (i.e., inverted) of an AAV cap partial ORF (pORF) located between int5 / fw and rep3 / rc and directly linked to rep3 / rc. Preferably, the inverted AAV cap pORF is located between recB / fw and rep3 / rc and is directly linked to rep3 / rc, in order to form a cap ORF with a 3' fragment of rep.

[0106] The results of Example 4 indicate that the method using the nucleic acid construct of the first aspect with rep from AAV2, allows producing rAAV particles with a capsid composition (cap) different from AAV2.

[0107] Thus, a cap ORF preferably comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 19, 21, and 48-67, and variants thereof, preferably SEQ ID NO: 19 or 21, more preferably SEQ ID NO: 19.

[0108] Alternatively, or in addition, a cap ORF preferably encodes for a VP1 protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 22, 68-89, and variants thereof, preferably SEQ ID NO: 20 or 22, more preferably SEQ ID NO: 20.

[0109] In one embodiment, a nucleic acid construct according to the first aspect comprises cap / rc, i.e., it has the 5' to 3' structure: rep5 / fw int5 / fw recA / fw recB / fw cap / rc - rep3 / rc int3 / rc recA / rc recB / rc.

[0110] Generally, — is a direct or an indirect link, - is a direct link, and ~ is an indirect link.

[0111] A "direct link" refers herein to a phosphodiester bond of the 3' end of the first element to the 5' end of the second element of a nucleic acid construct according to the first aspect.

[0112] An "indirect link" refers herein to a nucleic acid sequence comprised between the 3' end of the first element and the 5' end of the second element of a nucleic acid construct according to the first aspect.

[0113] The term "element" when referred to a nucleic acid construct according to the first aspect refers herein to rep5 / fw, int5 / fw, recA / fw, recB / fw, cap / rc, rep3 / rc, int3 / rc, recA / rc, or recB / rc.

[0114] Therein, preferably only recA / fw and recB / fw, recB / fw and cap / rc, recA / rc and recB / rc are linked indirectly, i.e., the nucleic acid construct preferably has the 5' to 3' structure: rep5 / fw - int5 / fw - recA / fw ~ recB / fw ~ cap / rc - rep3 / rc - int3 / rc - recA / rc ~ recB / rc.

[0115] In another embodiment, the nucleic acid construct according to the first aspect does not comprise cap / rc, i.e., it has the 5' to 3' structure: rep5 / fw — int5 / fw — recA / fw — recB / fw — rep3 / rc — int3 / rc — recA / rc — recB / rc. Therein, preferably only recA / fw and recB / fw, recB / fw and rep3 / rc, recA / rc and recB / rc are linked indirectly, i.e., the nucleic acid construct preferably has the 5' to 3' structure: rep5 / fw - int5 / fw - recA / fw ~ recB / fw ~ rep3 / rc - int3 / rc - recA / rc ~ recB / rc.

[0116] Therein, more preferably only recB / fw and cap / rc are linked indirectly, i.e., the nucleic acid construct preferably has the 5' to 3' structure: rep5 / fw - int5 / fw - recA / fw - recB / fw ~ cap / rc - rep3 / rc - int3 / rc - recA / rc - recB / rc

[0117] In another further preferred embodiment, the nucleic acid construct according to the first aspect has the 5' to 3' structure: rep5 / fw - int5 / fw - recA / fw - recB / fw ~ rep3 / rc - int3 / rc - recA / rc - recB / rc.

[0118] The indirect link between recA / fw and recB / fw preferably comprises a nucleic acid sequence of about 30 nt to about 100 nt in length, more preferably of about 62 nt in length. Alternatively, or in addition, this indirect link comprises a nucleic acid sequence of SEQ ID NO: 122, or a variant thereof.

[0119] The indirect link between recB / fw and cap / rc preferably comprises an AAV genomic sequence and an enzymatic restriction site, wherein the enzymatic restriction site is present at the end of the AAV genomic sequence, i.e., in the nucleic acid construct between recB / fw and cap / rc. The enzyme restriction site can be any known in the art, preferably it is an EcoRV restriction site. The restriction site is preferably inserted at the end of the AAV genomic sequence, i.e., at the 5' end of cap / rc. The AAV genomic sequence may be comprised between nucleotides 4400-4600, and more preferably 4411-4534 of SEQ ID NO: 29.

[0120] The indirect link between recB / fw and cap / rc preferably comprises an AAV genomic sequence of about 50 to about 200 nt in length, more preferably of about 130 nt, wherein the AAV genomic sequence is normally present in the AAV genome after the end of the cap ORF. Alternatively, or in addition, this indirect link comprises a nucleic acid sequence of SEQ ID NO: 123, or a variant thereof.

[0121] In the nucleic acid constructs not comprising cap / rc, the indirect link between recB / fw and rep3 / rc preferably comprises an AAV genomic sequence of about 5 to about 100 nt in length, more preferably of about 43 nt. Alternatively or in addition, this indirect link comprises the sequence according to SEQ ID NO: 124, or a variant thereof.

[0122] The indirect link between recA / rc and recB / rc preferably comprises a nucleic acid sequence of about 40 to about 100 nt in length, more preferably of about 70 nt in length. Alternatively, or in addition, this indirect link comprises a nucleic acid sequence of SEQ ID NO: 125, or a variant thereof. A nucleic acid construct according to the first aspect comprising cap / rc has a total length of about 5000 nt, i.e., between 4750 nt and 5250 nt. A nucleic acid construct according to the first aspect not comprising cap / rc has a total length of about 2770 nucleotides, i.e., between 2631.5 nt and 2908.5 nt.

[0123] The nucleic acid construct may be present in a vector within an expression cassette as defined herein or integrated in the genome of a cell. In either case, the 5' end of rep5 / fw is operably linked to a promoter capable of driving expression of the Rep78 and Rep68 proteins, and the nucleic acid construct is linked at the 3' end to signal sequences required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. The promoter may be an AAV promoter or a heterologous promoter, preferably an AAV promoter, more preferably an AAV p5 promoter of the same or different AAV serotype of rep ORF, still more preferably of the same AAV serotype of rep ORF.

[0124] Alternatively, or in addition, the AAV p5 promoter may be of an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13, more preferably of AAV2. More preferably, the p5 promoter comprises a nucleic acid sequence of SEQ ID NO: 123, or a variant thereof.

[0125] Recombinases

[0126] The term "a pair A of recombination sites" and "a pair B of recombination sites" as used herein refers to two different pairs of recombination sites specifically recognized by one or more recombinases, preferably by one recombinase (the same recombinase may recognize different recombination sites but recombine only between two identical sites). Preferably, the recombination sites of one pair (A or B) cannot recombine with the recombination sites of the other pair (B or A, respectively).

[0127] The phrase "each pair. ...allows for inversion of rep3 / rc int3 / rc and, if present, also cap / rc" means herein that when the nucleic acid construct of the first aspect is exposed to the one or more recombinases specific for the pairs A and / or B, the one or more recombinases may perform first an inversion of the DNA fragment flanked by recA / fw and recA / rc and / or by recB / fw and recB / rc, and then an excision of the DNA fragment flanked by the other pair, in order to restore a complete rep ORF and, if present, cap pORF in the 5' to 3' direction (Figure 1C). Therefore, each pair A and B of recombination sites allow for the rearrangement of the nucleic acid construct having the 5' to 3' structure (I): rep5 / fw int5 / fw recA / fw recB / fw (cap / rc — ) rep3 / rc int3 / rc recA / rc recB / rc into the nucleic acid construct having the 5' to 3' structure (II): rep5 / fw int5 / fw recA / fw int3 / fw rep3 / fw - (cap / fw) recB / rc, and after splicing of the intron, the 5' to 3' structure (III): rep5 / fw — rep3 / fw — (cap / fw) recB / rc, wherein rep5 / fw, rep3 / fw, int5 / fw, int3 / fw, and cap / fw indicate the sense sequences of rep5, rep3, int5, int3, and cap pORF respectively, and rep3 / rc, int3 / rc, and cap / rc indicate the reverse complementary sequences of rep3, int3, and cap pORF respectively.

[0128] An exemplary recombination site for Cre recombinase is loxP (locus of crossover (x) Pl) which is a 34 bp long sequence comprising two 13 bp long palindromic repeats (serving as the recombinase binding sites) flanking an 8 bp long asymmetric core spacer sequence. The asymmetry in the core sequence gives the loxP site directionality.

[0129] Herein, each pair comprises a first recombination site in sense direction (recA / fw or recB / fw) and a second recombination site in inverted direction (recA / rc or recB / rc), i.e., the second recombination site is the reverse complementary of the first one. In an embodiment, one recombination site has a nucleic acid sequence identical to or different from the reverse complement of the other recombination site of the same pair, preferably an identical nucleic acid sequence.

[0130] In a preferred embodiment, each pair A and B is specific for a recombinase independently selected from the group consisting of Cre recombinase, FLP recombinase, R recombinase, integrase X Int, recombinase of the GIN recombination system of the Mu phage, Bxbl recombinase, bacterial P recombinase, and variants thereof. The variant of a recombinase is preferably a functional variant that preserves the function of the wild-type recombinase. Pairs A and B can be specific for the same recombinase or each for a different recombinase, preferably for the same. For example, both pairs A and B may be specific for Cre recombinase, or a variant thereof, or one pair is specific for Cre recombinase, or a variant thereof, and the other for a recombinase selected from the group consisting of FLP recombinase, R recombinase, integrase X Int, recombinase of the GIN recombination system of the Mu phage, Bxbl recombinase bacterial P recombinase, and variants thereof. The variant of Cre recombinase preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to the parent Cre recombinase. The variant of FLP recombinase preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to the parent FLP recombinase. The variant of R recombinase preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to the parent R recombinase. The variant of integrase X Int preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to the parent Cre recombinase. The variant of the recombinase of the GIN recombination system of the Mu phage preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to the parent recombinase of the GIN recombination system of the Mu phage. The variant of recombinase of Bxbl recombinase preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to the parent Bxbl recombinase. The variant of recombinase of bacterial P recombinase preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to the parent bacterial P recombinase.

[0131] Cre (e.g. Genbank DQ023272, AAY56380.1) is a 38-kDa product of the cre (cyclization recombination) gene of bacteriophage Pl and is a site-specific DNA recombinase of the Int family. Cre recognizes the loxP site and efficiently catalyzes reciprocal conservative DNA recombination between pairs of loxP sites. Recombination mediated by Cre is freely reversible. The DNA substrates can be either linear or supercoiled. A number of mutant loxP sites have been described.

[0132] A FLP recombinase (e.g. Uniprot P03870) is a recombinase of Saccharomyces cerevisiae that catalyzes the recombination of DNA between two specific targeting-sites FLP recognition target (FRT) sites. An R recombinase (e.g. Uniprot P13785) of Zygosaccharomyces rouxii pSRl catalyzes the recombination of DNA between two specific targeting-sites pSR sites. An Int integrase (e.g. Uniprot P03700) is a recombinase of bacteriophage X that catalyzes the recombination of DNA between two specific targeting-sites selected from the group of pairs consisting of attB / attP, and attL / attR ("att" means attachment). A recombinase (e.g. Uniprot P03015) of the GIN recombination system of the Mu phage catalyzes the recombination of DNA between two specific targeting-sites "gix". A bacterial P recombinase (Diaz, Vicente et al., 1999. IBC, 274(10), 6634 - 6640) catalyzes the recombination of DNA between two specific targeting-sites "six". The P recombinase is encoded by the P gene of the Gram-positive broad host range plasmid pSM19035. A Bxbl recombinase (e.g. Uniprot Q9B086) catalyzes the recombination of DNA between two specific targeting-sites attP and attB.

[0133] Other examples of recombinases are known to those of skill in the art and can be used in the methods described herein, and also any new recombinase that is discovered or generated and is able to perform the same function.

[0134] In one preferred embodiment, the pairs A and B of recombination sites of the nucleic acid construct are independently selected from the group consisting of Lox P (SEQ ID NOs: 4 and 11), Lox2272 (SEQ ID NOs: 3 and 10), Lox 511 (SEQ ID NOs: 30 and 31), Lox5171 (SEQ ID NOs: 32 and 33), Lox 514 (SEQ ID NOs: 34 and 35), Lox 512 (SEQ ID NOs: 36 and 37), Lox 66 (SEQ ID NOs: 38 and 39), Lox 71 (SEQ ID NOs: 40 and 41), and variants thereof, preferably functional variants thereof. In a more preferred embodiment, the recA / fw and recA / rc are recombination sites selected from the group consisting of LoxP, Lox2272, Lox511, Lox5171, Lox514, Lox512, Lox66, Lox71, and variants thereof, and / or, the recB / fw and recB / rc are recombination sites selected from the group consisting of LoxP, Lox2272, Lox511, Lox5171, Lox514, Lox512, Lox66, Lox71, and variants thereof. The variant of a recombinase site of the pairs A and / or of the pair B is preferably a functional variant.

[0135] A "functional variant of a recombination site" refers herein to a variant recombination site that preserves the function of the wild-type recombination site, i.e., recognizability and cleavability by the respective recombinase. A functional variant of a recombination site differs from the wild-type sequence in one or more mutations that prevent neither binding nor cleavage of the site by the respective recombinase. For example, a loxP variant has one or more mutations in the 13 bp inverted repeats region and / or the 8 bp nonpalindromic spacer region.

[0136] In particular, recA / fw and recA / rc may be LoxP recombination sites having amino acid sequences SEQ ID NOs: 4 and 11, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 4 and 11, respectively. Alternatively, recA / fw and recA / rc may be Lox2272 recombination sites having amino acid sequences SEQ ID NOs: 3 and 10, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 3 and 10, respectively. Alternatively, recA / fw and recA / rc may be Lox 511 recombination sites having amino acid sequences SEQ ID NOs: 30 and 31, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 30 and 31, respectively. Alternatively, recA / fw and recA / rc may be Lox5171 recombination sites having amino acid sequences SEQ ID NOs: 32 and 33, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 32 and 33, respectively. Alternatively, recA / fw and recA / rc may be Lox514 recombination sites having amino acid sequences SEQ ID NOs: 34 and 35, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 34 and 35, respectively. Alternatively, recA / fw and recA / rc may be Lox512 recombination sites having amino acid sequences SEQ ID NOs: 36 and 37, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 36 and 37, respectively. Alternatively, recA / fw and recA / rc may be Lox66 recombination sites having amino acid sequences SEQ ID NOs: 38 and 39, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 38 and 39, respectively. Alternatively, recA / fw and recA / rc may be Lox71 recombination sites having amino acid sequences SEQ ID NOs: 40 and 41, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 40 and 41, respectively.

[0137] In particular, recB / fw and recB / rc may be LoxP recombination sites having amino acid sequences SEQ ID NOs: 4 and 11, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 4 and 11, respectively. Alternatively, recB / fw and recB / rc may be Lox2272 recombination sites having amino acid sequences SEQ ID NOs: 3 and 10, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 3 and 10, respectively. Alternatively, recB / fw and recB / rc may be Lox 511 recombination sites having amino acid sequences SEQ ID NOs: 30 and 31, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 30 and 31, respectively. Alternatively, recB / fw and recB / rc may be Lox5171 recombination sites having amino acid sequences SEQ ID NOs: 32 and 33, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 32 and 33, respectively. Alternatively, recB / fw and recB / rc may be Lox514 recombination sites having amino acid sequences SEQ ID NOs: 34 and 35, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 34 and 35, respectively. Alternatively, recB / fw and recB / rc may be Lox512 recombination sites having amino acid sequences SEQ ID NOs: 36 and 37, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 36 and 37, respectively. Alternatively, recB / fw and recB / rc may be Lox66 recombination sites having amino acid sequences SEQ ID NOs: 38 and 39, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 38 and 39, respectively. Alternatively, recB / fw and recB / rc may be Lox71 recombination sites having amino acid sequences SEQ ID NOs: 40 and 41, respectively, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NOs: 40 and 41, respectively.

[0138] Rep mutations

[0139] A rep ORF as used herein may comprise two or more silent mutations and / or a missense mutation.

[0140] Replication of hybrid adenoviruses (Ad) comprising an AAV rep ORF is inhibited. The inventors believe that this inhibition is in large parts due to a cis inhibitory sequence in the rep ORF at the 3' end termed "Rep inhibition sequence for adenoviral replication" (RIS-Ad), and that RIS-Ad functions independently of Rep protein expression.

[0141] Consequently, when the nucleic acid construct according to this invention is contained in an Ad vector, the rep ORF preferably comprises a mutated and inactivated RIS-Ad, meaning that the rep ORF comprises two or more silent mutations and / or a missense mutation in the RIS-Ad in order to prevent Rep expression in the Ad vector.

[0142] A "Rep Inhibition Sequence for Adenoviral replication (RIS-Ad)" refers herein to a nucleic acid sequence comprised between nt 1701 and nt 2186 of the AAV2 genome (SEQ ID NO: 29), which encompasses the Rep68 / 40 donor splice site, the p40 promoter, and the cap 5' UTR.

[0143] The term "silent mutation" refers herein to a modification of a nucleic acid sequence which does not affect the encoded amino acid sequence. Preferably, a silent mutation is a nucleotide substitution.

[0144] The term "missense mutation" refers herein to a nucleotide substitution that results in a different amino acid being encoded by the codon comprising the missense mutation. For example, a missense mutation may lead to a Ser->Cys substitution of the amino acid residue 503 in SEQ ID NO: 14 resulting in SEQ ID NO: 15.

[0145] Preferably, the rep ORF comprises two or more silent mutations and / or a missense mutation in a RIS-Ad comprised in the rep ORF. More preferably, rep3 / rc comprises two or more silent mutations and / or a missense mutation. Still more preferably, rep3 / rc comprises two or more silent mutations and / or a missense mutation in a Rep Inhibition Sequence for Adenoviral replication (RIS-Ad) comprised in the rep ORF.

[0146] Preferably, the number of silent mutations is between about 2 and about 485, about 2 and about 450, about 2 and about 400, about 2 and about 389, about 2 and about 350, about 2 and about 300, about 2 and about 260, about 2 and about 250, about 2 and about 200, about 2 and about 150, about 2 and about 130, about 2 and 100, about 2 and 50, or about 2 and about 35, preferably between about 2 and about 27, more preferably is about 27. A sequence comprising two or more silent mutations may also be termed "recoded nucleic acid sequence".

[0147] Preferably, the number of missense mutations is comprised between 1 and 30.

[0148] The two or more silent mutations and / or missense mutation may or may not disrupt or the p40 promoter in the rep ORF. When the p40 promoter is disrupted, the RNA polymerase cannot bind to the 3' end of the rep ORF, so that cap transcription and protein expression are prevented.

[0149] The position of a mutation in rep ORF is defined herein relative to the full-length rep nucleic acid sequence of SEQ ID NO: 12 starting from the 5' end (i.e., from the start codon "ATG"), also when the mutation is located in rep3 / rc, or relative to the encoded full-length Rep amino acid sequence of SEQ ID NO: 14 starting from the N-terminal.

[0150] The two or more silent mutations and / or missense mutation may be located between nt 1701 and nt 2186 of SEQ ID NO: 29, preferably nt 1787 and nt 2174, more preferably between nt 1787 and nt 1856 and between nt 2171 and nt 2174. In particular, a missense mutation may be located in the codon of nt 1827 to nt 1829 of SEQ ID NO: 29, preferably it is at nt 1827, for example A1827T as in SEQ ID NO: 128 v SEQ ID NO: 29.

[0151] Alternatively or in addition, the two or more silent mutations and / or the missense mutation may be comprised in a codon encoding for an amino acid residue selected from residues 489, 490, 492, 493, 494, 495, 496, 497, 498, 500, 501, 502, 503, 505, 506, 507, 509, 511, 512, 520, 521, and 618 of SEQ ID NO: 14. In particular, a missense mutation may be present in the codon encoding for the amino acid residue 503 of the wild-type Rep amino acid sequence of SEQ ID NO: 14. For example, a missense mutation leads to a Ser->Cys substitution of the amino acid residue 503 as in SEQ ID NO: 15 vs SEQ ID NO: 14.

[0152] The two or more silent mutations in rep or rep3 / rc are chosen in a way that a codon comprises 1 to 3 nucleotide substitutions.

[0153] Preferably, the rep ORF in the nucleic acid construct to be inserted in an Ad vector comprises the silent mutations and a missense mutation in the RIS-Ad such that the rep ORF has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13, 138, 139, 140, 141, 142, 143, 144, 145, and 146, more preferably SEQ ID NO: 139, or such that rep3 / rc has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7, 147, 148, 149, 150, 151, 152, 153, 154, and 155, more preferably SEQ ID NO: 148.

[0154] Additionally or alternatively, the rep ORF in the nucleic acid construct to be inserted in an Ad vector comprises the silent mutations and a missense mutation in the RIS-Ad such that the rep ORF comprises a Ris-Ad nucleic acid sequence selected from the group consisting of SEQ ID NOs: 156-165, and more preferably a nucleic acid sequence of SEQ ID NO: 158.

[0155] Sequences of the nucleic acid construct elements

[0156] In a preferred embodiment of the nucleic acid construct comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof; cap / rc comprises the nucleic acid sequence of SEQ ID NO: 6, or a variant thereof; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof; int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof.

[0157] In a preferred embodiment of the nucleic acid construct comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof having at least 80% sequence identity thereto; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof having at least 80% sequence identity thereto; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof having at least 80% sequence identity thereto; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof having at least 80% sequence identity thereto; cap / rc comprises the nucleic acid sequence of SEQ ID NO: 6, or a variant thereof having at least 80% sequence identity thereto; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof having at least 80% sequence identity thereto; int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof having at least 80% sequence identity thereto; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof having at least 80% sequence identity thereto; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof having at least 80% sequence identity thereto.

[0158] In a preferred embodiment of the nucleic acid construct comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof having at least 85% sequence identity thereto; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof having at least 85% sequence identity thereto; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof having at least 85% sequence identity thereto; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof having at least 85% sequence identity thereto; cap / rc comprises the nucleic acid sequence of SEQ ID NO: 6, or a variant thereof having at least 85% sequence identity thereto; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof having at least 85% sequence identity thereto; int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof having at least 85% sequence identity thereto; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof having at least 85% sequence identity thereto; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof having at least 85% sequence identity thereto.

[0159] In a preferred embodiment of the nucleic acid construct comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof having at least 90% sequence identity thereto; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof having at least 90% sequence identity thereto; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof having at least 90% sequence identity thereto; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof having at least 90% sequence identity thereto; cap / rc comprises the nucleic acid sequence of SEQ ID NO: 6, or a variant thereof having at least 90% sequence identity thereto; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof having at least 90% sequence identity thereto; int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof having at least 90% sequence identity thereto; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof having at least 90% sequence identity thereto; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof having at least 90% sequence identity thereto.

[0160] In a preferred embodiment of the nucleic acid construct comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof having at least 95% sequence identity thereto; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof having at least 95% sequence identity thereto; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof having at least 95% sequence identity thereto; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof having at least 95% sequence identity thereto; cap / rc comprises the nucleic acid sequence of SEQ ID NO: 6, or a variant thereof having at least 95% sequence identity thereto; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof; having at least 95% sequence identity thereto int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof having at least 95% sequence identity thereto; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof having at least 95% sequence identity thereto; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof having at least 95% sequence identity thereto.

[0161] In a preferred embodiment of the nucleic acid construct comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof having at least 98% sequence identity thereto; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof having at least 98% sequence identity thereto; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof having at least 98% sequence identity thereto; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof having at least 98% sequence identity thereto; cap / rc comprises the nucleic acid sequence of SEQ ID NO: 6, or a variant thereof having at least 98% sequence identity thereto; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof; having at least 98% sequence identity thereto int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof having at least 98% sequence identity thereto; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof having at least 98% sequence identity thereto; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof having at least 98% sequence identity thereto.

[0162] In a preferred embodiment of the nucleic acid construct not comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof; int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof.

[0163] In a preferred embodiment of the nucleic acid construct not comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof having at least 80% sequence identity thereto; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof having at least 80% sequence identity thereto; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof having at least 80% sequence identity thereto; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof having at least 80% sequence identity thereto; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof having at least 80% sequence identity thereto; int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof having at least 80% sequence identity thereto; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof having at least 80% sequence identity thereto; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof having at least 80% sequence identity thereto.

[0164] In a preferred embodiment of the nucleic acid construct not comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof having at least 85% sequence identity thereto; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof having at least 85% sequence identity thereto; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof having at least 85% sequence identity thereto; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof having at least 85% sequence identity thereto; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof having at least 85% sequence identity thereto; int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof having at least 85% sequence identity thereto; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof having at least 85% sequence identity thereto; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof having at least 85% sequence identity thereto.

[0165] In a preferred embodiment of the nucleic acid construct not comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof having at least 90% sequence identity thereto; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof having at least 90% sequence identity thereto; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof having at least 90% sequence identity thereto; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof having at least 90% sequence identity thereto; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof having at least 90% sequence identity thereto; int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof having at least 90% sequence identity thereto; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof having at least 90% sequence identity thereto; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof having at least 90% sequence identity thereto.

[0166] In a preferred embodiment of the nucleic acid construct not comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof having at least 95% sequence identity thereto; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof having at least 95% sequence identity thereto; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof having at least 95% sequence identity thereto; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof having at least 95% sequence identity thereto; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof having at least 95% sequence identity thereto; int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof having at least 95% sequence identity thereto; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof having at least 95% sequence identity thereto; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof having at least 95% sequence identity thereto.

[0167] In a preferred embodiment of the nucleic acid construct not comprising cap / rc, rep5 / fw comprises the nucleic acid sequence of SEQ ID NO: 1, or a variant thereof having at least 98% sequence identity thereto; int5 / fw comprises the nucleic acid sequence of SEQ ID NO: 2, or a variant thereof having at least 98% sequence identity thereto; recA / fw comprises the nucleic acid sequence of SEQ ID NO: 3, or a variant thereof having at least 98% sequence identity thereto; recB / fw comprises the nucleic acid sequence of SEQ ID NO: 4, or a variant thereof having at least 98% sequence identity thereto; rep3 / rc comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof having at least 98% sequence identity thereto; int3 / rc comprises the nucleic acid sequence of SEQ ID NO: 9, or a variant thereof having at least 98% sequence identity thereto; recA / rc comprises the nucleic acid sequence of SEQ ID NO: 10, or a variant thereof having at least 98% sequence identity thereto; and / or recB / rc comprises the nucleic acid sequence of SEQ ID NO: 11, or a variant thereof having at least 98% sequence identity thereto.

[0168] Additionally or alternatively, in a preferred embodiment of the nucleic acid construct not comprising cap / rc, rep3 / rc comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, 147, 148, 149, 150, 151, 152, 153, 154, and 155, more preferably SEQ ID NO: 148.

[0169] Additionally or alternatively, in a preferred embodiment of the nucleic acid construct comprising cap / rc, rep3 / rc comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, 147, 148, 149, 150, 151, 152, 153, 154, and 155, more preferably SEQ ID NO: 148.

[0170] Preferably, the nucleic acid construct according to the first aspect and comprising cap / rc has the nucleic acid sequence comprised in SEQ ID NO: 16, 17 or 133, or a variant thereof wherein rep5 / fw is operably linked to a p5 promoter and the cap ORF is operably linked, after inversion and splicing, to a 3' UTR sequence. Preferably, the nucleic acid construct according to the first aspect and comprising cap / rc has the nucleic acid sequence comprised in SEQ ID NO: 16, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NO: 16. Preferably, the nucleic acid construct according to the first aspect and comprising cap / rc has the nucleic acid sequence comprised in SEQ ID NO: 17, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NO: 17. Preferably, the nucleic acid construct according to the first aspect and comprising cap / rc has the nucleic acid sequence comprised in SEQ ID NO: 133, or a variant thereof, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NO: 133. Preferably, the nucleic acid construct according to the first aspect and not comprising cap / rc has the nucleic acid sequence comprised in SEQ ID NO: 18, or a variant thereof wherein rep5 / fw is operably linked to a p5 promoter and the cap ORF is operably linked, after inversion and splicing, to a 3' UTR sequence, wherein the variant preferably has at least 80% 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or at least 99.9% sequence identity to SEQ ID NO: 18. The nucleic acid constructs of SEQ ID NOs: 16 and 17 are suitable when the nucleic acid construct is provided in a rAd for rAAV production. The nucleic acid constructs of SEQ ID NOs: 133 and 18 are suitable when the nucleic acid construct is provided chromosomally in a cell for rAAV production.

[0171] Vectors

[0172] In a second aspect, the present invention relates to a vector comprising the nucleic acid construct according to the first aspect.

[0173] In one embodiment of the second aspect, the present invention relates to a vector comprising a nucleic acid construct comprising:

[0174] (i) an adeno-associated Virus (AAV) rep open reading frame (ORF) comprising a heterologous intron (int), wherein the rep ORF and the int are split within the intron into a 5' portion (rep5 / fw int5 / fw) and an inverted 3' portion (rep3 / rc int3 / rc),

[0175] (ii) an inverted AAV cap partial ORF (cap / rc) located between int5 / fw and rep3 / rc, and (iii) a pair A of recombination sites (recA / fw, recA / rc), and a different pair B of recombination sites (recB / fw, recB / rc), wherein each pair is specific for a recombinase and allows for inversion of rep3 / rc int3 / rc and cap / rc, wherein the nucleic acid construct has the 5' to 3' structure: rep5 / fw int5 / fw recA / fw recB / fw cap / rc -rep3 / rc int3 / rc recA / rc recB / rc, wherein — is a direct or an indirect link, and - is a direct link.

[0176] In another embodiment of the second aspect, the present invention relates to a vector comprising a nucleic acid construct comprising:

[0177] (i) an adeno-associated Virus (AAV) rep open reading frame (ORF) comprising a heterologous intron (int), wherein the rep ORF and the int are split within the intron into a 5' portion (rep5 / fw int5 / fw) and an inverted 3' portion (rep3 / rc int3 / rc), and

[0178] (ii) a pair A of recombination sites (recA / fw, recA / rc), and a different pair B of recombination sites (recB / fw, recB / rc), wherein each pair is specific for a recombinase and allows for inversion of rep3 / rc int3 / rc and, wherein the nucleic acid construct has the 5' to 3' structure: rep5 / fw — int5 / fw — recA / fw — recB / fw — rep3 / rc — int3 / rc — recA / rc — recB / rc, wherein — is a direct or an indirect link.

[0179] The nucleic acid construct is preferably present in the vector within an expression cassette, wherein the 5' end of rep5 / fw is operably linked to a promoter capable of driving expression of the Rep78 and Rep68 proteins. The promoter may be an AAV promoter or a heterologous promoter, preferably an AAV promoter, more preferably an AAV p5 promoter of the same or different AAV serotype, still more preferably of the same AAV serotype.

[0180] The term "vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus (Ad) vectors (e.g., non-replicating Ad2, Ad5, Ad6, Adi l, Ad26, Ad35, Ad49, GRAd23, GRAd32, ChAd3, ChAd4, ChAd5, ChAd7, ChAd8, ChAd9, ChAdlO, ChAdl l, ChAdl6, ChAdl7, ChAdl9, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82 vectors or replication-competent Ad4 and Ad7 vectors known e.g. from WO 2005 / 071093 A2), alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE), sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN chimeras), human cytomegalovirus vectors (HCMV), herpes simplex virus type 2 vectors (e.g. herpes simplex virus type-2, HSV-2), human herpes virus vectors (e.g. HHV-6), measles virus vectors, pox virus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MV A), NYVAC (derived from the Copenhagen strain of vaccinia), and avipox vectors: canarypox (ALVAC) and fowlpox (FPV) vectors), varicella zoster virus vectors (VZV), and vesicular stomatitis virus vectors, virus-like particles, or bacterial spores. A vector also includes expression vectors, cloning vectors and vectors that are useful to generate recombinant adenoviruses in host cells.

[0181] Preferably, the vector comprising the nucleic acid construct according to the first aspect is a recombinant virus.

[0182] The vector comprising the nucleic acid construct according to the first aspect may be a recombinant virus of the adenoviridae family, herpesvirus family or of the parvovirus family. In one preferred embodiment, the vector comprising the nucleic acid construct according to the first aspect is a recombinant virus selected from the group consisting of adenovirus, in particular human adenovirus or gorilla adenovirus, human cytomegalovirus (HCMV), herpes simplex virus (e.g., herpes simplex virus type-2, HSV-2), varicella zoster virus (VZV), and human herpes virus (e.g., HHV-6), human bocavirus 1, and baculovirus. Preferably, the vector comprising the nucleic acid construct according to the first aspect is a recombinant adenovirus (rAd), more preferably comprising E4, E2A and VA RNA.

[0183] The term "rAd vector" as used herein refers to an Ad virus particle whose genome has been modified by artificial means, e.g., by recombinant DNA technology via genetic engineering by using methods known in the art such as those described in Example 4 of WO 2022 / 003083 Al, to obtain a rAd particle that comprises a nucleic acid construct of the invention (repFLEX, repFLEXcap), cap, transgene, and / or recAB, and may be used as a vector to produce rAAV particles. The method of rAd production, well known in the art, foresees starting from the Bacterial Artificial Chromosome (BAC) containing the genome of the Adenovirus (preferentially a gorilla adenovirus, GRAd), which contains the sequence of interest. This BAC is linearized by means of digestion with appropriate restriction enzyme (e.g. Pmel), and the linearized DNA is transfected in wt HEK293 cells (i.e., without Cre expression) for the first viral growth that is typically called “rescue”. Usually, 14 days post transfection cells are harvested, if necessary, they are lysed, and the clarified lysate is used to infect subsequent passages of HEK293 cells (‘propagation’), that can grow either as adherent cells, or as suspension cells, preferably as suspension cells.

[0184] An adenovirus (Ad) is a non-enveloped, icosahedral virus that has been identified in several avian and mammalian hosts. Human adenoviruses (hAds) belong to the Mastadenovirus genus, which includes all known human, and many Ads of animal (e.g., bovine, porcine, canine, murine, equine, simian and ovine) origin. Human adenoviruses are generally divided into six subgroups (A-F) based on a number of biological, chemical, immunological, and structural criteria. The adenoviral genes are referred to as early (E) or late (L) genes according to whether transcription occurs prior to or after onset of DNA replication. In the early phase of transduction, the El A, E1B, E2A, E2B, E3 and E4 genes of adenovirus are expressed to prepare the host cell for viral replication. During the late phase of infection, expression of the late genes L1-L5, which encode for the structural components of the virus particles is activated. The VA (viral associated) RNA is a type of non-coding RNA found in adenovirus. It plays a role in regulating translation. There are two copies of this RNA called VAI or VA RNA I and VAII or VA RNA II, transcribed from two distinct VA RNA genes by PolIII. VA RNA I is the major species whereas VA RNA II is expressed at a lower level. Neither transcript is polyadenylated. Not limiting examples of vectors that can be used to deliver a nucleic acid according to the first aspect, a cap, transgene, and / or recAB are those according to SEQ ID NO: 166, 167, 169, 170, 171,172, 173, 174, 175, 176, 177, and 178.

[0185] Collection of vectors

[0186] In a third aspect, the present invention relates to a collection of vectors, comprising:

[0187] (i) a first vector according to the second aspect comprising cap / rc, and a second vector comprising a transgene flanked by AAV ITRs (inverted terminal repeats);

[0188] (ii) a first vector according to the second aspect comprising cap / rc and a transgene flanked by AAV ITRs, and a second vector comprising a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct comprised in the vector, or

[0189] (iii) a first vector according to the second aspect comprising cap / rc, a second vector comprising a transgene flanked by AAV ITRs, and a third vector comprising a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct comprised in the vector.

[0190] The phrase "vector according to the second aspect comprising cap / rc" refers to a vector comprising the nucleic acid construct of the first aspect comprising cap / rc directly linked to rep3 / rc.

[0191] Preferably, the second vector of (i) comprising a transgene flanked by AAV ITRs further comprises recAB.

[0192] Thus, the collection of vectors may comprise two vectors, e.g.: a first vector according to the second aspect comprising cap / rc, and a second vector comprising a transgene flanked by AAV ITRs and recAB.

[0193] The vector comprising a transgene flanked by AAV ITRs may comprise one or more transgenes, preferably one. The AAV 5' ITR may have the nucleic acid sequence of nt 1-145 of SEQ ID NO: 29 and the AAV 3' ITR may have the nucleic acid sequence of nt 4535-4679 of SEQ ID NO: 29.

[0194] The term "transgene" is used herein to refer to a nucleic acid or gene that is intended to be introduced into a mammalian cell. A transgene refers in particular to a heterologous nucleic acid or gene, i.e., a non-adenoviral and / or non-AAV nucleic acid or gene, encoding for a heterologous protein or peptide. The term "heterologous protein or peptide" refers herein to a non-adenoviral and / or non-AAV protein or peptide. A transgene preferably encodes for a therapeutic or diagnostic protein or peptide, more preferably for a therapeutic protein or peptide, for example an immunogen or antigen.

[0195] The transgene and / or recAB may be inserted in a recombinant virus within an expression cassette, as defined herein, comprising a promoter operably linked to the transgene and / or recAB.

[0196] The molecular size of the transgene or recAB is chosen such that the capsid can form around and package the genome, when the rAd or thereafter the AAV is produced in a packaging cell. Thus, preferably the one or more transgene or recAB comprise a nucleic acid sequence having a length of up to 8000 nt, preferably up to 7000 nt.

[0197] The term "nucleic acid recAB" or "nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites" is abbreviated herein to "recAB". RecAB may comprise one or more nucleic acids encoding for the one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct according to the first aspect or of the nucleic acid construct comprised in the vector according to the second aspect. Preferably, recAB encodes for one recombinase specific for both pairs A and B of recombination sites of the nucleic acid construct according to the first aspect or of the nucleic acid construct comprised in the vector according to the second aspect, and preferably for a Cre recombinase.

[0198] Cell

[0199] The term "cell" refers herein to a mammalian cell from a cell line that may be prepared by any transformation and selection method. The cell may be of a human cell line. Preferably, the cell is selected from the group consisting of a HEK293 cell, a A549 cell, a HeLa cell, a MRC5 cell, a VERO cell, a DF-1 cell, and a SK-OV-3 cell (available from the ATCC), preferably of HEK293 cell and HeLa cell. More preferably, the cell is a HEK293 cell. The cell as used herein may be an AAV producing and packaging cell, or a rAd producing and packaging cell.

[0200] The term "rAd producing and packaging cell" refers herein to a cell that expresses stably or transiently one or more viral genes required for viral packaging. A rAd producing and packaging cell preferably do not comprise recAB, i.e., does not express a recombinase specific for the pairs A and / or B of recombination sites of the nucleic acid construct of the first aspect.

[0201] The term "rAAV producing and packaging cell" refers herein to a cell that expresses AAV Rep and Cap stably (i.e., when the nucleic acid construct of the first aspect is present chromosomally) or transiently (i.e., when the nucleic acid construct of the first aspect is present extra-chromosomally, e.g. in a vector), and further comprises a recAB (chromosomally or extra-chromosomally), and optionally a transgene (extra-chromosomally). Of advantage, adenovirus production is inhibited during AAV production since Rep expression inhibits adenoviral replication.

[0202] Thus, in a fourth aspect, the present invention relates to a cell comprising:

[0203] (a) extra-chromosomally the nucleic acid construct of the first aspect comprising cap / rc, or extra-chromosomally the vector of the second aspect comprising cap / rc; or

[0204] (b) chromosomally the nucleic acid construct of the first aspect comprising cap / rc, or chromosomally the nucleic acid construct of the first aspect not comprising cap / rc.

[0205] The term "cell comprising extra-chromosomally" refers herein to a cell comprising the nucleic acid or vector not integrated in the genome of the cell. In particular, the nucleic acid according to the first aspect may be present in a plasmid, and preferably in an extra- chromosomally replicating plasmid, i.e., an autonomously replicating plasmid that is an extra- chromosomal entity and replicates independently of chromosomal replication.

[0206] The cell of the fourth aspect (a) may in particular comprise one of the following combinations of elements:

[0207] (i)l extra-chromosomally the nucleic acid construct of the first aspect comprising cap / rc, and chromosomally recAB;

[0208] (i)2 extra-chromosomally the nucleic acid construct of the first aspect comprising cap / rc and further a transgene flanked by AAV ITRs, and chromosomally recAB; (i)3 extra-chromosomally the vector of the second aspect comprising cap / rc, and chromosomally recAB;

[0209] (i)4 extra-chromosomally the vector of the second aspect comprising cap / rc and a transgene flanked by AAV ITRs, and chromosomally recAB;

[0210] (ii) 1 a first vector according to the second aspect comprising cap / rc, and a second vector comprising a transgene flanked by AAV ITRs;

[0211] (ii)2 a first vector according to the second aspect comprising cap / rc and a transgene flanked by AAV ITRs, and a second vector comprising recAB; or

[0212] (ii)3 a first vector according to the second aspect comprising cap / rc, a second vector comprising a transgene flanked by AAV ITRs, and a third vector comprising recAB.

[0213] When the cell comprises the collection of vectors defined in (ii)l, the second vector comprising a transgene flanked by AAV ITRs may further comprise recAB, or the cell may chromosomally comprise recAB.

[0214] The cell of the fourth aspect (b) comprising the nucleic acid construct comprising cap / rc may further comprise:

[0215] (i) a transgene flanked by AAV ITRs, and

[0216] (ii) recAB, wherein (i) and (ii) are comprised in one or more vectors. Preferably, (i) and (ii) are comprised in one vector. More preferably, the vector(s) is / are a recombinant virus. Still more preferably, the vector(s) is / are a rAd.

[0217] The cell of the fourth aspect (b) comprising the nucleic acid construct not comprising cap / rc may further comprise:

[0218] (i) a transgene flanked by AAV ITRs,

[0219] (ii) recAB, and

[0220] (iii) a nucleic acid comprising a cap ORF; wherein (i) to (iii) are comprised in one or more vectors. Preferably, (i) to (iii) are comprised in one vector. More preferably, the vector(s) is / are a recombinant virus. Still more preferably, the vector(s) is / are a rAd.

[0221] The nucleic acid of (iii) comprising a cap ORF may be included in a vector within an expression cassette as defined herein comprising an AAV cap promoter, i.e., an AAV cap 5' UTR and p40 promoter, or a heterologous (non-AAV) promoter operably linked to the cap ORF.

[0222] The expression cassette preferably comprises an AAV cap promoter, which for example may comprise the rep nucleic acid sequence between nt 1701 and nt 2202 of SEQ ID NO: 29.

[0223] If the nucleic acid of (iii) comprising a cap ORF is included in a rAd vector, the AAV cap promoter comprises two or more silent mutations and / or a missense mutation as defined for the first aspect, i.e., is a recoded cap promoter. Preferably, the recoded AAV cap promoter comprises the silent mutations and a missense mutation as comprised in the rep nucleic acid sequence of SEQ ID NO: 13, so that a nucleic acid sequence comprising the recoded AAV2 cap promoter, cap ORF and 3' UTR signal sequences has the nucleic acid sequence of SEQ ID NO: 23.

[0224] Kit

[0225] The fifth aspect of the present invention relates to a kit comprising the vector of the second aspect, the collection of vectors of the third aspect, or the cell of the fourth aspect, which can be useful to produce recombinant adeno-associated virus (rAAV) particles.

[0226] Specifically, the sixth aspect relates to a kit comprising:

[0227] (A) the collection of vectors of the third aspect, or the vector of the second aspect comprising cap / rc and a transgene flanked by AAV ITRs, and a cell; or

[0228] (B) the cell of the fourth aspect (b), and one or more vectors comprising:

[0229] (i) a transgene flanked by AAV ITRs,

[0230] (ii) a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct of the first aspect, and

[0231] (iii) a nucleic acid comprising a cap ORF, if a cap ORF is not present chromosomally.

[0232] One embodiment of the fifth aspect (fifth aspect (A)) relates to a kit providing the nucleic acid construct of the first aspect comprised in one or more vector(s), e.g.: the collection of vectors of the third aspect, and a cell; or the vector of the second aspect comprising cap / rc and a transgene flanked by AAV ITRs, and a cell.

[0233] Preferably, the cell chromosomally comprises recAB.

[0234] Another embodiment of the fifth aspect (fifth aspect (B)) relates to a kit providing the nucleic acid construct of the first aspect comprised in the chromosome of a cell, e.g., comprising: the cell of the fourth aspect (b), and one or more vectors comprising: (i) a transgene flanked by AAV ITRs,

[0235] (ii) recAB, and

[0236] (iii) a nucleic acid comprising a cap ORF.

[0237] Preferably, (i) to (iii) are comprised in one vector. More preferably, the vector(s) is / are a recombinant virus. Still more preferably, the vector(s) is / are a rAd.

[0238] The kit of the fifth aspect (A) may in particular comprise the following combinations of elements:

[0239] Al) a first vector comprising the nucleic acid construct of the first aspect comprising cap / rc, a second vector comprising a transgene flanked by AAV ITRs and recAB, and a cell;

[0240] A2) a first vector comprising the nucleic acid construct of the first aspect comprising cap / rc, a second vector comprising a transgene flanked by AAV ITRs, and a cell, wherein the cell chromosomally comprises recAB;

[0241] A3) a first vector comprising the nucleic acid construct of the first aspect comprising cap / rc and a transgene flanked by AAV ITRs, a second vector comprising recAB, and a cell;

[0242] A4) a first vector comprising the nucleic acid construct of the first aspect comprising cap / rc, a second vector comprising a transgene flanked by AAV ITRs, a third vector comprising recAB, and a cell;

[0243] A5) a vector comprising the nucleic acid construct of the first aspect comprising cap / rc and a transgene flanked by AAV ITRs, and a cell, wherein the cell chromosomally comprises recAB.

[0244] The kit of the fifth aspect (B) may comprise the following combinations of elements:

[0245] Bl) a cell comprising chromosomally the nucleic acid construct of the first aspect comprising cap / rc, and one or more vectors comprising:

[0246] (i) a transgene flanked by AAV ITRs,

[0247] (ii) recAB, or

[0248] B2) a cell comprising chromosomally the nucleic acid construct of the first aspect not comprising cap / rc, and one or more vectors comprising:

[0249] (i) a transgene flanked by AAV ITRs,

[0250] (ii) recAB, and

[0251] (iii) a nucleic acid comprising a cap ORF.

[0252] The kit may further comprise reagents and instructions for using the vector(s) to transduce a cell, preferably a rAAV producing and packaging cell, and to produce and optionally isolate the rAAV particles.

[0253] Production method and uses

[0254] The inventors have found that a rAAV production method based on the use of a cell comprising the nucleic acid construct of the first aspect and one or more vectors expressing an appropriate recombinase and a transgene allows to obtain high titers of rAAV with high purity, i.e., with minimal rAd contamination (Examples 4 and 5). Indeed, the method allows obtaining about 1-2 E+l l vg / ml (viral genome copies / ml) of rAAV starting from only 50 multiplicity of infection (MOI) of each rAd.

[0255] The sixth aspect of the present invention relates to the use of the nucleic acid construct of the first aspect, the vector of the second aspect, or the cell of the fourth aspect in a method for producing recombinant adeno-associated virus (rAAV) particles.

[0256] Specifically, the sixth aspect relates to a method for producing recombinant adeno- associated virus (rAAV) particles comprising a transgene, the method comprising the steps of:

[0257] (a) - providing the collection of vectors of the third aspect, or the vector of the second aspect comprising cap / rc and a transgene flanked by AAV ITRs, and a cell,

[0258] - transfecting the cell with the vector(s),

[0259] - culturing the cell to produce the rAAV particles; or

[0260] (b) - providing the cell of the fourth aspect (b), and one or more vectors comprising:

[0261] (i) a transgene flanked by AAV ITRs,

[0262] (ii) recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct of the first aspect, and

[0263] (iii) a nucleic acid comprising a cap ORF, if a cap ORF is not present chromosomally,

[0264] - transfecting the cell with the one or more vectors, and

[0265] - culturing the cell to produce the rAAV particles.

[0266] When the nucleic acid construct is provided in a vector, the method according to the sixth aspect (a) may comprise the steps of: - providing the collection of vectors of the third aspect, or the vector of the second aspect comprising cap / rc and a transgene flanked by AAV ITRs, and a cell,

[0267] - transfecting the cell with the vector(s), and

[0268] - culturing the cell to produce the rAAV particles.

[0269] The cell may chromosomally comprise recAB.

[0270] When the nucleic acid construct is provided chromosomally in a cell, the method according to the sixth aspect (b) may comprise the steps of:

[0271] - providing the cell of the fourth aspect (b), and one or more vectors comprising:

[0272] (i) a transgene flanked by AAV ITRs,

[0273] (ii) recAB, and

[0274] (iii) a nucleic acid comprising a cap ORF, if a cap ORF is not present chromosomally.

[0275] - transfecting the cell with the one or more vectors,

[0276] - culturing the cell to produce the rAAV particles.

[0277] Optionally, the method (a or b) further comprises a step of isolating the rAAV particles after the step of culturing the cell to produce the rAAV particles.

[0278] Herein, the term "recombinant adeno-associated virus (rAAV) particle" refers to an AAV particle whose genome has been modified by artificial means, e.g., by recombinant DNA technology via genetic engineering to obtain an AAV particle that comprises a transgene and may be used as a vector, e.g. in gene therapy.

[0279] Herein, the term "producing rAAV particles" refers to the generation of a rAAV particle by exploiting the protein biosynthesis machinery of a producer or packaging cell. The term "producing a rAAV particle" may also refer to the amplification of rAAV, e.g., increasing the amount of rAAV from a small amount of starter rAAV to titers high enough for e.g. clinical applications.

[0280] Herein, the term "isolating rAAV particles" refers to the collection of the supernatant of the cells after transfection and culture to allow the packaging of the rAAV particles, and separation from impurities, such as cell debris, and optionally rAd vectors. Optionally, "isolating rAAV particles" comprises a step for purifying rAAV, e.g., a step using heparin sulfate-based columns, or the double CsCI banding method.

[0281] The isolation step of the method of the present invention can further include steps of eliminating rAd from the produced rAAV particles. Thus, in one embodiment, the step of isolating rAAV particles further comprises exposing the cell lysate to a temperature sufficient for inactivation of rAd vectors. For example, adenovirus can be inactivated by heating host cell lysates at 55°C-65°C for 45-60 minutes, preferably a 56°C for 60 minutes. This treatment completely inactivates the rAd vectors while causing minimal reduction in rAAV titers.

[0282] Moreover, the cell transfected with the nucleic acids or vectors of the invention may be selected by using any technique known in the art. For example, the nucleic acid constructs and transgene used to transfect the cell may be introduced simultaneously with or operably linked to one or more detectable or selectable markers as is known in the art. For example, when using a drug resistance gene as a selectable marker, drug resistant cells can be picked-up and grown, and then tested for expression of the desired sequence (i.e., a product of the transgene).

[0283] When recAB is provided in a vector, the method according to the sixth aspect (a) may in particular comprise the steps of:

[0284] - providing a collection of vectors comprising:

[0285] (i) a first vector according to the second aspect comprising cap / rc, and a second vector comprising a transgene flanked by AAV ITRs and recAB,

[0286] (ii) a first vector according to the second aspect comprising cap / rc a transgene flanked by AAV ITRs, and a second vector comprising recAB, or

[0287] (iii) a first vector according to the second aspect comprising cap / rc, a second vector comprising a transgene flanked by AAV ITRs, and a third vector comprising recAB, and a cell,

[0288] - transfecting the cell with the vectors,

[0289] - culturing the cell to produce the rAAV particles.

[0290] When recAB is provided integrated chromosomally in a cell, and the nucleic acid construct with cap(rv) is provided in a different vector than the transgene, the method according to the sixth aspect (a) may in particular comprise the steps of:

[0291] - providing a collection of vectors comprising: a first vector according to the second aspect comprising cap / rc, and a second vector comprising a transgene flanked by AAV ITRs; and a cell, wherein the cell chromosomally comprises recAB,

[0292] - transfecting the cell with the vectors,

[0293] - culturing the cell to produce the rAAV particles.

[0294] Alternatively, when recAB is provided integrated chromosomally in a cell, and the nucleic acid construct with cap(rv) is provided in the same vector as the transgene, the method according to the sixth aspect (a) may comprise the steps of: - providing a vector comprising the nucleic acid of the first aspect comprising cap / rc and a transgene flanked by AAV ITRs, and a cell, wherein the cell chromosomally comprises recAB,

[0295] - transfecting the cell with the vector,

[0296] - culturing the cell to produce the rAAV particles.

[0297] When using the nucleic acid of the first aspect comprising cap / rc chromosomally integrated in the cell, the method according to the sixth aspect (b) may comprise the steps of:

[0298] - providing: a cell comprising chromosomally the nucleic acid construct of the first aspect comprising cap / rc, and one or more vectors comprising:

[0299] (i) a transgene flanked by AAV ITRs, and

[0300] (ii) recAB,

[0301] - transfecting the cell with the one or more vectors,

[0302] - culturing the cell to produce the rAAV particles.

[0303] When using the nucleic acid of the first aspect not comprising cap / rc chromosomally integrated in the cell, the method according to the sixth aspect (b) may comprise the steps of:

[0304] - providing: a cell comprising chromosomally the nucleic acid construct of the first aspect not comprising cap / rc, and one or more vectors comprising:

[0305] (i) a transgene flanked by AAV ITRs,

[0306] (ii) recAB, and

[0307] (iii) a nucleic acid comprising a cap ORF,

[0308] - transfecting the cell with the one or more vectors,

[0309] - culturing the cell to produce the rAAV particles.

[0310] The inventors found that the nucleic acid constructs of the first aspect allow to obtain high titers of rAd vectors for rAAV production, as the rep ORF is split and inverted and thus not expressed in protein during the first rAd production step.

[0311] Therefore, in one embodiment of the invention, the method comprises the preceding rAd production steps: a) providing a rAd genome, preferably a gorilla GRAd, comprising a nucleic acid construct according to the first aspect; b) providing a cell, wherein the cell does not comprise recAB; c) transfecting the cell with the rAd genome comprising the nucleic acid construct according to the first aspect; d) culturing the cell to produce the rAd particles, e) optionally isolating the rAd particles, f) optionally infecting a cell with the rAd particles obtained in e).

[0312] Preferably, the rAd genome is comprised in a Bacterial Artificial Chromosome (BAC).

[0313] The cell used above preferably is a rAd producing and packaging cell. The phrase "cell does not comprise recAB" means that the cell does not express a recombinase specific for the pairs A and / or B of recombination sites of the nucleic acid construct of the first aspect.

[0314] The isolated rAd particles can then be used as rAd vectors to produce the rAAV particles by performing the method of the sixth aspect.

[0315] Definitions and further embodiments of the invention

[0316] To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA techniques are employed which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2ndEdition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0317] The specification uses a variety of terms and phrases, which have certain meanings as defined below. Preferred meanings are to be construed as preferred embodiments of the aspects of the invention described herein. As such, they and also further embodiments described in the following can be combined with any embodiment of the aspects of the invention and in particular any preferred embodiment of the aspects of the invention described above.

[0318] The term "about" means approximately, and in the context of a numerical value means ±20%, ±10%, ±5%, or ±3% of the numerical value.

[0319] The term "polynucleotide" refers to a nucleic acid, i.e., a biological molecule made up of a plurality of nucleotides. It includes DNA, RNA, and synthetic analogs, e.g., PNA. DNA is preferred.

[0320] The term "open reading frame" (ORF) refers to a sequence of nucleotides that can be translated into amino acids to form a protein or peptide. Typically, an ORF contains a start codon, a subsequent region usually having a length which is a multiple of 3 nucleotides, but does not contain a stop codon (TAG, TAA, TGA, UAG, UAA, or UGA) in the given reading frame. An ORF codes for a protein where the amino acids into which it can be translated form a peptide-linked chain.

[0321] The term "restores" or "restoring" referred to a ORF herein refers to the process affecting a splitted or fragmented ORF (e.g., rep ORF in rep5 and rep3) or a partial ORF (e.g. CAP pORF) resulting in an ORF present in the nucleic acid construct in 5' to 3' direction, which can thus be expressed when operably linked to a promoter at its 5' and signal sequences at its 3'.

[0322] A "complementary" nucleic acid sequence refers herein to an antisense sequence represented in direction from its 3' to 5' end, i.e., in the same direction as the sense sequence.

[0323] A "forward" (fw or / fw) nucleic acid sequence refers herein to a sense sequence which is represented in direction from its 5' to 3' end.

[0324] A "reverse" nucleic acid sequence refers herein to a sense sequence which is represented in direction from its 3' to 5' end.

[0325] A "reverse complementary (rc or / rc)" or "inverted (in)" nucleic acid sequence are used interchangeably herein to refer to an antisense sequence represented in direction from its 5' to 3' end, i.e., in the opposite direction as the sense sequence. As example, if the sense sequence is: 5'-ACGTATAGGC-3', the complementary sequence is 3'-TGCATATCCG-5', the reverse sequence is 3'-CGGATATGCA-5', and the "reverse complementary" or "inverted" is 5'- GCCTATACGT-3'.

[0326] The term "inversion" of a nucleic acid sequence as used herein refers to a substitution of the nucleic acid sequence with its reverse complement sequence.

[0327] The term "portion" as used herein refers to a part of the nucleic acid construct of the first aspect comprising one or more elements, fragments or ORF, such as one or more of rep5, rep3, int5, int3, in sense direction or as inverted sequences. The term "fragment" as used herein refers to a part of a nucleic acid sequence of an open reading frame or intron or exon, or to a part of an amino acid sequence of a protein or peptide.

[0328] The term "expression cassette" refers to a nucleic acid molecule that comprises at least one nucleic acid sequence that is to be expressed, along with its transcription and translation control sequences. Because of the restriction sites being preferably engineered to be present at the 5' and 3' ends, the cassette can be easily inserted, removed, or replaced with another cassette. Preferably, an expression cassette includes cv.s-regulating elements for efficient expression of a given gene, such as promoter, initiation-site, polyadenylation-site, ribosome binding sites, and / or transcription termination, and optionally enhancers. More specific with respect to the present invention, an expression cassette contains all the elements required for the expression of the nucleic acid constructs of the first aspect, of the transgene, recAB or cap ORF. A typical expression cassette thus contains a promoter operably linked to rep5 / fw, transgene, recAB or cap ORF. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.

[0329] Methods to introduce an expression cassette into the genome of a virus are well known in the art. In one example, a rAd comprising a transgene or recAB within an expression cassette may be generated by replacing a genomic region of the adenovirus selected from E1A, E1B, E2A, E2B, E3 and E4 with the expression cassette comprising the transgene. The genomic regions E1A, E1B, E2A, E2B, E3 and E4 of the adenoviruses of the invention can easily be identified by an alignment with known and annotated adenoviral genomes (see: Birgitt Tauber and Thomas Dobner, Oncogene (2001) 20, p. 7847-7854; and also: Andrew J. Davison, et al., "Genetic content and evolution of adenoviruses", Journal of General Virology (2003), 84, p. 2895-2908).

[0330] The term "promoter" refers herein to a nucleic acid sequence that, when operably linked to a nucleic acid sequence to be expressed, is capable of controlling the transcription of the nucleic acid sequence into mRNA. A promoter is usually located 5' (i.e., upstream) of the nucleic acid sequence to be expressed and is specific bound by RNA polymerase and other transcription factors for initiation of transcription.

[0331] A nucleic acid sequence is "operably linked" to a promoter when said promoter is positioned in a functional location and direction in relation to said nucleic acid sequence so that the promoter is capable to control transcription of that sequence.

[0332] The term "heterologous promoter" refers herein to a promoter that has been artificially linked to the nucleic acid sequence to be expressed although said promoter does not normally control the expression of that nucleic acid sequence. A heterologous promoter may be constitutive or inducible. A heterologous promoter may also be cell- or tissue-specific promoter to ensure cell- or tissue-specific expression of the nucleic acid sequence to be expressed. The heterologous promoter used for the present invention may be any promoter suitable for the expression of the nucleic acid sequence (rep5 / fw, transgene, recAB or cap ORF) in a vector or in a cell. The heterologous promoter may be selected from the group consisting of P-Actin promoter (e.g. chicken P-Actin promoter (CBA)) (SEQ ID NO: 112), CAG promoter (SEQ ID NO: 113), CASI promoter (SEQ ID NO: 114), CMV (SEQ ID NO: 115), EFl alpha promoter (SEQ ID NO: 116), EGR1 promoter (SEQ ID NO: 117), PGK-1 promoter (SEQ ID NO: 118), RSV promoter (SEQ ID NO: 119), SV40 promoter (SEQ ID NO: 120), human thymidine kinase (TK) promoter (SEQ ID NO: 121), and Ubiquitin B promoter (SEQ ID NO: 122). A preferred heterologous promoter to be operably linked to rep5 / fw is a CAG promoter. A preferred heterologous promoter to be operably linked to recAB (e.g., Cre) is a SV40 or PGK-1 promoter.

[0333] The term "inducible promoter" as used herein refers to a promoter which activity of transcription control of an operably linked nucleic acid sequence is regulated by a stimulus, such as a chemical stimulus, so that the level of transcription of the operably linked nucleic acid sequence in the presence of the stimulus is different from the level of transcription in the absence of the stimulus. Example of inducible promoters are lac, tac, trc, ara, trp, X phage, T7 phage, and T5 phage promoter, and tetracycline inducible promoters. The term "constitutive" promoter refers herein to a promoter which is capable of directing transcription of an operably linked nucleic acid sequence independently of the presence of a stimulus.

[0334] The term “splicing” or “spliced” refers to the mechanism by which the exons of a transcript are joined together by the removal of one or more intron sequences from the primary transcript to form a mature messenger RNA. During this process, splice donor sites interact with splice acceptor sites to allow excision of the intron(s) bounded by the splice donor and acceptor sites. For each transcript the splice donor site splices with only splice acceptor site. “Spliced mRNA” or “spliced transcript” refers herein to mRNA or transcripts produced by either removal of one or more intron sequences from the primary transcript or by constructing a cDNA without introns.

[0335] The term "identity" or "identical" in the context of polynucleotide, polypeptide or protein sequences refers to the number of residues in the two sequences that are identical when aligned for maximum correspondence. Specifically, the percent sequence identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Alignment tools that can be used to align two sequences are well known to the person skilled in the art and can, for example, be obtained on the World Wide Web, e.g., Clustal Omega (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ) for polypeptide alignments or MUSCLE (http: / / www.ebi.ac.uk / Tools / msa / muscle / ) or MAFFT (http: / / www.ebi.ac.uk / Tools / msa / mafft / ) for polynucleotide alignments or WATER (http: / / www.ebi.ac.uk / Tools / psa / emboss water / ) for polynucleotide and polypeptide alignments. The alignments between two sequences may be carried out using default parameters settings, e.g., for MAFFT preferably: Matrix: Blosum62, Gap Open 1.53, Gap Extend 0.123, for WATER polynucleotides preferably: MATRIX: DNAFULL, Gap Open: 10.0, Gap Extend 0.5 and for WATER polypeptides preferably MATRIX: BLOSUM62, Gap Open: 10.0, Gap Extend: 0.5. Those skilled in the art understand that it may be necessary to introduce gaps in either sequence to produce a satisfactory alignment. The "best sequence alignment" is defined as the alignment that produces the largest number of aligned identical residues while having a minimal number of gaps. Preferably, it is a global alignment, which includes every residue in every sequence in the alignment.

[0336] The term "variant" refers, with respect to a polypeptide, generally to a modified version of the polypeptide, e.g., a mutation, so one or more amino acids of the polypeptide may be deleted, inserted, modified and / or substituted. Generally, the variant is functional, meaning that it preserves the function of the parent polypeptide. More specific functions are defined herein and have precedence over the general definition. A "mutation" or "amino acid mutation" can be an amino acid substitution, deletion and / or insertion ("and" may apply if there is more than one mutation). Preferably, it is a substitution (i.e., a conservative or non-conservative amino acid substitution), more preferably a conservative amino acid substitution. In some embodiments, a substitution also includes the exchange of a naturally occurring amino acid with a not naturally occurring amino acid. A conservative substitution comprises the substitution of an amino acid with another amino acid having a chemical property similar to the amino acid that is substituted. Preferably, the conservative substitution is a substitution selected from the group consisting of:

[0337] (i) a substitution of a basic amino acid with another, different basic amino acid;

[0338] (ii) a substitution of an acidic amino acid with another, different acidic amino acid;

[0339] (iii) a substitution of an aromatic amino acid with another, different aromatic amino acid;

[0340] (iv) a substitution of a non-polar, aliphatic amino acid with another, different non-polar, aliphatic amino acid; and

[0341] (v) a substitution of a polar, uncharged amino acid with another, different polar, uncharged amino acid.

[0342] A basic amino acid is preferably selected from the group consisting of arginine, histidine, and lysine. An acidic amino acid is preferably aspartate or glutamate. An aromatic amino acid is preferably selected from the group consisting of phenylalanine, tyrosine and tryptophane. A non-polar, aliphatic amino acid is preferably selected from the group consisting of glycine, alanine, valine, leucine, methionine, and isoleucine. A polar, uncharged amino acid is preferably selected from the group consisting of serine, threonine, cysteine, proline, asparagine, and glutamine. In contrast to a conservative amino acid substitution, a nonconservative amino acid substitution is the exchange of one amino acid with any amino acid that does not fall under the above-outlined conservative substitutions (i) through (v). Amino acids of a protein may also be modified, e.g., chemically modified. For example, the side chain or a free amino or carboxy-terminus of an amino acid of the protein or polypeptide may be modified by e.g., glycosylation, amidation, phosphorylation, ubiquitination, etc. The chemical modification can also take place in vivo, e.g., in a host-cell, as is well known in the art. For example, a suitable chemical modification motif, e.g., glycosylation sequence motif present in the amino acid sequence of the protein will cause the protein to be glycosylated. Unless a modification leads to a change in identity of a modified amino acid (e.g., a substitution or deletion), a modified polypeptide is within the scope of polypeptide as mentioned with respect to a certain SEQ ID NO, i.e., it is not a variant as defined herein.

[0343] Preferably the degree of identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence.

[0344] The term “parent” refers, with respect to a polypeptide, to the starting amino acid sequence for introduction of mutations to the sequence as described above, resulting in variants of the parent polypeptide amino acid sequence. A parent polypeptide may include: A wild-type polypeptide amino acid sequence or a synthetically generated polypeptide amino acid sequence that is used as starting sequence for introduction of further changes.

[0345] The term "variant" refers, with respect to a polynucleotide, generally to a modified version of the polynucleotide, e.g., a mutation, so one or more nucleotides of the polynucleotide may be deleted, inserted, modified and / or substituted. Generally, the variant is functional, meaning that it preserves the function of the parent polynucleotide. More specific functions are defined herein and have precedence over the general definition. A "mutation" can be a nucleotide substitution, deletion and / or insertion ("and" may apply if there is more than one mutation). Preferably, it is a substitution, more preferably it causes an amino acid substitution, most preferably a conservative amino acid substitution.

[0346] Preferably the degree of identity between a given nucleic acid sequence and a nucleic acid sequence which is a variant of said given nucleic acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is given preferably for a nucleic acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference nucleic acid sequence. For example, if the reference nucleic acid sequence consists of 200 nucleic acids, the degree of identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleic acids, in some embodiments, continuous nucleic acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference a nucleic acid sequence.

[0347] The term “parent” refers, with respect to a polynucleotide, to the starting nucleic acid sequence for introduction of mutations to the sequence as described above, resulting in variants of the parent polynucleotide sequence. A parent polynucleotide may include: A wild-type nucleic acid sequence or a synthetically generated nucleic acid sequence that is used as starting sequence for introduction of further changes. The term "recoded nucleic acid sequence" refers herein to a nucleic acid sequence that contains two or more silent mutations compared to a reference nucleic acid sequence, and thus encodes for the same amino acid sequence that is encoded by the reference nucleic acid sequence.

[0348] The term "AAV vector" or "AAV particle" as used herein refers to a complete virus particle, such as a wild-type ("wt") AAV virus particle, i.e., including a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat. In this regard, singlestranded AAV nucleic acid molecules of either complementary sense (i.e., "sense" or "antisense" strands) can be packaged into any one AAV virion; both strands are equally infectious. The AAV vector of the present invention may also be infectious and replicationdefective virus composed of an AAV protein shell, encapsidating a heterologous DNA molecule of interest that is flanked on both sides by an AAV ITR. The AAV 5' ITR has the nucleic acid sequence according to nucleotides 1-145 of SEQ ID NO: 29 and the AAV 3' ITR has the nucleic acid sequence of the complement of nucleotides 4535-4679 of SEQ ID NO: 29. The term "AAV genome" as used herein refers to any nucleic acid sequence derived from an adeno-associated virus serotype, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV9, AAV7, etc. AAV genome can have one or more of the AAV wild-type genes deleted in whole or in part, preferably the rep and / or cap genes, but retain functional flanking inverted terminal repeat ("ITR") sequences. Functional ITR sequences are generally necessary for the rescue, replication, and packaging of the AAV genome. Thus, an AAV genome is defined herein to include at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus. The ITRs need not be the wild-type nucleic acid sequences, and may be altered (e.g., by the insertion, deletion, or substitution of nucleotides) so long as the sequences provide for functional rescue, replication, and packaging.

[0349] The term "Ad genome" as used herein refers to any nucleic acid sequence derived from an adenovirus, including, without limitation, Ad2, Ad5, Ad6, Adi l, Ad26, Ad35, Ad49, GRAd32, ChAd3, ChAd4, ChAd5, ChAd7, ChAd8, ChAd9, ChAdlO, ChAdl l, ChAdl6, ChAdl7, ChAdl9, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 and ChAd82, etc. Adenovirus genomes are linear double-stranded DNA molecules of about 26-45 kb in length. An Adenovirus genome usually comprises genes encoding the major proteins required for viral DNA replication and major structural components. The genome has inverted repeat sequences of up to 150 bp in length located at the ends of the viral genome that function as DNA replication origins. The term "genomic sequence" as used herein refers to a chromosomal DNA sequence and is used to distinguish from coding DNA. As such, it includes exons, introns as well as regulatory sequences, in particular promoters, belonging to a gene.

[0350] The term "recombinant virus" as used herein refers to a virus particle whose genome has been modified by artificial means, e.g. by recombinant DNA technology via genetic engineering to obtain a virus particle that comprises a nucleic acid of interest, i.e., the nucleic acid construct of the first aspect, and / or a transgene, and / or a nucleic acid recAB, and / or cap ORF, and can be used to produce rAAV particles.

[0351] The term "recombinant adenovirus" (rAd) refers herein to an adenovirus that is modified to comprise a heterologous polynucleotide and / or polypeptide sequence. "Heterologous" can mean from another adenovirus strain, in particular a strain from a different host (e.g., a human host, so from a human adenovirus such as Ad3 or Ad5), or from a non-adenoviral organism such an antigen derived from a pathogen as described herein, or from human such as a human tumor antigen. The term "replication-competent" rAd refers to an adenovirus that can replicate in a host cell in the absence of any recombinant helper proteins comprised in the cell. Preferably, a "replication-competent" adenovirus comprises the following intact or functional essential early genes: E1A, E1B, E2A, E2B, E3 and E4. Wild type adenoviruses isolated from a particular animal will be replication competent in that animal.

[0352] The term "replication-defective" or "replication-incompetent" rAd refers to an adenovirus that has been rendered to be incapable of replication because it has been engineered to comprise at least a functional deletion, i.e., a deletion which impairs the function of a gene without removing it entirely, e.g. introduction of artificial stop codons, deletion or mutation of active sites or interaction domains, mutation or deletion of a regulatory sequence of a gene etc, or a complete removal of a gene encoding for a gene product that is essential for viral replication, such as one or more of the adenoviral genes selected from El, E2, E3 and E4. The recombinant adenoviral viruses used herein are preferably replication-defective.

[0353] The term "recombinase" refers to a viral or bacterial site-specific recombinase and has both endonuclease and ligase properties. Site-specific recombinases recognize specific sequences of bases in DNA and exchange the DNA segments flanking those segments. Sitespecific recombinases catalyze for example the following events: (1) deletion of a DNA fragment flanked by specific recombinase sites in the same direction (e.g., head-to-tail or tail- to-head); (b) inversion of a DNA fragment flanked by compatible recombinase sites in opposite direction (e.g. head-to-head or tail-to-tail). To perform those reactions, a site-specific recombinase typically performs the following activities: recognition of one or two specific DNA sequences; cleavage of said DNA sequence or sequences; DNA topoisomerase activity involved in strand exchange; and DNA ligase activity to reseal the cleaved strands of DNA. Numerous recombination systems from various organisms have been described.

[0354] ***

[0355] The invention is described by way of the following examples that are to be construed as merely illustrative and not limitative of the scope of the invention.

[0356] EXAMPLES

[0357] Example 1: General methods using repFLEX and repFLEXcap constructs

[0358] One embodiment of the nucleic acid construct of the present invention comprises a rep open reading frame (ORF) split into two parts, rep5 / fw and rep3 / rc. Rep3 is contained as inverse sequence, i.e., rep3 / rc and flanked by two fragments of an intron (int5 / fw and int3 / rc) and pairs A and B of recombination sites (recA / fw, recA / rc, recB / fw and recB / rc) (Figure 1A). The necessary AAV Cap protein can be either provided immediately downstream the end of the rep ORF in the repFLEXcap construct (Figure 1A) or can be provided in trans when using the repFLEX construct (Figure IB). Since the rep ORF in the nucleic acid constructs repFLEXcap or repFLEX of Figure 1 is splitted, a cell comprising such constructs chromosomally or extrachromosomally does not express rep during the initial rAd production step, so that rep cannot exert its cytotoxic effect. Therefore, these nucleic acid constructs allow for an efficient production of rAd particles containing the rep ORF in inactive splitted form. Upon exposure to a recombinase specific for the pairs A and / or B, the portion containing rep3 / rc-cap / rc or only rep3 / rc is inverted thus generating a complete intron which can then be processed by the cellular splicing machinery to restore a rep ORF and cap ORF, or only rep ORF for the repFLEX construct. Consequently, a recombinase is present only in the rAAV producing cell line (either stably integrated in the cellular genome or provided in trans) for the final rAAV production step, to allow production of rAAV particles.

[0359] Moreover, since replication of rAd may be inhibited in cis by a sequence near the 3' end of AAV rep, termed Rep inhibition sequence for adenoviral replication (RIS-Ad), the repFLEXcap constructs to be used for rAd transfection preferably comprise a recoded RIS-Ad (Figure ID), which allows production of a rAd that contains an AAV rep.

[0360] The nucleic acid construct according to the first aspect may be produced using standard molecular biology techniques that are known to a person skilled in the art.

[0361] Example 2: Method employing the repFLEXcap or repFLEX contructs

[0362] The production process of rAAV particles containing a transgene / gene of interest (GOI) (Figure 2A), using repFLEX or repFLEXcap according to the present invention can be implemented by a number of methods, wherein the repFLEX or repFLEXcap are either integrated in the genome of a producing and packaging cell line (Methods 2 A and 2B, see Table 2) or provided in trans, for example by a vector, preferably a rAd (Methods 1 A and IB, Figure 6A and B). These methods may be implemented according to various layouts (Table 2). Moreover, it is possible to include more than one transgene in one rAd vector.

[0363] Example 3: Control of rep expression by repFLEXcap and repLSLcap construct

[0364] A lox-Stop-lox (LSL) construct is a known construct (WO 2003 / 084977 Al; Qiao C. et al., 2002, J Virology, 76(4): 13015-13027) aiming at controlling rep expression. In the LSL construct, the rep ORF is split into two fragments by an intron, which is itself split by a transcriptional stop signal (TS) insert (multiple polyA signals) flanked by two LoxP recombination sites (Figure 3). In the LSL construct, the rep and intron fragments are all present in sense 5' to 3' direction.

[0365] In order to compare the repLSLcap construct head-to-head with the repFLEXcap (both with AAV2 cap) construct a quadruple transfection experiment of 293T cells was performed using the plasmids:

[0366] - pAAV-GFP (Cell Biolabs, VPK-400-DJ; SEQ ID NO: 166): encoding for GFP of SEQ ID NO: 27,

[0367] - pHelper (Cell Biolabs, VPK-400-DJ; SEQ ID NO: 167): providing adenoviral E4 and E2A proteins and the adenoviral VA (viral associated) RNA, which are necessary for rAAV production,

[0368] - pRepLSLcap (SEQ ID NO: 24 in a pBluescript plasmid; whole sequence: SEQ ID NO: 168) (panels 1 and 3 in Figure 4) or pRepFLEXcap (SEQ ID NO: 16 in a pBluescript plasmid; whole sequence: SEQ ID NO: 169) (panels 2 and 4 in Figure 4)

[0369] - + / - pSV40::Cre (SEQ ID NO: 170; Cre = SEQ ID NO: 131, encoding for SEQ ID NO: 132) (panels 1-2 / 3-4 in Figure 4, respectively)]. rAAV production was assessed by harvesting the transfected cells 3 days post transfection and the clarified cell lysate comprising rAAV was then added to fresh 293T cells (Figure 4). After 2-3 days, the transfected cells were analysed by fluorescence microscopy.

[0370] As shown in Figure 4 (3) and (4), infected cells showed a GFP signal when transfection had been performed including a pCre plasmid (i.e., Rep expression was unlocked, and therefore rAAV production was possible). However, the GFP signal was also visible for pRepLSLcap when pCre was omitted (Figure 4(1)), indicating Rep activity even without Cre expression. Instead, cells infected with the lysate obtained from cells transfected with the pRepFLEXcap construct only showed GFP expression when transfection included the pCre plasmid (Figure 4(4)), i.e., absence of leakiness when Cre is absent (Figure 4(2)). This finding suggests that RepLSLcap construct can be spliced also before the addition of the recombinase resulting in a functional rep coding sequence, i.e., is intrinsically leaky for rep expression by design.

[0371] Example 4: Generation of rAAV particles using repFLEXcap with cap being either AAV2 cap or AAV8 cap. rAd comprising the repFLEXcap construct SEQ ID NO: 16 (Ad-repFLEXcap) was prepared by cloning a rHAd5 genome including the repFLEXcap construct (SEQ ID NO: 172, 173) comprising a recoded rep ORF ( SEQ ID NO: 139) into a Bacterial Artificial Chromosome (BAC) as described in Example 4 of WO 2022 / 003083 Al. The BAC was then linearized by means of digestion with appropriate Pmel restriction enzyme, and the linearized DNA was transfected in wild-type (wt) HEK293 cells (i.e. without Cre expression) for the first viral growth. Approximatively 14 days post transfection, the cells were harvested and lysed, and the clarified lysate used to infect subsequent passages of HEK cells (‘propagation’). The productivity of this rAd was very high and comparable to that of a rAd not comprising an AAV rep, so that the amount of clarified lysate produced in a 500mL shake flask was for example sufficient to infect a 100L bioreactor of mammalian cells.

[0372] HEK293 cells with a cre integrated in the genome (i.e. chromosomally) and stably expressing the Cre recombinase under the control of a PGK-1 promoter, were obtained by infection with a lentivirus expressing the Cre following standard protocols and are referred to “HEK-iCre”. These HEK-iCre cells (2ml adherent culture) were transfected either with the 3 plasmids (pAAV-GFP of SEQ ID NO: 166, GFP of SEQ ID NO: 27, prepFLEXcap of SEQ ID NO: 169 with AAV2 cap or AAV8 cap, pHelper of SEQ ID NO: 167) as in Example 3 or infected with two El -deleted rAds, one containing a GFP transgene flanked by the AAV ITRs (GrAd23-GFP of SEQ ID NO: 171, comprising rAAV-GFP of SEQ ID NO: 28), the other containing the repFLEXcap construct of SEQ ID NO: 177, 178, 16 or 17 (Ad-repFLEXcap, Method IBb, Table 2). In both cases, pRepFLEXcap or Ad-repFLEXcap (SEQ ID NOs: 172 and 173), either AAV2 cap (SEQ ID NO: 177; SEQ ID NO: 16) or AAV8 cap (SEQ ID NO: 178; SEQ ID NO: 17) was present in the constructs. pRepFLEXcap with AAV8 cap was obtained by replacing in pRepFLEXcap of SEQ ID NO: 169 AAV2 cap with AAV8 cap (SEQ ID NO: 21).

[0373] After 5 days, the rAd infected cells were lysed, clarified by centrifugation at 2000rpm for 10 minutes and filtered through a 70um mesh.

[0374] The level of rAAV particles produced by the plasmid-transfected or the rAd-infected cells was determined by measuring the fluorescence from the GFP expressing rAAV particles by fluorescence microscopy and by digital droplet PCR (ddPCR) using primers / probes targeting the AAV ITRs.

[0375] For the assessment by fluorescence microscopy (Figure 5B-5E), the lysate of the rAd- infected cells was incubated at 56°C for Ih to inactivate the adenovirus and 15% of this clarified Ad-inactivated lysate was added to fresh standard HEK293T cells for 2-3 days.

[0376] For the assessment by digital droplet PCR (Figure 5A), the lysates of the rAd-infected cells were incubated at 56°C for Ih, the now non-incapsidated adenoviral genomes were removed by incubation with 50U / uL of SAN (Salt Active Nuclease) endonuclease for Ih at 37°C. Subsequently, the SAN was inactivated by 1 : 100 dilution, the rAAV viral capsids were opened by incubation with 0.1% SDS at 56°C for 10 minutes and the AAV DNA was subjected to droplet digital PCR using primers / probe targeting the AAV ITR (SEQ ID NOs: 45-47).

[0377] Level of rAAV particles was high (> 10E+04 viral particles per cell) for both methods (plasmid or adenovirus) and for both Cap proteins (AAV2 or AAV8) (Figure 5A). These results also indicate that the repFLEXcap based method, with rep from AAV2, does provide an efficient method to produce rAAV particles with a capsid composition (cap) different from AAV2.

[0378] Example 5: Generation of rAAV particles using repFLEXcap (e.g., method IBb) - large scale

[0379] IL of HEK-iCre cells (2e6 cells / mL) with ere integrated stably in the genome (see Example 4) were infected with rAd-GOI (SEQ ID NO: 28, transgene / GOI=GFP) at multiplicity of infection (MOI) 50 (2.5mL of an adenoviral stock concentrated at 4el0 vg / mL) and with rAd-repFLEXcap (SEQ ID NO: 16, AAV2 cap) at MOI 50 (3.85 mL of an adenoviral stock concentrated at 2.6el0 vg / ml (vg = viral genomes)) (Figure 6). 2.5 days post infection cells were lysed by three consecutive rounds of freeze / thawing at -80°C / 37°C of at least 30 minutes, followed by clarification through centrifugation at 2000rpm for 10 minutes and filtering through a 70um mesh. The non-encapsidated rAd and rAAV genomes were removed by incubation with 50U / uL of SAN for Ih at 37°C. In order to detect the ITR in the rAAV genome, previously protected from the SAN activity by the rAAV capsid, the SAN was inactivated by 1 : 100 dilution and the viral capsids were opened by incubation with 0.1% SDS at 56°C for 10 minutes. DNA was then subjected to droplet digital PCR using primers / probe targeting either the AAV ITR (SEQ ID NOs: 45-47) or the rAd hexon gene (SEQ ID NOs: 42-44). The lysate contained 4.4E+10 AAV vg / mL but only 1.7E+08 rAd vg / mL arising from a small fraction, about 0.5% of total, of SAN-resistant still encapsi dated residual adenoviral genomes (Table 3). The experiment shows that the repFLEXcap rAAV production method allows not only for a high level rAAV production in large volumes but provides also high purity rAAV preparations (low rAd particle content). Table 2: rAAV production methods utilizing the repFLEXcap or repFLEX construct can be implemented in various layouts, where the nucleic acid recAB is provided either integrated in the cellular genome or in trans and repFLEXcap or repFLEX is provided either integrated in the cellular genome or in trans. The transgene (GOI), the recombinase and the cap elements can be provided by individual vectors or by two or three vectors comprising different combinations thereof.

[0380] Table 3: Results of Example 5 showing generation of rAAV particles utilizing two rAds (one containing the transgene (GOI) GFP, the other repFLEXcap) and a packaging cell line with ere integrated in its genome (Method IBb). vg = viral genomes

[0381] Example 6: Generation of rAAV particles using repFLEXcap (e.g., method IBb)

[0382] - large scale

[0383] The experiments described in Example 5 to generate rAAV particles using repFLEXcap have been repeated using a different procedure to collect the packaging cells and purify the rAAV particles.

[0384] 500mL of HEK-iCre cells (2e6 cells / mL), with ere integrated stably in the genome and thus stably expressing Cre recombinase (see Example 4), were co-infected with rAd-GOI (SEQ ID NO: 28, transgene / GOI=GFP) at MOI 50 and with rAd-repFLExCap (SEQ ID NO: 16, AAV2 cap) at MOI 200. 2.5 days after incubation, cells were lysed with IX AAV-MAX Lysis Buffer (Thermofisher), followed by clarification through centrifugation at 2000rpm for 20 minutes and filtration of the supernatant through a 0.45 pm filter. The clarified lysate was concentrated via Tangential Flow Filtration (TFF) with no buffer exchange, and the retentate applied to a POROS GoPure AAVX affinity column (0.5 x 5 cm, 1 mL).

[0385] In-process samples were analyzed to assess rAAV production and impurity clearance across purification steps. Specifically, residual adenovirus particles were quantified by droplet digital PCR (ddPCR) targeting the rAd hexon gene (SEQ ID NOs: 42-44), after SAN digestion to remove non-encapsidated genomes and SDS treatment to release encapsidated DNA, while rAAV vector genomes (vg) were quantified using primers and probes targeting the rAAV ITR region (SEQ ID NOs: 45-47).

[0386] The results of ddPCR in Table 4 show successful rAAV production by using repFLEXcap. Moreover, they show that additional purification steps can be performed to further increase the rAAV preparation purity. In this example the adenoviral contamination in the bulk lysate was very low, i.e. 0.05% (9.55E+07 / 2.11E+11 vg / mL), and could be decreased to 0.03% (7.10E+08 / 2.66E+12) by purification with POROS AAVX.

[0387] Table 4: ddPCR to measure rAAV production and rAd contamination

[0388] Example 7: Generation of rAAV particles using repFLExcap (e.g. method IBB) - small scale

[0389] 30mL of HEK-iCre cells (2e6 cells / mL) stably expressing the Cre recombinase (see Example 4) were infected with rAd-GOI (SEQ ID NO: 171, SEQ ID NO: 28, transgene / GOI=GFP) at MOI 50 (l luL of a dilution 1 : 10 of an adenoviral stock concentrated at 2.7el2 vg / mL) and with GrAd23 -repFLExCap (SEQ ID NO: 174, comprising SEQ ID NO: 16 with AAV2 cap) at MOI 200 (15uL of an adenoviral stock concentrated at 8.1el lvg / ml) (method exemplified in Figure 6B). 2.5 days post infection cells were lysed with IX AAV- MAX Lysis Buffer (Thermofisher), followed by clarification through centrifugation at 2000rpm for 20 minutes and filtration of the supernatant through a 0.45 pm filter. 18mL of lysate were loaded into a Vivapure Q Maxi M spin column containing an ion exchange membrane adsorber that binds adenoviral particles, and centrifugated 5 min at 1500rpm. The flow-through containing rAAV was collected and used for rAAV purification with magnetic beads. 500uL of Vivapure Q Maxi M spin column flow-through were purified through an overnight incubation at 4°C with 40uL Dynabeads CaptureSelect AAV Magnetic Beads from Thermo Scientific.

[0390] To monitor the production of rAAV and residual recombinant Adenovirus (rAd) presence, in-process samples were analyzed by droplet digital PCR (ddPCR), and re-infection of cultured cells as described below.

[0391] For the droplet digital PCR, the non-encapsidated rAd and rAAV genomes were removed by incubation with 50U / uL of SAN for Ih at 37°C, after which the SAN was inactivated by 1 :100 dilution, and the viral capsids opened by incubation with 0.1% SDS at 56°C for 10 minutes. DNA was then subjected to droplet digital PCR using primers and probes targeting either the rAAV ITR region (SEQ ID NOs: 45-47) or the rAd hexon gene (SEQ ID NOs: 42-44). the results reported in Table 5 demonstrate the successful production of rAAV by using repFLExCap. Moreover, they show that additional purification steps can be performed to further increase the rAAV preparation purity. In this example the adenoviral contamination in the bulk lysate was very low, i.e. 0.04% (1.02E+08 / 2.78E+11 vg / mL) and could be decreased to 0.01% (2.81E+07 / 3.00E+11) by purification with Vivapure Q Maxi M spin column and magnetic beads.

[0392] Table 5: ddPCR to measure rAAV production and rAd contamination

[0393] Finally, the in-process samples were also applied to cultured cells, to monitor the infectivity of the residual adenoviral particles (Figure 13), alongside the parental rAds as control (Figure 12). HEK293T cells were infected with:

[0394] CPE positive control rAd-GOI (GOI=GFP) (Figure 12);

[0395] CPE positive control rAd-repFLEXCap (without expression of a fluorescent marker);

[0396] CPE test sample: produced rAAV (Figure 13): the volume of post-AAVX sample was considered to contain 1 MOI of contaminating rAd and approximately 14,000 MOI of rAAV based on the ddPCR results ofTable 5. Two weeks after infection, as few as 0.01 MOI of the parental rAd (rAd-GOI and rAd- RepFLExCap) induced a cytopathic effect (Figure 12), whereas the rAAV sample, which was thought to contain 1 MOI of contaminating rAd, did not (Figure 13). This finding demonstrates that the post-AAVX sample contained no contaminating adenovirus and that the rAd Hexon ddPCR signal shown in Table 5 was a false positive. This is also suggested by the fact that the ddPCR value was close to the negative control (not shown) and below the Lower Limit Of Quantification.

Claims

CLAIMS1. A nucleic acid construct comprising:(i) an adeno-associated Virus (AAV) rep open reading frame (ORF) comprising a heterologous intron (int), wherein the rep ORF and the int are split within the intron into a 5' portion (rep5 / fw int5 / fw) and an inverted 3' portion (rep3 / rc int3 / rc),(ii) optionally an inverted AAV cap partial ORF (cap / rc) located between int5 / fw and rep3 / rc, and(iii) a pair A of recombination sites (recA / fw, recA / rc), and a different pair B of recombination sites (recB / fw, recB / rc), wherein each pair is specific for a recombinase and allows for inversion of rep3 / rc int3 / rc and, if present, also cap / rc, wherein the nucleic acid construct has the 5' to 3' structure: rep5 / fw int5 / fw recA / fw recB / fw (cap / rc -) rep3 / rc int3 / rc recA / rc recB / rc, wherein — is a direct or an indirect link, and - is a direct link.

2. The nucleic acid construct of claim 1, wherein each pair A and B is specific for a recombinase independently selected from the group consisting of Cre recombinase, FLP recombinase, R recombinase, integrase X Int, recombinase of the GIN recombination system of the Mu phage, Bxbl recombinase, bacterial P recombinase, and a variant thereof.

3. The nucleic acid construct of claims 1 or 2, wherein pairs A and B are specific for the same recombinase, preferably wherein both pairs A and B are specific for Cre recombinase, or a variant thereof.

4. The nucleic acid construct of any one of claims 1 to 3, wherein recA / fw and recA / rc are recombination sites independently selected from the group consisting of LoxP, Lox2272, Lox511, Lox5171, Lox514, Lox512, Lox66, Lox71, and variants thereof, and / or wherein recB / fw and recB / rc are recombination sites independently selected from the group consisting of LoxP, Lox2272, Lox511, Lox5171, Lox514, Lox512, Lox 66, Lox71, and variants thereof.

5. The nucleic acid construct of any one of claims 1 to 4, wherein the rep ORF, preferably rep3 / rc, comprises two or more silent mutations and / or a missense mutation, preferablyin a Rep Inhibition Sequence for Adenoviral replication (RIS-Ad) comprised in the rep ORF.

6. The nucleic acid construct of any one of claims 1 to 5, wherein the nucleic acid construct comprises cap / rc, and preferably wherein rep5 / fw comprises a nucleic acid sequence of SEQ ID NO: 1, or a variant thereof; int5 / fw comprises a nucleic acid sequence of SEQ ID NO: 2, or a variant thereof; recA / fw comprises a nucleic acid sequence of SEQ ID NO: 3, or a variant thereof; recB / fw comprises a nucleic acid sequence of SEQ ID NO: 4, or a variant thereof; cap / rc comprises a nucleic acid sequence of SEQ ID NO: 6, or a variant thereof; rep3 / rc comprises a nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a variant thereof; int3 / rc comprises a nucleic acid sequence of SEQ ID NO: 9, or a variant thereof; recA / rc comprises a nucleic acid sequence of SEQ ID NO: 10, or a variant thereof; and recB / rc comprises a nucleic acid sequence of SEQ ID NO: 11, or a variant thereof.

7. The nucleic acid construct of any one of claims 1 to 5, wherein the nucleic acid construct does not comprise cap / rc, and preferably wherein rep5 / fw comprises a nucleic acid sequence of SEQ ID NO: 1, or a variant thereof; int5 / fw comprises a nucleic acid sequence of SEQ ID NO: 2, or a variant thereof; recA / fw comprises a nucleic acid sequence of SEQ ID NO: 3, or a variant thereof; recB / fw comprises a nucleic acid sequence of SEQ ID NO: 4, or a variant thereof; rep3 / rc comprises a nucleic acid sequence of SEQ ID NO: 8, or a variant thereof; int3 / rc comprises a nucleic acid sequence of SEQ ID NO: 9, or a variant thereof; recA / rc comprises a nucleic acid sequence of SEQ ID NO: 10, or a variant thereof; and recB / rc comprises a nucleic acid sequence of SEQ ID NO: 11, or a variant thereof.

8. A vector comprising the nucleic acid construct according to any one of claims 1 to 6.

9. A collection of vectors, comprising:(i) a first vector according to claim 8 comprising cap / rc and a second vector comprising a transgene flanked by AAV ITRs (inverted terminal repeats), and optionally a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct comprised in the vector according to claim 8,(ii) a first vector according to claim 8 comprising cap / rc and a transgene flanked by AAV ITRs and a second vector comprising a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct comprised in the vector according to claim 8, or(iii) a first vector according to claim 8 comprising cap / rc, a second vector comprising a transgene flanked by AAV ITRs, and a third vector comprising a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct comprised in the vector according to claim 8.

10. The vector of claim 8 or the collection of vectors of claim 9, wherein the vector(s) is / are a recombinant virus, preferably a recombinant adenovirus (rAd).

11. A cell comprising:(a) extra-chromosomally the nucleic acid construct of claim 6, or the vector of claim 8 or 10 comprising cap / rc, wherein the nucleic acid construct of claim 6 or the vector of claim 8 or 10 optionally further comprises a transgene flanked by AAV ITRs; or(b) chromosomally the nucleic acid construct of claim 6 or 7.

12. The cell of claim 11 (a), comprising(i) chromosomally a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct of claim 6 or of the nucleic acid construct comprised in the vector of claim 8, and / or(ii) the collection of vectors of claim 9 or 10.

13. The cell of claim 11 (b), further comprising extra-chromosomally(i) a transgene flanked by AAV ITRs,(ii) a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct of claim 6 or 7, and / or(iii) a nucleic acid comprising a cap ORF, if a cap ORF is not present chromosomally, wherein (i) to (iii) are optionally comprised in one or more vectors, preferably one vector, wherein the vector(s) preferably is / are a recombinant virus, more preferably a rAd.

14. A kit comprising:(A) the collection of vectors of claim 9 or 10, or the vector of claim 8 comprising cap / rc and a transgene flanked by AAV ITRs, and a cell, wherein the cell optionally chromosomally comprises a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct comprised in the vector of claim 8; or(B) the cell of claim 11 (b), and one or more vectors, preferably one vector, wherein the vector(s) preferably is / are a recombinant virus, more preferably a rAd, comprising:(i) a transgene flanked by AAV ITRs,(ii) a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct of claim 6 or 7, and / or(iii) a nucleic acid comprising a cap ORF, if a cap ORF is not present chromosomally.

15. A method for producing recombinant adeno-associated virus (rAAV) particles comprising a transgene, the method comprising the steps of:(a) - providing the collection of vectors of claim 9 or 10, or the vector of claim 8 comprising cap / rc and a transgene flanked by AAV ITRs, and a cell, wherein the cell optionally chromosomally comprises a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct comprised in the vector of claim 8,- transfecting the cell with the vector(s),- culturing the cell to produce the rAAV particles, and- optionally isolating the rAAV particles; or(b) - providing the cell of claim 11 (b), and one or more vectors, preferably one vector, wherein the vector(s) preferably is / are a recombinant virus, more preferably a rAd, comprising:(i) a transgene flanked by AAV ITRs,(ii) a nucleic acid recAB encoding for one or more recombinases specific for the pairs A and B of recombination sites of the nucleic acid construct of claim 6 or 7, and(iii) a nucleic acid comprising a cap ORF, if a cap ORF is not present chromosomally.- transfecting the cell with the one or more vectors,- culturing the cell to produce the rAAV particles, and- optionally isolating the rAAV particles.

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