DNA Amplification Methods

Abstract

The present invention relates to a method for amplifying a DNA molecule operably linked to a CARE element in a host cell. The method comprises the step of culturing a host cell comprising a CARE element operably linked to the DNA molecule, a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof, a nucleic acid molecule comprising a nucleotide sequence encoding an AAV Rep polypeptide or a variant thereof, and optionally one or more additional nucleic acid molecules. The present invention also relates to a nucleic acid molecule encoding an L4 22K polypeptide or a variant thereof operably linked to a heterologous promoter; a nucleic acid molecule encoding a CARE element operably linked to a viral gene; a method for producing adenoviral vectors and host cells; and a method for producing viral particles, more preferably AAV particles, in a host cell.

Description

Technical Field

[0001] The present invention relates to a method for amplifying a DNA molecule operably linked to a CARE element in a host cell. The method comprises the step of culturing a host cell comprising a CARE element operably linked to the DNA molecule, a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof, a nucleic acid molecule comprising a nucleotide sequence encoding an AAV Rep polypeptide or a variant thereof, and optionally one or more additional nucleic acid molecules.

[0002] The present invention also relates to a nucleic acid molecule encoding an L4 22K polypeptide or a variant thereof operably linked to a heterologous promoter; a nucleic acid molecule encoding a CARE element operably linked to a viral gene; a method for producing adenoviral vectors and host cells; and a method for producing viral particles (more preferably AAV particles) in a host cell. Background Art

[0003] Adeno-associated virus (AAV) is a single-stranded DNA virus belonging to the Parvoviridae family. This virus is able to infect a wide range of host cells, including both dividing and non-dividing cells. In addition, it is a non-pathogenic virus that produces only a limited immune response in most patients.

[0004] Over the past few years, vectors derived from AAV have emerged as a very useful and promising mode of gene delivery. This is due to the following properties of these vectors:

[0005] -AAVs are small, non-enveloped viruses, and they have only two native genes (rep and cap). Therefore, they can be easily manipulated to develop vectors for different gene therapies. This is achieved by removing the rep and cap genes in the AAV genome and replacing these sequences with foreign sequences that may provide therapeutic benefits to patients.

[0006] -AAV particles are not easily degraded by shear forces, enzymes or solvents. This facilitates the simple purification and final formulation of these viral vectors.

[0007] -AAV is non-pathogenic and has low immunogenicity. The use of these vectors further reduces the risk of adverse inflammatory responses. Unlike other viral vectors (such as lentiviruses, herpes viruses, and adenoviruses), AAV is harmless and is not believed to cause any human disease.

[0008] -Gene sequences up to about 4500 bp can be delivered to patients using AAV vectors.

[0009] -While wild-type AAV vectors have been shown to sometimes insert genetic material into human chromosome 19, most AAV gene therapy vectors generally eliminate this property by removing the rep and cap genes from the viral genome. In this case, the virus remains in an episomal form within the host cell. These episomes remain intact in non-dividing cells, whereas in dividing cells they are lost during cell division.

[0010] The native AAV genome contains two genes, each encoding multiple open reading frames (ORFs): the rep gene encodes nonstructural proteins required for the AAV life cycle and site-specific integration of the viral genome; and the cap gene encodes the structural capsid protein.

[0011] In addition, the two genes are flanked by 145 bases of inverted terminal repeat (ITR) sequences that form hairpin structures. These hairpin sequences are required for the primase-independent synthesis of the second DNA strand and for the integration of the viral DNA into the host cell genome.

[0012] To eliminate any integration ability of the virus, the recombinant AAV vector has rep and cap removed from the DNA of the viral genome. To produce such a vector, the desired transgene is inserted between the inverted terminal repeats (ITRs) along with a promoter that drives transgene transcription; and the rep and cap genes are provided in trans. Auxiliary genes, such as adenovirus E4, E2a, and VA genes, are also provided. Rep, cap, and auxiliary genes can be provided on a separate plasmid that is transfected into the cell.

[0013] Traditionally, the production of AAV vectors has been achieved through a number of different approaches. Initially, AAV was produced using wild-type (WT) adenovirus serotype 5, and cells were transfected with plasmids encoding the rep and cap genes as well as the AAV genome. This allows the WT adenovirus to provide many trans factors that promote viral replication. However, there are many limitations to this approach: for example, each batch of AAV must be separated from the Ad5 particles after manufacturing to provide a pure product, and ensuring the removal of all Ad5 is challenging. In addition, the fact that cells devote a lot of resources to producing adenoviral particles rather than AAV during the production process is also undesirable.

[0014] In other systems, stable packaging cell lines expressing rep and cap genes have been used. In such systems, rep and cap genes are integrated into the cell genome, thus eliminating the need for plasmid-based rep and cap genes. However, due to their inherent toxicity, these genes are usually only integrated at low frequencies (e.g., 1-2 copies per cell). These systems require infection with adenoviral vectors.

[0015] Recently, adenovirus-based systems have been replaced by plasmids encoding the portions of the adenoviral genome required for AAV production. While this has addressed some concerns about the presence of adenoviral particles in the final viral preparation, a number of issues remain. These include the need to pre-manufacture sufficient plasmid for transfection into production cell lines, and the inherently inefficient process of transfection itself. The yields from these systems are also lower than those obtained using Ad5-based approaches.

[0016] Recently, adenovirus-based systems have been replaced by plasmids encoding the portions of the adenoviral genome required for AAV production. While this has addressed some concerns about the presence of adenoviral particles in the final viral preparation, a number of issues remain. These include the need to pre-manufacture sufficient plasmid for transfection into production cell lines, and the inherently inefficient process of transfection itself. The yields from these systems are also lower than those obtained using Ad5-based approaches.

[0017] It has been previously reported (Tessier, J., et al. J. Virol. 2001; 375-383; Chadeuf, G., et al. J. Gene Med. 2000; 2: 260-268) that transfection of Hela cell lines in which rep and cap genes have been integrated on the chromosome using AAV vectors and adenovirus infection resulted in a 100-fold amplification of the integrated rep-cap sequences. These amplified sequences exist in an extrachromosomal form. Adenovirus DNA binding protein (DBP) is said to be essential for this amplification.

[0018] This phenomenon is further described in US2004 / 0014031, where a cis-acting replication element (CARE) was identified. The CARE element is said to be located in a 171 nucleotide region corresponding to nucleotides 190-361 of the AAV-2 genome (Example 12); this includes the AAV p5 promoter. The CARE element is said to be bound by a "CARE-dependent replication inducer" ("CARE-DRI"). Such inducers are said to include (all) adenoviruses and herpes viruses. More specifically, a DNA binding protein (DBP) encoded by the adenovirus E2a gene was identified as a specific inducer of CARE-dependent amplification of the rep and cap genes (US2004 / 0014031, Example 4).

[0019] In addition, it was reported in US2004 / 0014031 that the CARE element can induce the amplification of an adjacent heterologous gene (Example 11), ie, the CARE element can serve as a replication origin.

[0020] It has now been found that the description of "CARE-dependent replication inducer (CARE-DRI)" in US2004 / 0014031 as being necessary and sufficient for CARE-dependent replication is incorrect. In US2004 / 0014031, the inducer is described as adenovirus DNA binding protein (DBP), the gene product of the E2a expression cassette. It has been found that while DBP may be involved in CARE-dependent replication, it is not sufficient to mediate this effect. Essentially, the product of one of the adenovirus late genes, namely L4 22K, is required. This 22K protein was previously only known to be involved in viral encapsidation.

[0021] The identification of this specific inducer and its precise mechanism of action facilitates the generation of a new method for amplifying genes juxtaposed to CARE elements, namely by providing the L4 22K polypeptide. Summary of the invention

[0022] It is therefore an object of the present invention to provide a method for amplifying a gene of interest in juxtaposition with a CARE element.

[0023] In one of the applicant's earlier patent publications (WO2019 / 020992, the contents of which are specifically incorporated herein in their entirety), the applicant disclosed that transcription of late adenoviral genes can be regulated (e.g., inhibited) by inserting a repressor element into the major late promoter. By "turning off" the expression of adenoviral late genes, the protein manufacturing capacity of the cell can be diverted to the production of desired recombinant proteins or AAV particles.

[0024] The ability to "turn off" the production of adenoviral late (i.e. structural) proteins means that no or substantially no adenoviral particles are produced during the process. Thus, economic savings can be achieved due to the reduced need to remove adenoviral particles from the purified product.

[0025] In particular, the invention also has the potential to provide a simple, cost-effective method for manufacturing AAV particles, in which the Rep and Cap proteins of AAV are integrated and encoded in the cellular genome to provide the high expression levels required for manufacturing AAV particles by maintaining the replication of the adenoviral genome while preventing the production of adenoviral particles in the final AAV preparation.

[0026] However, the applicant subsequently discovered that suppression of late adenoviral genes by inhibiting the major late promoter in the manner described in WO2019 / 020992 had the undesirable effect of inhibiting DNA amplification of the rep and cap genes in host cells by inhibiting the CARE-dependent replication mechanism. This was a completely unexpected result, as there were no reports of adenoviral late gene products being involved in CARE-dependent replication.

[0027] The identification of the L4 22K polypeptide as a CARE element inducer thus enables the use of an AAV production system utilizing the invention described in WO2019 / 020992, wherein the L4 22K polypeptide is provided in cis or trans.

[0028] Therefore, another object of the present invention is to provide a method for AAV production in which high expression levels of Rep and Cap polypeptides are obtained to prepare AAV particles while also inhibiting or preventing the production of adenovirus particles.

[0029] In one embodiment, the present invention provides a method for amplifying a DNA molecule in a host cell, wherein the DNA molecule is operably linked to a CARE element, the method comprising the step of culturing a host cell under certain conditions, the host cell comprising:

[0030] (a) a first nucleic acid molecule comprising the DNA molecule operably linked to a CARE element;

[0031] (b) a second nucleic acid molecule comprising a heterologous promoter operably associated with a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof;

[0032] (c) a third nucleic acid molecule comprising a nucleotide sequence encoding an AAV Rep polypeptide or a variant thereof;

[0033] and optionally in addition,

[0034] (d) one or more additional nucleic acid molecules comprising one or more promoters operably associated with one or more adenoviral early gene products,

[0035] The conditions allow expression of the second and third and optionally one or more additional nucleic acid molecules, thereby promoting amplification of the DNA molecule.

[0036] Preferably, the one or more adenoviral early gene products are selected from the group consisting of E2A, VARNA and E4 gene products.

[0037] The first, second, third and (when present) further nucleic acid molecules are preferably present in the host cell in the following manner:

[0038] (i) in an adenoviral vector;

[0039] (ii) stably integrated into the host cell genome; or

[0040] (iii) In an episomal vector or plasmid.

[0041] In another embodiment, the present invention provides a method for producing viral particles, the method comprising the steps of:

[0042] (a) introducing an adenoviral vector into a host cell, wherein the adenoviral vector comprises:

[0043] (i) a second nucleic acid molecule of the invention comprising a heterologous promoter operably associated with a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof;

[0044] (ii) a transfer plasmid containing the 5′- and 3′-viral ITRs flanking the transgene;

[0045] (iii) sufficient accessory genes for packaging into viral transfer plasmids,

[0046] The host cell comprises:

[0047] CARE element, which is operably connected to

[0048] (i) AAV cap gene; and

[0049] (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a viral Rep polypeptide, preferably wherein said nucleotide sequence is not operably associated with a functional promoter,

[0050] (b) culturing the host cell under conditions that allow assembly of viral particles within the host cell; and

[0051] (c) Harvesting the packaged viral particles from the cells or from the culture medium.

[0052] Preferably, the host cell is a virus packaging cell. Preferably, the virus is AAV.

[0053] In another embodiment, the present invention provides a method for producing viral particles, the method comprising the steps of:

[0054] (a) introducing an adenoviral vector into a host cell, wherein the adenoviral vector comprises:

[0055] (i) a second nucleic acid molecule of the invention comprising a heterologous promoter operably associated with a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof;

[0056] (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a viral Rep polypeptide, preferably wherein said nucleotide sequence is not operably associated with a functional promoter,

[0057] (iii) sufficient accessory genes for packaging into viral transfer plasmids,

[0058] The host cell comprises stably integrated into the host cell genome:

[0059] (i) a CARE element operably linked to an AAV cap gene; and

[0060] (ii) a transfer plasmid comprising 5'- and 3'-viral ITRs flanking the transgene, wherein the transfer plasmid may or may not be operably linked to the CARE element;

[0061] (b) culturing the host cell under conditions that allow assembly of viral particles within the host cell; and

[0062] (c) harvesting the packaged viral particles from the host cells or from the culture medium.

[0063] In some preferred embodiments, the AAV cap gene is integrated into the host cell genome under the control of a promoter that is activated by a polypeptide encoded within the adenoviral vector.

[0064] The DNA molecule operably linked to the CARE element can generally be any DNA molecule that is desired to be amplified.

[0065] CARE amplification can be bidirectional. Thus, the DNA molecule can be located at the 5' or 3' of the CARE element. In some embodiments, the length of the nucleotide sequence from the 3'-end of the CARE element to the 3'-end of the DNA molecule is 1-5Kb, 5-10Kb, 10-15Kb, 15-50Kb or 50-100Kb. In other embodiments, the length of the nucleotide sequence from the 5'-end of the CARE element to the 5'-end of the DNA molecule is 1-5Kb, 5-10Kb, 10-15Kb, 15-50Kb or 50-100Kb.

[0066] The DNA molecule can be a coding or non-coding sequence. It can be genomic DNA or cDNA. Preferably, the DNA sequence encodes a polypeptide or a fragment thereof. Preferably, the DNA molecule is operably associated with one or more transcription and / or translation control elements (e.g., enhancer, promoter, terminator sequence, etc.).

[0067] In some embodiments, the DNA molecule encodes a therapeutic polypeptide or a fragment thereof. Examples of preferred therapeutic polypeptides include antibodies, CAR-T molecules, scFV, BiTE, DARPins, and T cell receptors. In some embodiments, the therapeutic polypeptide is a G-protein coupled receptor (GPCR), such as DRD1. In some embodiments, the therapeutic polypeptide is a functional copy of a gene related to human vision or retinal function, such as RPE65 or REP. In some embodiments, the therapeutic polypeptide is a functional copy of a gene related to human blood production, or a blood component, such as factor IX, or those related to β and α thalassemia or sickle cell anemia. In some embodiments, the therapeutic polypeptide is a functional copy of a gene related to immune function (such as those in severe combined immunodeficiency (SCID) or adenosine deaminase deficiency (ADA-SCID)).

[0068] In some embodiments, the therapeutic polypeptide is a protein that increases / decreases cell proliferation, such as a growth factor receptor. In some embodiments, the therapeutic polypeptide is an ion channel polypeptide. In some preferred embodiments, the therapeutic polypeptide is an immune checkpoint molecule.

[0069] Preferably, the immune checkpoint molecule is PD1, PDL1, CTLA4, Lag1 or GITR.

[0070] In some preferred embodiments, the DNA molecule encodes a CRISPR enzyme (e.g., Cas9, dCas9, Cpf1, or a variant or derivative thereof) or a CRISPR sgRNA.

[0071] In some embodiments, the DNA molecule comprises genes from viruses known to infect mammals. The genes encoded in the DNA molecule can encode polypeptides that can self-assemble into virus-like particles, which may or may not be used as vaccines. In a preferred embodiment, the DNA molecule encodes a norovirus capsid protein.

[0072] In other embodiments, the DNA molecule may encode one or more polypeptides known to induce an immune response in humans as a vaccine that can self-assemble into a multimeric complex. A preferred embodiment encodes the five genes required for the cytomegalovirus (CMV) pentamer complex; these include CMV gH / gL / UL128 / UL130 / UL131.

[0073] In other embodiments, the gene may encode a protein known to induce an immune response in humans as a vaccine that does not self-assemble into virus-like particles. A preferred embodiment encodes Ebola virus F protein, influenza virus F and H proteins, or coronavirus S, E or M proteins.

[0074] In some embodiments, the DNA molecule comprises genes from a retrovirus, more preferably a lentivirus. Such genes include, but are not limited to, Gag-Pol genes, Rev genes, and Env genes.

[0075] In some embodiments, the DNA molecule comprises a gene from a rhabdovirus, more preferably a vesicular stomatitis virus (VSV). Such genes include, but are not limited to, a VSV glycoprotein gene (ie, a VSV G gene).

[0076] In some embodiments, the DNA molecule comprises genes required for making a viral packaging cell line encoding genes required for assembling a gene therapy viral vector or encoding a gene therapy transfer vector.

[0077] In some embodiments, the DNA molecule contains the genes required to make a viral producer cell line that encodes all the genes required to produce a gene therapy vector and a transfer vector.

[0078] In other embodiments, the DNA molecule may comprise one or more genes for a lentiviral vector (e.g., Gag-pol, REV, VSV-G, RD114) or one or more genes for an adenoviral vector (e.g., Hexon, Fibre, Penton, pVII, or pVI).

[0079] In some embodiments, the DNA molecule comprises a rep gene sequence and / or a cap gene sequence and / or a transfer vector comprising flanking AAV inverted terminal repeats (ITRs) or fragments thereof. Preferably, the rep and cap genes are AAV genes.

[0080] In other embodiments, the DNA molecule does not comprise an AAV rep gene sequence or does not comprise an AAV cap gene sequence or does not comprise an AAV inverted terminal repeat (ITR) sequence. In other embodiments, the DNA molecule does not comprise an AAV sequence. In some embodiments, the CARE element is not connected (continuously or non-continuously) to an AAV rep or cap gene.

[0081] In some embodiments, a third nucleic acid comprising a nucleotide sequence encoding an AAV Rep polypeptide or a variant thereof is not required.

[0082] As used herein, the term "rep gene" refers to a gene encoding one or more open reading frames (ORFs), wherein each of the ORFs encodes an AAV Rep nonstructural protein or a variant or derivative thereof. These AAV Rep nonstructural proteins (or variants or derivatives thereof) are involved in AAV genome replication and / or AAV genome packaging.

[0083] The wild-type rep gene contains three promoters: p5, p19, and p40. p5 and p19 can produce two overlapping messenger RNAs (mRNAs) of different lengths. Each of these mRNAs contains introns, which can be spliced ​​or unspliced ​​using a single splice donor site and two different splice acceptor sites. Therefore, six different mRNAs can be formed, only four of which are functional. The two mRNAs that fail to remove the introns (one transcribed from p5 and one from p19) read through a shared polyadenylation terminator sequence and encode Rep78 and Rep52, respectively. Removing the introns and using the 5'-most splice acceptor site does not produce any functional Rep protein - it cannot produce the correct Rep68 or Rep40 protein because the frame of the rest of the sequence is frameshifted, and it cannot produce the correct C-terminus of Rep78 or Rep52 because their terminators are spliced. In contrast, removal of the intron and use of the 3' splice acceptor will include the correct C-terminus of Rep68 and Rep40, while the terminators of Rep78 and Rep52 are spliced ​​in. Thus, the only functional splices either completely avoid splicing the intron (producing Rep78 and Rep52) or use the 3' splice acceptor (producing Rep68 and Rep40). Thus, four different functional Rep proteins with overlapping sequences can be synthesized from these promoters.

[0084] In the wild-type rep gene, the p40 promoter is located at the 3' end. Transcription of the Cap proteins (VP1, VP2, and VP3) is initiated from this promoter in the wild-type AAV genome.

[0085] The four wild-type Rep proteins are Rep78, Rep68, Rep52 and Rep40. Therefore, the wild-type rep gene is a gene encoding the four Rep proteins Rep78, Rep68, Rep52 and Rep40. As used herein, the term "rep gene" includes wild-type rep genes and their derivatives; and artificial rep genes with equivalent functions.

[0086] In one embodiment, the rep gene encodes functional Rep78, Rep68, Rep52 and Rep40 polypeptides. In another embodiment, the rep gene encodes functional Rep78 and Rep68 polypeptides. In some embodiments, the rep gene p19 promoter is non-functional. In another embodiment, the rep gene encodes non-functional Rep52 and Rep40 polypeptides.

[0087] The wild-type AAV (serotype 2) rep gene nucleotide sequence is given in SEQ ID NO:1.

[0088] In one embodiment, the term "rep gene" refers to a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 1 and which encodes one or more Rep78, Rep68, Rep52 and Rep40 polypeptides.

[0089] As used herein, the term "cap gene" refers to a gene encoding one or more open reading frames (ORFs), wherein each of the ORFs encodes an AAV Cap structural protein or a variant or derivative thereof. These AAV Cap structural proteins (or variants or derivatives thereof) form the AAV capsid.

[0090] All three Cap proteins must be functional to produce infectious AAV viral particles capable of infecting appropriate cells.

[0091] The three Cap proteins are VP1, VP2 and VP3, and their sizes are generally 87 kDa, 72 kDa and 62 kDa, respectively. Therefore, the cap gene is a gene encoding the three Cap proteins VP1, VP2 and VP3.

[0092] In wild-type AAV, these three proteins are translated from the p40 promoter to form a single mRNA. After this mRNA is synthesized, long or short introns can be excised to form a 2.3 kb or 2.6 kb mRNA.

[0093] The AAV capsid is composed of 60 capsid protein subunits (VP1, VP2, and VP3) arranged in an icosahedral symmetry in a ratio of 1:1:10, with an estimated size of 3.9 MDa.

[0094] As used herein, the term "cap gene" includes wild-type cap genes and their derivatives, as well as artificial cap genes with equivalent functions. The AAV (serotype 2) cap gene nucleotide sequence and Cap polypeptide sequence are given in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0095] As used herein, the term "cap gene" preferably refers to a nucleotide sequence having the sequence given in SEQ ID NO:2 or a nucleotide sequence encoding SEQ ID 3:11; or a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:2 or at least 80%, 90%, 95% or 99% nucleotide sequence identity with a nucleotide sequence encoding SEQ ID NO:3, and which encodes VP1, VP2 and VP3 polypeptides.

[0096] The rep and cap genes are preferably viral genes or derived from viral genes. More preferably, they are AAV genes or derived from AAV genes. In some embodiments, AAV is adeno-associated parvovirus A. In other embodiments, AAV is adeno-associated parvovirus B.

[0097] There are 11 different AAV serotypes known. All known serotypes can infect cells from a variety of different tissue types. Tissue specificity is determined by the capsid serotype.

[0098] The AAV may be from serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11. Preferably, the AAV is serotype 1, 2, 5, 6, 7, 8 or 9. Most preferably, the AAV serotype is 5 (ie, AAV5).

[0099] The rep and cap genes (and each protein encoding ORF therein) can be from one or more different viruses (e.g., 2, 3, or 4 different viruses). For example, the rep gene can be from AAV2, and the cap gene can be from AAV5. Those skilled in the art recognize that the rep and cap genes of AAV vary from clade to clade and isolate to isolate. Sequences of these genes from all such clades and isolates and their derivatives are included herein.

[0100] As used herein, the term "CARE" element refers to a cis-acting replication element. A CARE element is a DNA element that is capable of promoting replication of an operably linked DNA molecule. This replication is dependent on the presence of an adenovirus L4 22K polypeptide or variant thereof and optionally an E2A polypeptide or variant thereof. Without being bound by theory, it is believed that the L4 22K polypeptide or variant thereof can bind to a CARE element at one or more tttg motifs.

[0101] The CARE element comprises a Rep binding site (RBS; gcccgagtgagcacgc SEQ ID NO: 4) and a trs-like element.

[0102] The wild-type AAV CARE element comprises the AAV p5 promoter. The TATA box in the CARE element has been shown to be essential for CARE amplification. The CARE element is preferably an AAV CARE element.

[0103] In the wild-type AAV genome, the CARE element includes the AAV p5 promoter, the Rep binding site, the trs element, and the 5' portion of the AAV rep gene. Examples of such CARE elements have been described previously: Tessier, J., et al. J. Virol. 2001; 375-383; Chadeuf, G., et al. J. Gene Med. 2000; 2: 260-268; and in US 2004 / 0014031, among others. The AAV CARE element has been reported to be located between nucleotides 190 and 540 of wild-type AAV2 (Nony, P. et al. J Virol. 2001).

[0104] In some preferred embodiments, the CARE element is a 171 nucleotide region corresponding to nucleotides 190-361 of the AAV-2 genome. Preferably, the CARE element has a nucleotide sequence as given in SEQ ID NO: 5, or a variant thereof having at least 50%, 60%, 70%, 80%, 90% or 95% sequence identity thereto, and is capable of promoting amplification of an operably linked DNA molecule in the presence of an L4 22K polypeptide and optionally an E2A polypeptide.

[0105] The binding ability of L4 22K polypeptide to CARE element (or its variant) can be determined by chromatin immunoprecipitation (ChIP) assay. The ability of L4 22K polypeptide or its variant to promote amplification of DNA molecules operably linked to CARE element can be determined by polymerase chain reaction (PCR) or quantitative PCR (as described in Examples 3, 5 and 6 herein). In any variant of SEQ ID NO: 5, the sequences of RBS, TATA box and trs element are preferably retained.

[0106] As used herein, the terms "AAV genome", "AAV transfer vector" and "transfer plasmid" are used interchangeably herein. They all refer to vectors containing 5'- and 3'-viral (preferably AAV) inverted terminal repeats (ITRs) flanking a transgene.

[0107] The CARE element and the DNA molecule are operably linked. As used herein, the term "operably linked" (in the case of a CARE element and a DNA molecule) means that the CARE element and the DNA molecule are linked in such a way that the CARE element promotes amplification of the DNA molecule in the presence of the L4 22K polypeptide and optionally an additional adenovirus E2A polypeptide. This means that the CARE element and the DNA molecule are present in the same DNA molecule, for example, they are juxtaposed, adjacent or continuously linked.

[0108] The CARE element can be located at the 5' or 3', preferably 5', of the DNA molecule to be amplified. The direction of the CARE element sequence is defined according to its natural (wild type) environment. The CARE element can be able to act in a forward or reverse direction (upstream or downstream relative to the DNA molecule of interest). The distance between the 3'-end of the CARE element and the 5'-end of the DNA molecule is preferably 1-1000 nucleotides, more preferably 1-500 nucleotides. In some embodiments, the distance is less than 1000 nucleotides, preferably or less than 50 nucleotides.

[0109] The CARE element is contacted with the L4 22K polypeptide or variant thereof, and optionally additionally with the E2A polypeptide or variant thereof, within the host cell.

[0110] Adenoviral genes are divided into early (E1-4) and late (L1-5) transcripts with multiple protein isoforms driven by a series of splicing events.

[0111] The early region is divided into E1, E2, E3 and E4. E1 is essential for converting cells to a cell cycle phase that is conducive to viral replication and inhibits apoptosis and promotes cell division. The E2 region is mainly responsible for the replication of the DNA genome. It contains the E2A region encoding DNA binding protein (DBP) and the E2B region that mainly encodes terminal protein, DNA polymerase (Pol) and IVa2 protein. E3 contains genes involved in immune regulation of host response, and E4 contains a series of genes involved in regulating cell pathways (such as non-homologous end joining (NHEJ) and complexing with E1B-55K to mediate p53 degradation).

[0112] Adenovirus late genes are all transcribed from the same promoter (the major late promoter) and all share the same 5' mRNA end, which contains three exons that together form a tripartite leader sequence. The late genes are expressed through a series of splicing events that allow the expression of approximately 13 proteins that form part of the viral particle (e.g., Hexon and Fibre) or participate in its assembly (e.g., the 100K protein).

[0113] The transcripts of the L4 series encode 100K, 33K, 22K, and pVII proteins. These proteins are involved in a range of functions. The 100K protein is involved in helping viral hexon assembly and nuclear import, but may also play a role in converting cellular mRNA translation to cap-independent translation. In one embodiment of the invention, the 100K protein can be provided in trans in the cell rather than from within the viral genome. The 22K protein is known to be involved in viral encapsidation. The L4 gene is required for successful viral assembly, but not for genomic DNA replication.

[0114] It has now been discovered that the L4 22K polypeptide is involved in promoting amplification of operably linked DNA molecules in a CARE-dependent manner.

[0115] As used herein, the term "L4 22K polypeptide" refers to the gene product of the adenovirus L4 22K gene, or a variant or derivative thereof. Most preferably, the L4 22K polypeptide is an adenovirus L4 22K polypeptide. The molecular weight of the wild-type adenovirus L4 22K polypeptide is 22 kDa.

[0116] Preferably, the adenovirus is a human adenovirus from group A, B, C, D, E, F or G. More preferably, the adenovirus is a human adenovirus from group B or C or D. Even more preferably, the adenovirus is a human adenovirus from group B or C. Group C is preferred because Ad5 and Ad2 (both group C) are commonly used as helper viruses for AAV production. Ad5 is the most preferred adenovirus.

[0117] The human adenovirus D serotype 9 (HAdV-9) L4 22K protein sequence is available from UniProtKB-Q5TJ00. It is given herein in SEQ ID NO: 6. The Ad5 DNA sequence is given herein as SEQ ID NO: 7. The Ad5 amino acid sequence is given in SEQ ID NO: 8.

[0118] The nucleotide sequence encoding the L4 22K polypeptide is preferably the nucleotide sequence given in SEQ ID NO:7 or the nucleotide sequence encoding the polypeptide of SEQ ID NO:6 or SEQ ID NO:8; or a variant thereof, the variant having a nucleotide sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO:7 or at least 80%, 90%, 95% or 99% nucleotide sequence identity with the nucleotide sequence encoding the polypeptide of SEQ ID NO:6 or 8 and encoding a DNA binding protein.

[0119] Preferably, the L4 22K polypeptide has the amino acid sequence given in SEQ ID NO:6 or SEQ ID NO:8, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% amino acid sequence identity with SEQ ID NO:6 or SEQ ID NO:8 and is capable of promoting the amplification of a DNA molecule operably linked to a CARE element.

[0120] In some embodiments, the second nucleic acid molecule is provided in the form of a vector or plasmid. A vector or plasmid can be in a host cell (free) or introduced into a host cell. In other embodiments, the second nucleic acid molecule is integrated into the host cell genome. In other embodiments, the second nucleic acid molecule is present in a viral vector, such as a herpes virus or a slow virus vector, preferably an adenovirus vector. The viral vector can be in a host cell or introduced into a host cell.

[0121] Most preferably, the second nucleic acid molecule is inserted into an adenoviral vector, wherein expression of the L4 22K polypeptide or variant thereof is substantially independent of (ie, not associated with) the adenoviral major late promoter (MLP).The adenoviral vector may be within or introduced into a host cell.

[0122] Preferably, the second nucleic acid molecule of the present invention comprising a heterologous promoter operably associated with the nucleotide sequence encoding the L4 22K polypeptide is located in the E1 or E3 region or the E1 / E3 deletion region of the adenoviral vector. It can also be inserted in the L5 region.

[0123] The host cell may additionally comprise (d) one or more additional nucleic acid molecules comprising one or more promoters operably associated with one or more adenoviral early gene products.In order to enhance CARE-dependent amplification of DNA molecules, one or more adenoviral early gene products may be required.

[0124] In embodiments of the invention involving AAV production, one or more adenoviral early gene products may be required to achieve packaging of AAV. Preferably, the adenoviral early gene products are selected from adenoviral E1A, E1B, E2A, VA RNA and E4. These gene products are preferably present in an adenoviral vector in a host cell.

[0125] The E2A polypeptide encodes a viral DNA binding protein (DBP).Most preferably, the E2A polypeptide is an adenovirus E2A polypeptide.

[0126] Preferably, the adenovirus is a human adenovirus from group A, B, C, D, E, F or G. More preferably, the adenovirus is a human adenovirus from group B or C or D. Even more preferably, the adenovirus is a human adenovirus from group B or C. Group C is preferred because Ad5 and Ad2 (both group C) are commonly used as helper viruses for AAV production. Ad5 is the most preferred adenovirus.

[0127] Preferably, the nucleotide sequence encoding the E2A polypeptide has the sequence given in SEQ ID NO: 9 (adenovirus type 5). Preferably, the E2A polypeptide has the amino acid sequence given in SEQ ID NO: 10 (adenovirus type 5).

[0128] The nucleic acid molecule encoding the E2A polypeptide is preferably a nucleic acid molecule having the nucleotide sequence given in SEQ ID NO: 9 or a nucleotide sequence encoding SEQ ID NO: 10; or a variant thereof, the variant having a nucleotide sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 9 or at least 80%, 90%, 95% or 99% nucleotide sequence identity with the nucleotide sequence encoding SEQ ID NO: 10, and which encodes a DNA binding protein.

[0129] Preferably, the E2A polypeptide has the amino acid sequence given in SEQ ID NO: 10 or a variant thereof having at least 80%, 85%, 90%, 95% or 99% amino acid sequence identity to SEQ ID NO: 10 and is a DNA binding protein.

[0130] In some embodiments, the nucleic acid molecule encoding the E2A polypeptide is provided in the form of a vector or plasmid. The vector or plasmid can be present in the host cell (free form) or introduced into the host cell. In other embodiments, the nucleic acid molecule encoding the E2A polypeptide is stably integrated into the host cell genome. In other embodiments, the nucleic acid molecule encoding the E2A polypeptide is present in a viral vector, such as a herpes virus or a lentiviral vector, preferably an adenoviral vector. The viral vector can be present in the host cell or introduced into the host cell.

[0131] Preferably, the nucleic acid molecule encoding the E2A polypeptide is provided in an adenoviral vector (more preferably in its natural location). Preferably, the nucleic acid molecule encoding the E2A polypeptide is operably associated with its natural promoter or a heterologous constitutive promoter.

[0132] In some embodiments, the second and additional nucleic acid molecules are provided on the same plasmid or vector, or are present in the same viral vector.

[0133] In some embodiments, the first nucleic acid molecule and the third nucleic acid molecule are linked such that the nucleotide sequence encoding the AAV Rep polypeptide is operably linked to the CARE element (and thereby amplifies the nucleotide sequence encoding the AAV Rep polypeptide).

[0134] The second nucleic acid molecule comprises a heterologous promoter operably associated with a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof. As used herein, the term "heterologous promoter" refers to a promoter that is not naturally associated with the L4 22K gene. In wild-type adenovirus, expression of the L4 22K gene is driven by the major late promoter. Thus, the term "heterologous promoter" refers to a promoter that is not the major late promoter of an adenovirus.

[0135] In some embodiments, the heterologous promoter is not an adenovirus promoter, is not a herpes virus promoter, or is not a viral promoter. In some embodiments, the heterologous promoter is a mammalian promoter. In some embodiments, the heterologous promoter has less than 90%, 80%, 70%, 60% or 50% sequence identity with the wild-type adenovirus major late promoter (MLP), preferably with the sequence of SEQ ID NO: 14.

[0136] The nucleotide sequence of wild-type Ad5 MLP is as follows:

[0137]

[0138] The TATA box is underlined in the above sequence, and the last base (in bold) indicates the transcription start position (ie, the +1 position).

[0139] In some embodiments, the promoter is a constitutive promoter. In other embodiments, the promoter is inducible or repressible. Examples of constitutive promoters include CMV, SV40, PGK (human or mouse), HSV TK, SFFV, ubiquitin, elongation factor α, CHEF-1, FerH, Grp78, RSV, adenovirus E1A, CAG or CMV-β-globin promoters, or promoters derived therefrom. Preferably, the promoter is a cytomegalovirus immediate early (CMV) promoter, or a promoter derived therefrom, or a promoter with equal or increased strength compared to the CMV promoter in human cells and human cell lines (e.g., HEK-293 cells).

[0140] In some embodiments, the promoter is inducible or repressible by comprising an inducible or repressible regulatory (promoter) element. For example, the promoter can be a promoter inducible by doxycycline, tetracycline, IPTG or lactose (preferably tetracycline).

[0141] In some preferred embodiments, the nucleotide sequence encoding the AAV Rep polypeptide or the rep gene is not operably associated with a functional promoter. In this way, low expression levels of the Rep polypeptide are obtained, wherein the expression level is low enough not to prevent adenovirus growth and is not toxic enough to cells to prevent AAV production.

[0142] In wild-type AAV, expression of the rep gene product is driven by the p5 and p19 promoters. As used herein, the term "rep gene is not operably associated with a functional promoter" means that the rep gene does not contain a functional p5 or functional p19 promoter, and the rep gene is not operably associated with any other functional promoter, so that only a baseline or minimal transcription of the rep gene is obtained.

[0143] In some preferred embodiments, the AAV cap gene is integrated into the host cell genome under the control of a promoter that can be activated by a polypeptide (activator) encoded within the adenoviral vector.

[0144] In another embodiment of the present invention, the adenoviral vector of the present invention comprises a nucleic acid molecule of the present invention, which encodes a polypeptide capable of transcriptionally activating a (remote) promoter (e.g., a promoter present in a host cell). Preferably, the promoter in the host cell is a promoter that is operably associated with (i.e., drives its expression) an AAV cap gene.

[0145] In some embodiments, the adenoviral vector encodes a polypeptide capable of transcriptionally activating a promoter that is not present in the adenoviral vector. Examples of such activators include the VP16 transcriptional activator from herpes simplex virus and the transactivator domain from the p53 protein. Such activators can be linked to a DNA binding domain, such as those that bind to the cumate binding site or tetracycline binding site in the cap gene promoter. This allows inducing transcription of the cap gene only when the adenoviral vector is present in the host cell, thereby reducing the burden of expressing the AAV cap gene during adenovirus.

[0146] Host cells can be isolated cells, e.g., they are not present in living animals or mammals. Preferably, host cells are mammalian cells. Examples of mammalian cells include cells from any organ or tissue of humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cattle, and apes. Preferably, cells are human cells. Cells can be primary or immortalized cells.

[0147] Preferred cells include HEK-293, HEK 293T, HEK-293E, HEK-293FT, HEK-293S, HEK-293SG, HEK-293FTM, HEK-293SGGD, HEK-293A, MDCK, C127, A549, HeLa, CHO, mouse myeloma, PerC6, 911 and Vero cell lines. HEK-293 cells have been modified to include E1A and E1B proteins, and this eliminates the need to provide these proteins on a helper plasmid or in an adenovirus vector used in the present invention. Similarly, PerC6 and 911 cells include similar modifications and can also be used. Most preferably, human cells are HEK293, HEK293T, HEK293A, PerC6 or 911. Other preferred cells include Hela, CHO and VERO cells.

[0148] The host cell is cultured under conditions such that the second, third and optionally additional nucleic acid molecules are expressed (in a suitable culture medium). Suitable culture conditions for host cells are well known in the art (e.g., "Molecular Cloning: A Laboratory Manual" (4th edition), Green, MR and Sambrook, J., (2014 update)). In some embodiments, the host cell will be cultured in a culture medium, preferably a liquid culture medium.

[0149] In some embodiments of the invention, the second nucleic acid molecule does not comprise a nucleotide sequence encoding one or more of an adenovirus L4 33K polypeptide, an adenovirus L4 100K polypeptide, or an adenovirus pVIII polypeptide. In some embodiments of the invention, the additional nucleic acid molecule does not comprise a nucleotide sequence encoding an E2B polypeptide. In some embodiments of the invention, the host cell does not comprise an adenovirus or a herpes virus.

[0150] The CARE element can promote the amplification of the DNA molecule that is operably connected. In this regard, the CARE element serves as a replication origin. As used herein, the term "amplification" refers to the generation of multiple DNA molecules. Multiple DNA molecules may include DNA molecules of different lengths. Each DNA molecule in the multiple DNA molecules will have a nucleotide sequence that includes all or part of the nucleotide sequence of the CARE element, preferably the entire nucleotide sequence of the CARE element. Each DNA molecule in the multiple DNA molecules will have a nucleotide sequence that includes all or part of the nucleotide sequence of the DNA molecule that is operably connected. In some embodiments, multiple (amplified) DNA molecules can be composed of 50-1000 or more discrete DNA molecules.

[0151] The plurality of amplified DNA molecules are double-stranded DNA molecules.The plurality of amplified DNA molecules are linear extrachromosomal molecules.

[0152] In some embodiments, the method of the present invention further comprises the steps of: separating and / or purifying the amplified DNA molecule and / or its gene product. For example, the amplified DNA product can be purified by using a silica resin for DNA purification in the presence of ethanol. The gene product (e.g., polypeptide) of the amplified DNA product can be purified by any method suitable for purifying the particular product, such as affinity chromatography.

[0153] The DNA molecules, plasmids and vectors of the present invention can be prepared by any suitable technology. The recombinant methods for producing nucleic acid molecules and packaging cells of the present invention are well known in the art (e.g., "Molecular Cloning: A Laboratory Manual" (4th edition), Green, MR and Sambrook, J., (2014 update)). The expression of rep and cap genes and L4 22K genes from the DNA molecules of the present invention can be measured in any suitable assay, such as by qPCR to measure the genome copy number per ml (as described in the Examples herein).

[0154] In a further embodiment, the present invention provides a method for amplifying a DNA molecule in a host cell, wherein the DNA molecule is operably linked to a CARE element, the method comprising the step of culturing the host cell under certain conditions, the host cell comprising:

[0155] (a) a first nucleic acid molecule comprising the DNA molecule operably linked to a CARE element, wherein the DNA molecule comprises an AAV rep gene and an AAV cap gene;

[0156] (b) a second nucleic acid molecule comprising a heterologous promoter operably associated with a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof;

[0157] and optionally in addition,

[0158] (c) one or more additional nucleic acid molecules comprising one or more promoters operably associated with one or more adenoviral early gene products,

[0159] The conditions allow expression of the second and optionally one or more additional nucleic acid molecules, thereby promoting amplification of the DNA molecule.

[0160] In some embodiments, the second nucleic acid molecule is present in the host cell in an adenoviral vector. In some embodiments, the adenoviral vector further comprises an AAV transfer plasmid.

[0161] In a further embodiment, the present invention provides a method for amplifying a DNA molecule in a host cell, wherein the DNA molecule is operably linked to a CARE element, the method comprising the step of culturing the host cell under certain conditions, the host cell comprising:

[0162] (a) a first nucleic acid molecule comprising the DNA molecule operably linked to a CARE element, wherein the DNA molecule comprises a cap gene and optionally an additional AAV transfer plasmid;

[0163] (b) a second nucleic acid molecule comprising a heterologous promoter operably associated with a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof;

[0164] (c) a third nucleic acid molecule comprising a nucleotide sequence encoding an AAV Rep polypeptide or a variant thereof;

[0165] and optionally in addition,

[0166] (d) one or more additional nucleic acid molecules comprising one or more promoters operably associated with one or more adenoviral early gene products,

[0167] The conditions allow expression of the second and third and optionally one or more additional nucleic acid molecules, thereby promoting amplification of the DNA molecule.

[0168] In some embodiments, the second nucleic acid molecule and / or the third nucleic acid molecule is present in the host cell in an adenoviral vector.

[0169] Preferably, the rep gene is not operably associated with a functional promoter. Preferably, the rep gene is inserted into the E1 region of an E1 / E3-deleted adenoviral vector. Preferably, when located in the E1 region, the rep gene coding sequence is encoded in the same DNA strand as the E2B, E2A and E4 transcription units.

[0170] In a further embodiment, a method for producing a modified host cell is provided, the method comprising step (a) and / or step (b):

[0171] (a) introducing a first nucleic acid molecule of the present invention into a host cell, wherein the first nucleic acid molecule comprises a DNA molecule encoding an AAV cap gene operably linked to a CARE element;

[0172] (b) introducing a second nucleic acid molecule of the invention into a host cell; and optionally:

[0173] (c) introducing the third nucleic acid molecule of the present invention into a host cell;

[0174] Such that the first, second, and (when present) third nucleic acid molecules independently:

[0175] (i) stably integrated into the host cell genome, or

[0176] (ii) Existing freely in the host cell.

[0177] In some embodiments, the host cell is a cell that expresses or is capable of expressing an AAV Rep polypeptide and / or a Cap polypeptide and / or an AAV genome.

[0178] For example, the host cell can be a host cell in which one or more DNA molecules comprising nucleotide sequences encoding AAV Rep polypeptides and / or Cap polypeptides and / or AAV genomes are stably integrated. The nucleotide sequences encoding Rep polypeptides and / or Cap polypeptides and / or AAV genomes are preferably operably associated with suitable regulatory elements (e.g., inducible or constitutive promoters).

[0179] For example, the host cell can be a host cell comprising one or more DNA plasmids or vectors, the DNA plasmids or vectors comprising nucleotide sequences encoding AAV Rep polypeptides and / or Cap polypeptides and / or AAV genomes. The nucleotide sequences encoding Rep polypeptides and / or Cap polypeptides and / or AAV genomes are preferably operably associated with suitable regulatory elements (e.g., inducible or constitutive promoters). The host cell can be an AAV packaging cell or an AAV production cell.

[0180] In yet another embodiment, the present invention also provides a method for producing a modified adenoviral vector, the method comprising the following steps:

[0181] (a) introducing a nucleic acid molecule comprising a heterologous promoter operably associated with a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof into an adenoviral vector;

[0182] and optionally

[0183] (b) introducing a nucleic acid molecule comprising a nucleotide sequence encoding an AAV Rep polypeptide into an adenoviral vector, wherein the nucleic acid molecule encoding the AAV Rep polypeptide is not operably associated with a functional promoter in the adenoviral vector.

[0184] In yet another embodiment, the present invention provides a method for producing viral particles, the method comprising the steps of:

[0185] (a) introducing a transfer plasmid comprising 5'- and 3'-viral ITRs flanking a transgene into a host cell, said host cell comprising:

[0186] (i) a second nucleic acid molecule of the invention comprising a heterologous promoter operably associated with a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof;

[0187] (ii) AAV rep and cap genes, which are present in a free plasmid in the packaging cell or integrated into the packaging cell genome, and which are operably linked to the CARE element;

[0188] (iii) sufficient helper genes for packaging transfer plasmids, which are present in free helper plasmids in the cells, in adenoviral vectors or integrated in the packaging cell genome;

[0189] (b) culturing the host cell under conditions that allow the host cell to assemble viral particles; and

[0190] (c) harvesting the packaged viral particles from the host cells or from the culture medium.

[0191] The culture medium is the culture medium surrounding the host cells. Preferably, the virus is AAV. Preferably, the host cells are virus packaging cells. Preferably, the harvested viral particles are subsequently purified.

[0192] The auxiliary gene is preferably selected from one or more of (adenovirus) E1A, E1B, E2A, E4 and VA genes. In some embodiments of the present invention, the auxiliary gene also includes the E2A gene. In other embodiments, the auxiliary gene does not include the E2A gene.

[0193] As used herein, the term "introducing" one or more plasmids or vectors into a cell includes transformation, as well as any form of electroporation, conjugation, infection, transduction or transfection, etc. Methods for such introduction are well known in the art (e.g., Proc. Natl. Acad. Sci. USA. 1995 Aug 1; 92(16): 7297-301).

[0194] In some preferred embodiments, the transgene encodes a CRISPR enzyme (e.g., Cas9, Cpf1) or a CRISPR sgRNA. In other embodiments, the transgene is a gene involved in hemophilia (e.g., factor VIII or IX).

[0195] WO2019 / 020992 discloses that the transcription of late adenoviral genes can be regulated (e.g., inhibited) by inserting a repressor element into the major late promoter. By "turning off" the expression of adenoviral late genes, the protein manufacturing capacity of the cell can be diverted to the production of desired recombinant proteins or AAV particles. However, the applicant subsequently discovered that if those genes are integrated into the host cell genome, the suppression of late adenoviral genes by repressing the major late promoter in the manner described in WO2019 / 020992 has the undesirable effect of inhibiting the CARE-dependent replication of rep and cap genes. The identification of the L4 22K polypeptide as a CARE element-inducing polypeptide enables the use of an AAV production system utilizing the invention described in WO2019 / 020992 (the contents of which are specifically incorporated herein in their entirety), wherein the L4 22K polypeptide is provided in cis or trans.

[0196] In another embodiment, the present invention provides a method for producing viral particles, the method comprising the steps of:

[0197] (a) introducing an adenoviral vector into a host cell, wherein the adenoviral vector comprises:

[0198] (i) a second nucleic acid molecule of the invention comprising a heterologous promoter operably associated with a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof;

[0199] (ii) a transfer plasmid containing the 5′- and 3′-viral ITRs flanking the transgene;

[0200] (iii) sufficient accessory genes for packaging into viral transfer plasmids,

[0201] The host cell comprises:

[0202] CARE element, which is operably connected to

[0203] (i) AAV cap gene; and

[0204] (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a viral Rep polypeptide, preferably wherein said nucleotide sequence is not operably associated with a functional promoter,

[0205] (b) culturing the host cell under conditions that allow assembly of viral particles within the host cell; and

[0206] (c) harvesting the packaged viral particles from the host cells or from the culture medium.

[0207] In another embodiment, the present invention provides a method for producing viral particles, the method comprising the steps of:

[0208] (a) introducing an adenoviral vector into a host cell, wherein the adenoviral vector comprises:

[0209] (i) a second nucleic acid molecule of the invention comprising a heterologous promoter operably associated with a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof;

[0210] (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a viral Rep polypeptide, preferably wherein said nucleotide sequence is not operably associated with a functional promoter,

[0211] (iii) sufficient accessory genes for packaging into viral transfer plasmids,

[0212] The host cell comprises stably integrated into the host cell genome:

[0213] (i) a CARE element operably linked to an AAV cap gene; and

[0214] (ii) a transfer plasmid comprising 5'- and 3'-viral ITRs flanking the transgene, wherein the transfer plasmid may or may not be operably linked to the CARE element;

[0215] (b) culturing the host cell under conditions that allow assembly of viral particles within the host cell; and

[0216] (c) harvesting the packaged viral particles from the host cells or from the culture medium.

[0217] In some preferred embodiments, the AAV cap gene is integrated into the host cell genome under the control of a promoter that is activated by a polypeptide encoded within the adenoviral vector. Preferably, the virus is AAV. Preferably, the host cell is a virus packaging cell.

[0218] Preferably, the adenoviral vector comprises a repressible major late promoter (MLP), more preferably, wherein the MLP comprises one or more repressor elements capable of regulating or controlling transcription of adenoviral late genes, and wherein the one or more repressor elements are inserted downstream of the MLP TATA box.

[0219] Preferably, the CARE element, the AAV cap gene; and the transfer plasmid:

[0220] (i) stably integrated into the host cell genome; or

[0221] (ii) It exists in the host cell as an episomal plasmid or vector.

[0222] Preferred features of the method for producing viral (preferably AAV) particles include the following:

[0223] - wherein one or more repressor elements are inserted between the MLP TATA box and the +1 position of transcription.

[0224] - wherein the repressor element is an element capable of being bound by a repressor protein.

[0225] - wherein a gene encoding a repressor protein capable of binding to the repressor element is encoded within the adenoviral genome.

[0226] -wherein the repressor protein is transcribed under the control of MLP.

[0227] - wherein the repressor protein is tetracycline repressor, lactose repressor or ecdysone repressor, preferably tetracycline repressor (TetR).

[0228] - wherein the repressor element is a tetracycline repressor binding site comprising or consisting of the sequence shown in SEQ ID NO: 11.

[0229] - wherein the nucleotide sequence of MLP comprises or consists of the sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13.

[0230] - wherein the presence of the repressor element does not affect the production of adenoviral E2B protein.

[0231] - wherein the adenoviral vector encodes adenoviral L4 100K protein and wherein the L4 100K protein is not under the control of MLP.

[0232] - wherein the transgene is inserted into one of the adenoviral early regions, preferably into the adenoviral E1 region and not into a transfer plasmid.

[0233] - wherein the transgene comprises a triplet leader sequence (TPL) in its 5'-UTR.

[0234] - wherein the transgene encodes a therapeutic polypeptide.

[0235] - wherein the transgene encodes a viral protein, preferably a protein that can be assembled intracellularly or extracellularly to produce virus-like particles, preferably wherein the transgene encodes Norovirus VP1 or Hepatitis B HBsAG.

[0236] In another embodiment, the present invention provides a DNA molecule encoding L4 22K polypeptide:

[0237] (i) wherein the DNA molecule is operably associated with a promoter that is not an adenovirus major late promoter;

[0238] (ii) wherein the DNA molecule is operably associated with a mammalian promoter;

[0239] (iii) wherein the DNA molecule does not additionally encode an adenovirus L4 100K, L4 33K or pVII polypeptide;

[0240] (iv) wherein the DNA molecule is operably associated with a CMV, PGK or SV40 promoter.

[0241] In another embodiment, the present invention provides a host cell comprising:

[0242] (a) a second nucleic acid molecule of the invention comprising a heterologous promoter operably associated with a nucleotide sequence encoding an L4 22K polypeptide or a variant thereof,

[0243] The nucleic acid molecule is stably integrated into the genome of the host cell or exists in an episomal plasmid or vector.

[0244] Preferably,

[0245] (i) the heterologous promoter is not the adenovirus major late promoter;

[0246] (ii) the promoter is a mammalian promoter;

[0247] (iii) the nucleic acid molecule does not additionally encode an adenovirus L4 100K, L4 33K or pVII polypeptide; or

[0248] iv) wherein the DNA molecule is operably associated with a CMV, PGK or SV40 promoter.

[0249] Preferably, the host cell is one as defined herein.

[0250] In some embodiments, the host cell further comprises:

[0251] (b) AAV rep and cap genes, which are present in a free plasmid in the host cell or stably integrated into the cell genome, and which are operably linked to the CARE element.

[0252] In other embodiments, the host cell further comprises one or both of the following:

[0253] (c) an AAV transfer plasmid comprising a transgene flanked by ITRs; and

[0254] (d) an adenoviral helper plasmid for AAV production, which comprises one or more genes selected from E1A, E1B, E2A, E4 and VA RNA.

[0255] Such cells are often referred to as packaging cells.

[0256] In some embodiments of the invention, the helper plasmid additionally comprises an E2A gene. In other embodiments, the helper plasmid does not comprise an E2A gene. In the latter case, the deletion of the E2A gene significantly reduces the amount of DNA required in the helper plasmid.

[0257] In another embodiment, the present invention provides a host cell comprising:

[0258] (a) A first nucleic acid molecule of the present invention, comprising a DNA molecule operably linked to a CARE element, wherein the DNA molecule encodes one or more of the following:

[0259] (i) AAV Cap polypeptide,

[0260] (ii) AAV Rep polypeptide, and

[0261] (iii) AAV transfer vector,

[0262] The first nucleic acid molecule is stably integrated into the genome of the host cell or exists in an episomal plasmid.

[0263] In yet another embodiment, the present invention provides an adenoviral vector comprising a nucleic acid molecule encoding an adenoviral L4 22K polypeptide or a variant thereof, wherein the L4 22K polypeptide or variant thereof encoding sequence is not operably associated with an adenoviral MLP.

[0264] In some such embodiments, it may be preferred to insert a new L422K coding sequence into the adenoviral vector in addition to the native L422K coding sequence.

[0265] In such embodiments, the adenoviral vector comprises:

[0266] (i) a nucleic acid molecule encoding an adenovirus L4 22K polypeptide or a variant thereof, wherein the L4 22K polypeptide or variant thereof encoding sequence is not operably associated with an adenovirus MLP; and

[0267] (ii) a nucleic acid molecule encoding an adenovirus L4 22K polypeptide, wherein the L4 22K polypeptide coding sequence is operably associated with an adenovirus MLP.

[0268] Preferably, the adenoviral MLP is a repressible MLP (eg, as defined herein).

[0269] Preferably, the adenoviral vector further comprises a nucleic acid molecule encoding an AAV Rep polypeptide, more preferably, wherein the nucleic acid molecule is not operably associated with a functional promoter.

[0270] Preferably, the L4 22K polypeptide coding sequence is inserted into the adenovirus E1 or E3 region.

[0271] The present invention also provides a kit, comprising:

[0272] (A) a host cell, the host cell comprising:

[0273] (a) a first nucleic acid molecule of the present invention, comprising a DNA molecule operably linked to a CARE element, wherein the DNA molecule encodes one or more of the following:

[0274] (i) AAV Cap polypeptide, and

[0275] (ii) AAV Rep polypeptide,

[0276] wherein the first nucleic acid molecule is stably integrated into the genome of the host cell or exists in an episomal plasmid; and

[0277] (B) an adenoviral vector, the adenoviral vector comprising:

[0278] (i) a nucleic acid molecule encoding an adenovirus L4 22K polypeptide or a variant thereof, wherein the L4 22K polypeptide or variant encoding sequence is not operably associated with an adenovirus MLP, and

[0279] (ii) A nucleic acid molecule encoding an AAV transfer vector.

[0280] The present invention also provides a kit comprising:

[0281] (A) a host cell, the host cell comprising:

[0282] (a) a first nucleic acid molecule of the present invention, comprising a DNA molecule operably linked to a CARE element, wherein the DNA molecule encodes one or more of the following:

[0283] (i) AAV Cap polypeptide, and optionally

[0284] (ii) AAV transfer vector,

[0285] wherein the first nucleic acid molecule is stably integrated into the genome of the host cell or exists in an episomal plasmid; and

[0286] (B) an adenoviral vector, the adenoviral vector comprising:

[0287] (i) a nucleic acid molecule encoding an adenovirus L4 22K polypeptide or a variant thereof, wherein the L4 22K polypeptide or variant encoding sequence is not operably associated with an adenovirus MLP, and

[0288] (ii) a nucleic acid molecule encoding an AAV Rep polypeptide, preferably wherein the nucleic acid molecule is not operably associated with a functional promoter.

[0289] The kit may also include materials for purifying AAV particles, such as those involved in density banding and purification of viral particles, such as one or more of centrifuge tubes, iodixanol, dialysis buffer, and dialysis cassettes.

[0290] There are many established algorithms that can be used for comparing two amino acid or nucleotide sequences.Usually, a sequence serves as a reference sequence, and a test sequence can be compared thereto.The sequence comparison algorithm calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.The comparison for the amino acid or nucleotide sequence compared can be carried out, for example, by a computer-implemented algorithm (such as GAP, BESTFIT, FASTA or TFASTA) or BLAST and BLAST 2.0 algorithms.

[0291] Percent amino acid sequence identity and nucleotide sequence identity can be obtained using the BLAST alignment method (Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; and http: / / www.ncbi.nlm.nih.gov / BLAST). Preferably, standard or default alignment parameters are used.

[0292] Standard protein-protein BLAST (blastp) can be used to find similar sequences in protein databases. Like other BLAST programs, blastp is designed to find local regions of similarity. When sequence similarity spans the entire sequence, blastp will also report a global alignment, which is a preferred result for protein identification purposes. It is preferred to use standard or default alignment parameters. In some cases, the "low complexity filter" can be disabled.

[0293] BLAST protein searches can also be performed using the BLASTX program with score = 50 and word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389 can be used. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search to detect long-range relationships between molecules. (See Altschul et al. (1997), supra). When using BLAST, Gapped BLAST, or PSI-BLAST, the default parameters of each program can be used.

[0294] About nucleotide sequence comparison, MEGABLAST, discontinuous-megablast and blastn can be used to achieve this goal.Preferably use standard or default comparison parameters.MEGABLAST is specially designed to effectively find long comparisons between very similar sequences.Discontinuous MEGABLAST can be used to find nucleotide sequences similar but not identical to nucleic acid of the present invention.

[0295] The BLAST nucleotide algorithm finds similar sequences by breaking down the query into short subsequences called words. The program first identifies exact matches (word hits) to the query word. The BLAST program then expands these word hits in multiple steps to generate the final gap comparison. In some embodiments, the BLAST nucleotide search can be performed with the BLASTN program, score = 100, word length = 12.

[0296] One of the important parameters that controls the sensitivity of a BLAST search is the word length. The most important reason that blastn is more sensitive than MEGABLAST is that it uses a shorter default word length (11). Because of this, blastn is superior to MEGABLAST in finding alignments with related nucleotide sequences from other organisms. The word length is adjustable in blastn and can be reduced from the default to a minimum of 7 to increase search sensitivity.

[0297] By using the newly introduced discontinuous megablast page (www.ncbi.nlm.nih.gov / Web / Newsltr / FallWinter02 / blastlab.html), more sensitive searches can be achieved. The algorithm used on this page is similar to the algorithm reported by Ma et al. (Bioinformatics. 2002 March; 18(3): 440-5). Discontinuous megablast does not require exact word matches as seeds for alignment extension, but instead uses discontinuous words within a longer window of the template. In encoding mode, the third base wobble is taken into account by focusing on finding matches at the first and second codon positions and ignoring mismatches at the third position. Searching in discontinuous MEGABLAST using the same word length is more sensitive and more efficient than standard blastn using the same word length. The unique parameters of discontinuous megablast are: word length: 11 or 12; template: 16, 18 or 21; template type: encoding (0), non-coding (1) or both (2).

[0298] In some embodiments, the BLASTP 2.5.0+ algorithm (eg, available from NCBI) can be used using default parameters.

[0299] In other embodiments, the BLAST global alignment program (eg, available from NCBI) can be used using a Needleman-Wunsch alignment of two protein sequences with the following gap penalties: extension 11 and extension 1.

[0300] The disclosure of each reference identified herein is specifically incorporated by reference in its entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0301] Figure 1 : Production of AAV2 vectors from the HelaRC32 cell line requires adenoviral L4-22K expression.

[0302] Figure 2 : Superinfection with TERA-E1 cannot induce AAV2 production in the stable packaging cell line HelaRC32.

[0303] Figure 3 : DNA amplification of stably integrated AAV Rep and Cap genes requires transcription from adenovirus L4-22K.

[0304] Figure 4 : siRNA knockdown of the adenoviral late transcript L4 encoding adenovirus 22K inhibits AAV2 replication in the stable packaging cell line HelaRC32.

[0305] Figure 5Adenovirus late protein L4-22K induces CARE-dependent amplification of the AAV Cap gene in HelaRC32 cells.

[0306] Figure 6 : Adenovirus late protein L4-22K induces CARE-dependent amplification of the AAVCap gene in HelaRC32 cells in the absence of L4-100K. DETAILED DESCRIPTION

[0307] Example

[0308] The present invention is further illustrated by the following examples, wherein parts and percentages are by weight, and degrees are degrees Celsius, unless otherwise stated. It should be understood that these examples, although showing preferred embodiments of the present invention, are given only by way of illustration. Through the above discussion and these examples, those skilled in the art can determine the essential characteristics of the present invention, and without departing from its spirit and scope, the present invention can be subjected to various changes and modifications to adapt it to various uses and conditions. Therefore, according to the foregoing description, in addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art. Such modifications are also intended to fall within the scope of the appended claims.

[0309] Example 1: Production of AAV2 vectors from the HelaRC32 cell line requires adenoviral L4-22K expression.

[0310] L4 22K is expressed from the adenovirus major late promoter late in infection, and transcriptional inhibition by MLP inhibits AAV2 production by HelaRC32 cells. Control adenoviruses Ad5-E1 and TERA-E1 (a recombinant replicating adenovirus in which its modified major late promoter transcribes the repressor protein TetR, and in which transcription from the modified major late promoter is repressed by TetR) were produced by molecular cloning methods and produced from HEK293 cells. HELARC32 cells were seeded at 9e4 cells / well in 48-well tissue culture plates for 24 hours, then transfected with plasmid pSF-AAV-EGFP and infected with Ad5-E1 or TERA-E1 in the presence of doxycycline 0.5ug / mL or DMSO. AAV2 particles were harvested 96 hours after production and quantified by QPCR. The results are shown in Figure 1 shown.

[0311] Example 2: Superinfection with TERA-E1 cannot induce the stable packaging cell line HelaRC32 to produce AAV2.

[0312] Control adenoviruses Ad5-E1 and TERA-E1 (a recombinant replicating adenovirus in which its modified major late promoter transcribes the repressor protein TetR, and in which transcription from the modified major late promoter is repressed by TetR) were generated by molecular cloning methods and produced from HEK293 cells. HeLaRC32 cells were seeded at 9e4 cells / well in 48-well tissue culture plates for 24 hours, then transfected with plasmid pSF-AAV-EGFP and infected with Ad5-E1 or TERA-E1 at the indicated multiplicity of infection in the absence of doxycycline 0.5ug / mL or DMSO. AAV2 particles were harvested 96 hours after production and quantified by QPCR. The results are shown in Figure 2 shown.

[0313] Example 3: DNA amplification of stably integrated AAV Rep and Cap genes requires transcription of L4-22K from adenovirus.

[0314] TERA-E1 (a recombinant replicating adenovirus in which its modified major late promoter transcribes the repressor protein TetR, and in which transcription from the modified major late promoter is repressed by TetR) was generated by molecular cloning methods and produced from HEK293 cells. HEK293 cells were seeded in 48-well tissue culture plates at 9e4 cells / well for 24 hours and infected with TERA-E1 at MOI50 in the presence of doxycycline 0.5ug / mL or DMSO. Total DNA was extracted 96 hours after infection, and AAV Rep and Cap DNA were amplified by PCR. AAV Rep and Cap amplicon DNA were resolved by agarose gel electrophoresis. The results are shown in Figure 3 shown.

[0315] Example 4: siRNA knockdown of adenoviral late transcript L4 encoding adenovirus 22K inhibits AAV2 replication in the stable packaging cell line HelaRC32

[0316] The MLP-repressible adenovirus TERA-E1 (a recombinant replicating adenovirus in which its modified major late promoter transcribes the repressor protein TetR, and in which transcription from the modified major late promoter is repressed by TetR) was generated by standard molecular cloning methods and produced from HEK293 cells. HELARC32 cells were seeded in 48-well tissue culture plates at 1.5e4 cells / well and transfected with siRNA targeting adenoviral primary mRNA transcripts L1, L2, L3, L4 or L5 for 24 hours. HelaRC32 cells were transfected with plasmid pSF-AAV-EGFP and infected with TERA-E1 at MOI50 in the presence of doxycycline 0.5ug / mL or DMSO. AAV2 was quantified by QPCR 96 hours after infection. The results are shown in Figure 4 shown.

[0317] Example 5: Adenovirus late protein L4-22K induces CARE-dependent amplification of AAV Cap gene in HelaRC32 cells.

[0318] The MLP-repressible adenovirus TERA-E1 (a recombinant replicating adenovirus in which its modified major late promoter transcribes the repressor protein TetR, and in which transcription from the modified major late promoter is repressed by TetR) was generated by molecular cloning methods and produced from HEK293 cells. HeLaRC32 cells were seeded at 9.0e4 cells / well in 48-well tissue culture plates for 24 hours, then transfected with a plasmid that transcribes the adenoviral L4 gene under the control of the CMV promoter and infected with TERA-E1 at MOI50. Total DNA was extracted 96 hours after infection, and AAV Cap DNA was quantified by QPCR. The results are shown in Figure 5 shown.

[0319] Example 6: Adenovirus late protein L4-22K induces CARE-dependent amplification of the AAV Cap gene in HelaRC32 cells in the absence of L4-100K.

[0320] The MLP-repressible adenovirus TERA-E1 (a recombinant replicating adenovirus in which its modified major late promoter transcribes the repressor protein TetR, and in which transcription from the modified major late promoter is repressed by TetR) was generated by molecular cloning methods and produced from HEK293 cells. HeLaRC32 cells were seeded at 9.0e4 cells / well in 48-well tissue culture plates for 24 hours, then co-transfected with a CMV promoter plasmid transcribing adenovirus L4-22K and CMV-driven L4-100K or filler DNA, and infected with TERA-E1 at MOI50. Total DNA was extracted 96 hours after infection, and AAV Cap DNA was quantified by QPCR. The results are shown in Figure 6 shown.

[0321] sequence

[0322] SEQ ID NO:1

[0323] Rep nucleotide sequence (AAV serotype 2)

[0324]

[0325] SEQ ID NO:2

[0326] Cap nucleotide sequence (AAV serotype 2)

[0327]

[0328] SEQ ID NO:3

[0329] Cap amino acid sequence (AAV serotype 2)

[0330] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL*

[0331] SEQ ID NO:4

[0332] Rep binding site (RBS)

[0333] gcccgagtgagcacgc

[0334] SEQ ID NO:5

[0335] CAKE element AAV2 gtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaagcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgccatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgacagctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccgtggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccc

[0336] SEQ ID NO:6 (UniProtKB-Q5TJ00)

[0337] L4 22K

[0338] Protein name: Human adenovirus D serotype 9 (HAdV-9)

[0339] MPRKKQEPLV EEMEEEWDSQ AEEDEWEEET EEEELEEVEE EQATEQPVAAPSAPAAPAVTDTTSAAPAKP PRRWDRVKGD GKHERQGYRS WRAHKAAIIACLQDCGGNIA FARRYLLFHR GVNIPRNVLHYYRHLHS

[0340] SEQ ID NO:7

[0341] Ad 5 L4 22K

[0342] atggcacccaaaaagaagctgcagctgccgccgccacccacggacgaggaggaatactgggacagtcaggcagaggaggttttggacgaggaggaggaggacatgatggaagactgggagagcctagacgaggaagcttccgaggtcgaagaggtgtcagacgaaacaccgtcaccctcggtcgcattcccctcgccggcgccccagaaatcggcaaccggttccagcatggctacaacctccgctcctcaggcgccgccggcactgcccgttcgccgacccaaccgtagatgggacaccactggaaccagggccggtaagtccaagcagccgccgccgttagcccaagagcaacaacagcgccaaggctaccgctcatggcgcgggcacaagaacgccatagttgcttgcttgcaagactgtgggggcaacatctccttcgcccgccgctttcttctctaccatcacggcgtggccttcccccgtaacatcctgcattactaccgtcatctctacagcccatactgcaccggcggcagcggcagcaacagcagcggccacacagaagcaaaggcgaccggatag

[0343] SEQ ID NO:8

[0344] Ad 5 L422K

[0345] MAPKKKLQLPPPPTDEEEYWDSQAEEVLDEEEEDMMEDWESLDEEASEVEEVSDETPSPSVAFPSPAPQKSATGSSMATTSAPQAPPALPVRRPNRRWDTTGTRAGKSKQPPPLAQEQQQRQGYRSWRGHKNAIVACLQDCGGNISFARRFLLYHHGVAFPRNILHYYRHLYSPYCTGGSGSNSSGHTEAKATG*

[0346] SEQ ID NO:9

[0347] E2A polypeptide nucleotide sequence (Adenovirus type 5)

[0348]

[0349] SEQ ID NO:10

[0350] Amino acid sequence of E2A polypeptide (adenovirus type 5)

[0351] MASREEEQRETTPERGRGAARRPPTMEDVSSPSPSPPPPRAPPKKRMRRRIESEDEEDSSQDALVPRTPSPRPSTSAADLAIAPKKKKKRPSPKPERPPSPEVIVDSEEEREDVALQMVGFSNPPVLIKHGKGGKRTVRRLNEDDPVARGMRTQEEEEEPSEAESEITVMNPLSVPIVSAWEKGMEAARALMDKYHVDNDLKANFKLLPDQVEALAAVCKTWLNEEHRGLQLTFTSNKTFVTMMGRFLQAYLQSFAEVTYKHHEPTGCALWLHRCAEIEGELKCLHGSIMINKEHVIEMDVTSENGQRALKEQSSKAKIVKNRWGRNVVQISNTDARCCVHDAACPANQFSGKSCGMFFSEGAKAQVAFKQIKAFMQALYPNAQTGHGHLLMPLRCECNSKPGHAPFLGRQLPKLTPFALSNAEDLDADLISDKSVLASVHHPALIVFQCCNPVYRNSRAQGGGPNCDFKISAPDLLNALVMVRSLWSENFTELPRMVVPEFKWSTKHQYRNVSLPVAHSDARQNPFDF

[0352] SEQ ID NO:11

[0353] TetR binding site

[0354] tccctatcag tgatagaga

[0355] SEQ ID NO:12

[0356] Modified MLP

[0357] cgccctcttc ggcatcaagg aaggtgattg gtttgtaggt gtaggccacg tgaccgggtgttcctgaagg ggggctataa aaggtcccta tcagtgatag agactca

[0358] SEQ ID NO:13

[0359] Modified MLP

[0360] cgccctcttc ggcatcaagg aaggtgattg gtttgtaggt gtaggccacg tgactccctatcagtgatag agaactataa aaggtcccta tcagtgatag agactca

[0361] SEQ ID NO:14

[0362] Nucleotide sequence of wild-type Ad5 MLP

[0363] cgccctcttcggcatcaaggaaggtgattggtttgtaggtgtaggccacgtgaccgggtgttcctgaaggggggctataaaagggggtgggggcgcgttcgtcctca

[0364] Sequence Listing Free Text

[0365] <210 1

[0366] <213> Rep nucleotide sequence (adeno-associated virus 2)

[0367] <210 2

[0368] <213> Cap nucleotide sequence (adeno-associated virus 2)

[0369] <210 3

[0370] <213> Cap amino acid sequence (adeno-associated virus 2)

[0371] <210 4

[0372] <223> Rep binding site (RBS)

[0373] <210 5

[0374] <213> CARE element (adeno-associated virus 2)

[0375] <210 6

[0376] <223> L4 22K (Human adenovirus D serotype 9 (HAdV-9))

[0377] <210 7

[0378] <223> Ad 5L4 22K

[0379] <210 8

[0380] <223> Ad 5L4 22K

[0381] <210 9

[0382] <223> E2A polypeptide nucleotide sequence (adenovirus type 5)

[0383] <210 10

[0384] <223> E2A polypeptide amino acid sequence (adenovirus type 5)

[0385] <210> 11

[0386] <223> TetR binding site

[0387] <210> 12

[0388] <223> Modified MLP

[0389] <210> 13

[0390] <223> Modified MLP

[0391] <210> 14

[0392] <223> Nucleotide sequence of wild-type Ad5 MLP

Claims

1. A method for amplifying a DNA molecule in a host cell, wherein the DNA molecule is operably linked to a CARE element, the method comprising the step of culturing the host cell under certain conditions, the host cell comprising: (a) a first nucleic acid molecule comprising a CARE element and the DNA molecule operably linked thereto, wherein the nucleotide sequence of the CARE element is the nucleotide sequence given in SEQ ID NO:5 and is capable of promoting the amplification of the operably linked DNA molecule in the presence of the L4 22K polypeptide shown in SEQ ID NO:6 or SEQ ID NO:8; (b) a second nucleic acid molecule comprising a nucleotide sequence encoding an L4 22K polypeptide and a heterologous promoter operably associated therewith, wherein the nucleotide sequence encoding the L4 22K polypeptide is: (i) the nucleotide sequence given in SEQ ID NO:7 and encoding a DNA binding protein capable of promoting the amplification of a DNA molecule operably linked to the CARE element of SEQ ID NO:5; or (ii) a nucleotide sequence encoding the polypeptide of SEQ ID NO:6 or SEQ ID NO:8 and encoding a DNA binding protein capable of promoting the amplification of a DNA molecule operably linked to the CARE element of SEQ ID NO:5; and (c) a third nucleic acid molecule comprising a nucleotide sequence encoding an AAV Rep polypeptide; The conditions allow expression of the second and third nucleic acid molecules, thereby promoting amplification of the DNA molecule.

2. The method of claim 1, wherein the host cell further comprises: (d) one or more additional nucleic acid molecules comprising one or more adenoviral early gene products and one or more promoters operably associated therewith, The conditions allow expression of the second and third nucleic acid molecules and the additional nucleic acid molecules, thereby promoting amplification of the DNA molecule.

3. The method of claim 1, wherein the first, second and third nucleic acid molecules are independently present in the host cell in the following manner: (i) in an adenoviral vector; (ii) stably integrated into the genome of the host cell; or (iii) In an episomal vector or plasmid.

4. The method of claim 2, wherein the first, second and third nucleic acid molecules and the additional nucleic acid molecules are independently present in the host cell in the following manner: (i) in an adenoviral vector; (ii) stably integrated into the genome of the host cell; or (iii) In an episomal vector or plasmid.

5. The method of claim 1 or 2, wherein the DNA molecule encodes a therapeutic polypeptide or a viral polypeptide.

6. The method of claim 5, wherein the DNA molecule encodes a rep gene sequence and / or a cap gene sequence and / or a viral transfer vector comprising flanking AAV inverted terminal repeats (ITRs).

7. A method for producing viral particles, the method comprising the following steps: (a) introducing an adenoviral vector into a host cell, The adenoviral vector comprises: (i) a second nucleic acid molecule as defined in claim 1; (ii) a transfer plasmid containing the 5′- and 3′-viral ITRs flanking the transgene; (iii) sufficient accessory genes for packaging into viral transfer plasmids, The host cell comprises: A CARE element as defined in claim 1 and an apparatus operatively connected thereto (i) AAV cap gene; and (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a viral Rep polypeptide, (b) culturing the host cell under conditions that allow assembly of viral particles within the host cell; and (c) harvesting the packaged viral particles from the host cells or from the culture medium.

8. A method for producing viral particles, the method comprising the following steps: (a) introducing an adenoviral vector into a host cell, The adenoviral vector comprises: (i) a second nucleic acid molecule as defined in claim 1; (ii) a nucleic acid molecule comprising a nucleotide sequence encoding a viral Rep polypeptide; and (iii) sufficient accessory genes for packaging into viral transfer plasmids, The host cell comprises stably integrated into the genome of the host cell: (i) a CARE element as defined in claim 1 and an AAV cap gene operably linked thereto; and (ii) a transfer plasmid containing the 5′- and 3′-viral ITRs flanking the transgene; (b) culturing the host cell under conditions that allow assembly of viral particles within the host cell; as well as (c) harvesting the packaged viral particles from the host cells or from the culture medium.

9. The method of claim 7, wherein in step (a), the nucleotide sequence encoding the viral Rep polypeptide is not operably associated with a functional promoter.

10. The method of claim 7, wherein the AAV cap gene is integrated into the genome of the host cell under the control of a promoter that is activated by a polypeptide encoded within the adenoviral vector.

11. The method of claim 7, wherein the adenoviral vector comprises a repressible major late promoter (MLP).

12. The method of claim 11, wherein the MLP comprises one or more repressor elements capable of regulating or controlling transcription of adenoviral late genes, and wherein one or more of the repressor elements are inserted downstream of the MLP TATA box.

13. The method of claim 7, wherein the nucleotide sequence encoding the viral Rep polypeptide is inserted into the E1 region of an E1 / E3-deleted adenoviral vector.

14. A method for producing a modified host cell, the method comprising steps (a) and (b): (a) introducing a first nucleic acid molecule into a host cell, wherein the first nucleic acid molecule comprises a CARE element as defined in claim 1 and an AAV cap gene operably linked thereto; and (b) introducing a second nucleic acid molecule into the host cell, the second nucleic acid molecule being a second nucleic acid molecule as defined in claim 1; Such that the first and second nucleic acid molecules independently: (i) stably integrated into the genome of the host cell, or (ii) exists freely in the host cell.

15. The method according to claim 14, further comprising step (c): (c) introducing a third nucleic acid molecule into the host cell, wherein the third nucleic acid molecule comprises a nucleotide sequence encoding an AAV Rep polypeptide; Such that the first, second and third nucleic acid molecules independently: (i) stably integrated into the genome of the host cell, or (ii) exists freely in the host cell.

16. A kit comprising: (A) a host cell, the host cell comprising: (a) a first nucleic acid molecule comprising a CARE element as defined in claim 1 and a DNA molecule operably linked thereto, wherein the DNA molecule encodes one or more of the following: (i) AAV Cap polypeptide, and (ii) AAV Rep polypeptide, wherein the first nucleic acid molecule is stably integrated into the genome of the host cell or exists in an episomal plasmid; and (B) an adenoviral vector, the adenoviral vector comprising: (i) a nucleic acid molecule encoding an adenovirus L4 22K polypeptide as defined in claim 1, wherein the L4 22K polypeptide encoding sequence is not operably associated with an adenovirus MLP, and (ii) A nucleic acid molecule encoding an AAV transfer vector.

17. A kit comprising: (A) a host cell, the host cell comprising: (a) a first nucleic acid molecule comprising a CARE element as defined in claim 1 and a DNA molecule operably linked thereto; wherein the DNA molecule encodes (i) AAV Cap polypeptide wherein the first nucleic acid molecule is stably integrated into the genome of the host cell or exists in an episomal plasmid; and (B) an adenoviral vector, the adenoviral vector comprising: (i) a nucleic acid molecule encoding an adenovirus L4 22K polypeptide as defined in claim 1, wherein the L4 22K polypeptide encoding sequence is not operably associated with an adenovirus MLP, and (ii) A nucleic acid molecule encoding an AAV Rep polypeptide.

18. The kit of claim 17, wherein the DNA molecule further encodes (ii) AAV transfer vectors.

19. The kit of claim 17 or 18, wherein the nucleic acid molecule encoding the AAV Rep polypeptide is not operably associated with a functional promoter.

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