Nucleic acid sequences

Abstract

The invention relates to the field of recombinant polynucleotides comprising adenoviral genome sequences. It also relates to methods and processes comprising such nucleic acid sequences.

Description

[0001] NUCLEIC ACID SEQUENCES

[0002] Field of the invention

[0003] The invention relates to the field of recombinant polynucleotides comprising adenoviral genome sequences. It also relates to methods and processes comprising such recombinant polynucleotides.

[0004] Background of the invention

[0005] Recombinant adeno-associated viruses (rAAV) are commonly used to deliver gene therapy products for the treatment of human diseases. Their production requires the preliminary stage of transfection of cells with different plasmids, including the plasmid comprising a heterologous nucleic acid (e.g. a transgene) to be expressed. Additional components, typically brought by additional plasmids, are required for the production of rAAVs. The additional components include the AAV rep and the cap proteins, needed to form the virus itself, as well as sequences needed to help replicate and propagate the different elements of the virus. The latest, i.e. helper sequences, can be provided by helper viruses, such as adenovirus, a herpesvirus or vaccina virus.

[0006] The helper sequences can be derived from viruses, for instance provided by adenovirus helper nucleic acids (Ad helpers) which comprise the required components. The use of Ad helpers is considered safer than the use of other types of helper viruses because the Ad helpers only produce proteins for the production of rAAV and, in particular, do not produce infectious helper virus. The most common Ad helpers are typically based on Adenovirus 2 (Ad2) or Adenovirus 5 (Ad5). rAAV production requires the incorporation and expression of up to three large plasmids into one single cell. Successfully delivering two or three plasmids to one cell is a relatively inefficient process. For larger-scale manufacturing efforts, transient delivery of plasmid requires excess quantities of DNA, adding to the overall cost of production and purification.

[0007] Optimisation of the production of rAAV can result in more efficient manufacture and flexibility in the development of cell culture. Therefore, there is a need for optimisation of the production of rAAV and in particular there is a need for optimisation of the helper sequences, preferably safer helper sequences where unnecessary natural viral sequences have been removed.

[0008] When it comes to producing rAAV for gene therapy, the cost of goods is a critical aspect. These high costs of goods linked to manufacturing even raise concerns about the economic viability of gene therapy treatments, and importantly could result in medicine access challenges for patients. One of the main materials impacting the cost of good is the helper plasmid. Therefore, any optimisation of the helper sequences leading to a less expensive material, without negatively impacting the yield and quality of the final rAAV, would provide a deep commercial advantage.

[0009] Summary of the invention

[0010] The invention provides a recombinant polynucleotide comprising adenoviral genome sequences comprising or consisting of: (a) a VA Fragment comprising or consisting of at least a E2B region comprising the VA RNA promoter region and at least a portion of the VA RNA,

[0011] (b) a E2A Fragment comprising or consisting of at least a portion of E2A region encoding DBP protein, and at least a fragment of the gene encoding pVIII protein, and

[0012] (c) at least a portion of E4 region comprising or consisting of the E4 ORF6 / 7 region, and wherein the recombinant polynucleotide does not comprise any adenoviral genome sequences encoding any one of:

[0013] 1) pllla protein,

[0014] 2) HEXON protein,

[0015] 3) U exon, and

[0016] 4) Fiber protein.

[0017] (hereinafter referred to as the recombinant polynucleotide of the invention).

[0018] In a further aspect the invention provides a process for the production of AAV using recombinant polynucleotides of the invention as a helper plasmid for the introduction of adenovirus genes to host cells.

[0019] Detailed description of the invention

[0020] Definitions

[0021] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

[0022] The term “and / or” used in a phrase such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A”, and “B”.

[0023] The forms “a”, “an”, and “the” include both single and plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” includes both “an antibody” and “antibodies”. As used herein, the term “between” includes values within the stated range as well as the endpoints of the stated range, i.e. the ranges are inclusive. Thus, for example, reference to “between 10 and 50 nucleotides” refers to both values within the range and the endpoints of 10 and 50 nucleotides.

[0024] The term “comprising” does not exclude other elements. For the purpose of the present disclosure, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. Polypeptides are organic polymers consisting of a number of amino acid residues bonded together in a chain. As used herein, ‘polypeptide’ is used interchangeably with ‘protein’ and ‘peptide’.

[0025] As used herein, the terms “nucleotide sequence”, "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multistranded DNA, genomic DNA, or cDNA. DNA refers to deoxyribonucleic acid, which is a polymer typically composed of two polynucleotide chains that coil around each other to form a double helix (double stranded DNA). A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein. A "gene product" or, alternatively, a "gene expression product" refers typically to the amino acid sequence (e.g., peptide or polypeptide) generated when a gene is transcribed and translated. It may happen that a “gene product” is an RNA which is not further translated.

[0026] Suitably, the polynucleotides used in the present invention are isolated. An “isolated” polynucleotide is one that is removed from its original environment. For example, a naturally occurring polynucleotide is isolated if it is separated from some or all of the coexisting materials in the natural system. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of its natural environment or if it is comprised within cDNA.

[0027] As used herein, "expression" refers to the two-step process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0028] The term "encode" as it is applied to polynucleotides refers to a polynucleotide which is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom (which can also be referred as the ‘sense’ strand.

[0029] The term “regulatory sequence” refers to one or more sequences that direct and / or are involved in the expression of a gene (herein of the transgene). Typically said one or more regulatory sequences are selected to direct, drive, assist and / or control the expression of a given gene. Non limiting example of regulatory sequences are for instance: transcription initiation sequences (such as a promoter), translation initiation sequences, mRNA stability sequences, transcription termination sequences (such as polyadenylation sequences), secretory sequences, enhancer sequences, introns, TATA boxes, microRNA targeted sequences, polylinker sequences facilitating the insertion of a DNA fragment within a vector, signal sequences, or yet posttranscriptional regulatory elements.

[0030] The term "promoter" as used herein means a control sequence that is a region of a polynucleotide sequence at which the initiation and rate of transcription of a coding sequence, such as a gene or a transgene, are controlled. Promoters may be constitutive, inducible, repressible, and / or tissue specific. Promoters may contain genetic elements at which regulatory proteins and molecules such as RNA polymerase and transcription factors may bind. It is known in the art that the nucleotide sequences of such promoters may be modified in order to increase or decrease the efficiency of mRNA transcription. See, e.g., Gao et al. (2018). Synthetically derived promoters may be used for ubiquitous or tissue specific expression. In embodiments, the promoter is used together with an enhancer to increase the transcription efficiency.

[0031] An “enhancer” is a regulatory element that increases the expression of a target sequence. The enhancer or promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer or promoter is one which is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" enhancer or promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e. , molecular biological techniques) such that transcription of that gene is directed by the linked enhancer or promoter. It is understood in the art that enhancers can operate from a distance and irrespective of their orientation relative to the location of an endogenous or heterologous promoter. It is thus further understood that an enhancer operating at a distance from a promoter is thus “operably linked” to that promoter irrespective of its location in the vector or its orientation relative to the location of the promoter.

[0032] A “heterologous nucleic acid “ or “transgene” is a gene or an RNA that has, or is intended for, transfer from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code.

[0033] A “therapeutic transgene” is a transgene encoding a correctly functioning protein which may be used in transduction of cells in a subject suffering from a genetic or other disorder in which a defective version of the protein is produced or in subject where the protein is not expressed. Alternatively the “therapeutic transgene” can be an inhibitory element (including siRNA) which may be used in transduction of cells in a subject suffering from a genetic or other disorder in which overexpression or misexpression of the gene and / or protein occurs.

[0034] A ‘functional fragment’ in relation to a nucleic acid sequence or protein is a portion of a reference nucleic acid or protein sequence which maintains the function / biological activity of the reference nucleic acid or protein sequence, if said sequence codes for a protein. A ‘functional variant’ is a mutated version of a reference nucleic acid or protein sequence which maintains the function / biological activity of the reference nucleic acid or protein sequence. A functional variant may be a variant of a functional fragment. The term “active portion” is used to refer to a functional fragment or variant. Suitably the functional fragment or functional variant comprises a sequence having at least 70% identity, such as at least 80% identity, such as at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity with the reference nucleic acid sequence or protein. Suitably the functional fragment or functional variant comprises at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99% of the length, of the reference nucleic acid sequence or protein. In one embodiment, any amino acid substitutions in a functional variant relative to the reference sequence of a protein are conservative amino acid substitutions. In one embodiment, any substitutions in a functional variant relative to the reference sequence of a nucleic acid sequence which encodes a protein are (i) nucleotide substitutions which are degenerate in the genetic code and thus encode the same protein and (ii) nucleotide substitutions which encode a protein containing conservative amino acid substitutions. An “active protein”, such as used in the term “an active Fiber protein” and similarly by reference to other proteins, refers to a functional fragment or functional variant of said reference protein which is produced by the translation of an mRNA molecule encoding said functional variant or functional fragment.

[0035] By “maintains the function” of a nucleic acid or protein means maintains at least one function / biological activity, such as the principal function / biological activity, of said nucleic acid or protein, particularly a function / biological activity relevant in the context of providing helper genes for AAV replication.

[0036] The phrase “at least a portion of” in relation to a nucleic acid sequence requires the presence of a specified minimal number of contiguous nucleotides from the specified sequence.

[0037] By “contiguous nucleotides”, we include the meaning of a continuous set of nucleotides as found in a naturally occurring sequence. Specifically, nucleotides found within the set are all present in order.

[0038] The term “vector”, as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid” or “plasmid construct”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g. non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. Expression vectors include viral vectors (e.g., replication defective retroviruses, lentiviral vectors, adenoviruses, Sendai viruses and adeno-associated viruses), which serve equivalent functions, and also bacteriophage and phagemid systems. Another type of vector includes RNA molecules, e.g., mRNA and stabilised RNA, to carry coding genetic information to the cells. Yet a further type of vector includes “Doggybone DNA™” or “dbDNA™”, which refers to a linear double-stranded DNA construct.

[0039] The term “host cell”, as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. Such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell, for example, when said progeny are employed to make a cell line or cell bank which is then optionally stored, provided, sold, transferred, or employed to manufacture a polypeptide, antibody or fragment thereof as described herein.

[0040] With respect to general recombinant techniques, vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available. Alternatively, they can be prepared on demand, in house. In order to optimize expression and / or in vitro transcription, it may be necessary to remove, add or alter 5' and / or 3' untranslated portions of cloned transgenes to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites (Kozak Sequence) can be inserted immediately upstream of the start codon to enhance expression.

[0041] A "viral vector" is defined as a recombinantly produced virus or viral particle that contains a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenovirus vectors, alphavirus vectors and the like. A viral vector is capable of infecting and transducing a cell.

[0042] “AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or modifications, derivatives, or pseudotypes thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. AAVs are parvoviruses. They depend on other viruses to replicate. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" or “rAAV” includes AAV type 1 (AAV1 ; accession numbers e.g. NC_002077, AF063497), AAV type 2 (AAV2; accession number e.g. NC_001401), AAV type 3 (AAV3; accession numbers e.g. NC_001729, NC_001863), AAV type 4 (AAV4; accession number e.g. NC_001829), AAV type 5 (AAV5; accession number e.g. NC_006152), AAV type 6 (AAV 6; accession number e.g. NC_001862), AAV type 7 (AAV7; accession number e.g. NC_006260), AAV type 8 (AAV8; accession number e.g. NC_006261), AAV type 9 (AAV9; accession number e.g. AY53057), and AAV10 (AAVrHIO; accession number e.g. AY243015), as well as recombinant serotypes, or yet avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and modifications, derivatives (such as AAV-TT (alternatively spelt AAVTT); W02015121501), or pseudotypes thereof. "Primate AAV" refers to AAV that infect primates, "non-primate AAV" refers to AAV that infect non-primate mammals, "bovine AAV" refers to AAV that infect bovine mammals, etc. "Recombinant", by reference to an AAV particle means that the AAV particle is the product of one or more non-natural procedures that result in an AAV particle construct that is distinct from an AAV particle in nature.

[0043] A recombinant adeno-associated virus vector, or "rAAV vector", refers to a viral vector or a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide comprising a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell). In this description, the terms “rAAV vector” or simply “rAAV” (such as in “rAAV manufacturing...”), will be used interchangeably. The rAAV vector may be of any AAV serotype, including any modification, derivative or pseudotype (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or derivatives / modifications / pseudotypes thereof, such as AAV-TT). Such AAV serotypes and derivatives / modifications / pseudotypes, and methods of producing such serotypes / derivatives / modifications / pseudotypes are known in the art (see, e.g., Asokan et al., Mol. Ther. 20(4):699-708 (2012).

[0044] A "composition" is intended to mean a combination of active polypeptide, polynucleotide or antibody, and at least one other compound, inert (e.g., a detectable label) or active.

[0045] As used herein, the term “about” includes up to and including 10% greater and up to and including 10% lower than the value specified, suitably up to and including 5% greater and up to and including 5% lower than the value specified, especially the value specified. The term “between”, includes all the values of the specified boundaries.

[0046] For the purposes of comparing two closely related polypeptide sequences, the “% sequence identity” (or sometime the “% identity”) between a first polypeptide sequence and a second polypeptide sequence may be calculated using NCBI BLAST v2.0 for example, using standard settings for polypeptide sequences (BLASTP).

[0047] For the purposes of comparing two closely related polynucleotide sequences, the “% sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated using NCBI BLAST v2.0 for example, using standard settings for nucleotide sequences (BLASTN).

[0048] Polypeptide or polynucleotide sequences are said to be the same as or “identical” to other polypeptide or polynucleotide sequences if they share 100% sequence identity over their entire length. Residues in sequences are numbered from left to right, i.e. from N- to C- terminus for polypeptides; from 5’ to 3’ terminus for polynucleotides.

[0049] A “difference” between sequences refers to an insertion, deletion or substitution of a single amino acid residue or nucleotide in a position of the second sequence, compared to the first sequence. Two sequences can contain one, two or more such amino acid / nucleotide differences. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity. For example, if the identical sequences are 9 amino acid residues long, one substitution in the second sequence results in a sequence identity of 88.9%. If the first and second polypeptide sequences are 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences share greater than 66% identity (the first and second polypeptide sequences share 66.7% identity).

[0050] It is noted that because of the degeneracy of the genetic code (see Asubel 2003, Appendix 1 C), some substitutions of nucleotides will be silent once translated in amino acids.

[0051] Using the three letter and one letter codes, the naturally occurring amino acids may be referred to as follows: glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or lie), proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp), lysine (K or Lys), arginine (R or Arg), histidine (H or His), aspartic acid (D or Asp), glutamic acid (E or Glu), asparagine (N or Asn), glutamine (Q or Gin), cysteine (C or Cys), methionine (M or Met), serine (S or Ser) and Threonine (T or Thr). Where a residue may be aspartic acid or asparagine, the symbols Asx or B may be used. Where a residue may be glutamic acid or glutamine, the symbols Glx or Z may be used. References to aspartic acid include aspartate, and glutamic acid include glutamate, unless the context specifies otherwise.

[0052] A “conservative” amino acid substitution is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue of similar chemical structure, and which is expected to have little influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions suitably are substitutions in which one amino acid within the following groups is substituted by another amino acid residue from within the same group, as shown in Table 1 below.

[0053] Table 1: Amino acids

[0054] Suitably, a hydrophobic amino acid residue is a non-polar amino acid. More suitably, a hydrophobic amino acid residue is selected from V, I, L, M, F, W or C. In some embodiments, a hydrophobic amino acid residue is selected from glycine, alanine, valine, methionine, leucine, isoleucine, phenylalanine, tyrosine, or tryptophan. Suitably, any residues in a sequence which do not correspond to the residues provided in a reference sequence are conservative amino acid substitutions with respect to the residues of the reference sequence.

[0055] Mutations can be made to the DNA or cDNA that encode polypeptides which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli and S. cerevisiae, as well as mammalian, specifically human, are known. This process is referred to as codon optimisation.

[0056] Adenovirus genome

[0057] Although all sequences herein specifically exemplified were based on the sequences as found in native Human Adenovirus 5, accession AC_000008.1 (SEQ ID NO: 1), the skilled person understands that the equivalent sequences from other well-known types of adenoviruses can be used instead. In non-limiting examples, the sequences of native Human Adenovirus 2 (accession NO: J01917), or yet native Human Adenovirus C serotype 5 (accession NO: AY339865.1), or other adenoviruses can be used instead. It should be understood that reference to an adenoviral sequence also includes homologues of said sequence, such as the equivalent sequence as found in different adenoviruses. Furthermore, all references to adenoviral sequences includes reference to sequences with at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or yet 100% identity to the sequence provided.

[0058] Main Regions of adenoviral genome

[0059] E2B region refers to a region that is critical for the adenoviral genome replication. It comprises the VA RNA region (including its promoter), pTP (including its intron and exons to enable splicing) and Pol (Saha and Parks, 2017). VA RNA refers to a region of the adenoviral genome which is non-protein coding, i.e. when transcribed it produces non-coding RNA. The natural VA RNA region and the one from most if not all the adenovirus-based helper plasmid transcribes two copies of VA RNA: VA RNA I and VA RNA II.

[0060] E2A region refers to a transcriptional unit found in any adenoviral genome. In native adenoviruses, the E2A region comprises a nucleotide sequence encoding DBP. DBP protein, also known as singlestranded DNA-binding protein, refers to a DNA binding protein found in adenoviruses. E2A region typically also comprising coding sequences for 100K, 33K and 22K proteins, as well as pVIII protein which refers to a capsid protein precursor found in adenoviruses. pVIII protein is cleaved by the L3 protease after production to produce the mature viral protein VIII.

[0061] E3 region refers to a transcriptional unit found in any adenoviral genome. In native adenoviruses, the E3 region comprises nucleotide sequences encoding control proteins including for instance E3 12.5K. E4 region refers to a transcriptional unit found in any adenoviral genome. In native adenoviruses, the E4 region comprises nucleotide sequences encoding the following control proteins: E4ORF1 (protein reprogram cellular signalling pathways in infected cells to shape an optimal microenvironment for viral replication), E4ORFB, E4ORF3 (involved in efficient replication and late protein synthesis during adenoviral infection), E4ORF4 (involved in efficient replication during adenoviral infection), and E4ORF6 / 7 (interacts with virus packaging components during adenoviral infection and promotes transcription of the E2 promoter; note that ORF6 is also called E4 34K).

[0062] The major late transcription unit (MLTU) refers to a transcriptional unit found in any adenoviral genome. It is transcribed in the late phase of an adenoviral infection. Once transcribed, the MLTU is spliced in five regions: L1 , L2, L3, L4 and L5. In native adenoviruses, the L1 region comprises nucleotide sequences encoding for example the encapsidation protein 52K and capsid protein precursor pl Ila.

[0063] 23K endopeptidase refers to a protease protein found in adenoviruses and encoded in the L3 transcription unit of an adenovirus. Hexon protein (also known as “HEXON”) refers to a capsid protein found in any adenoviruses, and is also encoded in the L3 transcription unit of an adenovirus.

[0064] U exon refers to a DNA region found in any adenoviral genome. An example of an adenoviral genome sequence which encodes the U exon is provided as SEQ ID NO: 22.

[0065] Fiber protein refers to a capsid protein found in adenoviruses, and is encoded in the L5 transcription unit of any adenovirus. Inverted Terminal Repeats, or ITRs, refers to portions of DNA which promote bi-directional transcription of the viral genome. ITRs are localised at the 5’ and 3’ of the viral genome, which are referred to as the 5’ ITR and 3’ ITR respectively. All viruses comprise ITRs, including adenoviruses (AdV) and adeno-associated viruses (AAV).

[0066] The present invention is based on the need for optimisation of the production of rAAV and in particular the optimisation of the helper sequences. This invention describes a class of nucleotide sequences (more especially recombinant nucleotide sequences) with adenovirus origin that can serve, among others, as helper sequences, such as helper plasmids, and provide flexibility in plasmid production methods and support manufacturing of safer recombinant adeno-associated viruses (rAAV) due to unnecessary adenovirus sequences being removed, enable increase of successful transfection and rAAV production titres, boost rAAV product quality by reducing unwanted packaging of plasmid backbone and host cell DNA (in other term, mispackaging), without any penalty in potency of the drug substance.

[0067] Although it is already known that only a few elements from adenoviruses are sufficient to get helper function, such as E1A / B (typically brought by the host cell, such as HEK293 cell), E2A, E4 and VA RNA, as will be detailed herein, it has surprisingly been shown by the inventors that these regions are not needed in full to provide a helper function. It has further been shown that only partial sequences of some adenoviral genome regions are sufficient to provide such helper function. Further, as will be detailed in the following sections, the recombinant polynucleotides of the invention, when used as helper plasmids, can also help improving the yield and the quality of rAAV manufacturing processes (titres of more than 1e11viral capsids / mL were reached for the sequences according to the invention that have been assessed). Last but not least, as the recombinant polynucleotides of the invention can be used as helper plasmids of limited size, they will provide the great commercial advantage of being less expensive to produce and thus using such sequences in rAAV manufacturing processes will allow for the decrease of the cost of goods of said manufacturing processes in which there are used (in order to produce rAAV for gene therapy applications for instance).

[0068] The systematic construct design approach followed by the inventors was based in the following strategy:

[0069] - Viral elements maintained in the order they exist in wild type adenovirus (whereas most of the helper plasmids currently available contain sequences in inverted directions compared to the natural viral sequence).

[0070] - No addition of any new fragments to induce additional function (with the exception of the necessary elements in the backbones, such as Origin and antibiotic resistance).

[0071] - Removal of sequences that are not necessary for AAV packaging, to reduce helper plasmid size and to possibly increase safety.

[0072] It is noted that although the sequences described below relate to the sequences as found in native Human Adenovirus 5, accession # AC_000008.1 , which has been chosen to exemplify the invention, it should be understood that the concept of the invention is applicable to any viruses from the family Adenoviridae, especially adenoviruses belonging to genus Mastadenovirus, which is originated from the mammals and their genomes contain all regions E1 , E2B, E2A, E3 and E4. Non limiting examples of such human adenoviruses that can be used according to the invention are Human Adenovirus 2 (accession # J01917), Human Adenovirus C serotype 5 (accession # AY339865.1), and any others from the same genus.

[0073] Contrary to many recombinant polynucleotides in the art, such as helper plasmid in the art, it is noted that in the context of the present invention as a whole, the polynucleotides comprise adenoviral sequences assembled in the order of wild type adenovirus genome.

[0074] The main object of the present invention is to provide a recombinant polynucleotide comprising adenoviral genome sequences comprising or consisting of:

[0075] (a) a VA Fragment comprising or consisting of at least a E2B region comprising the VA RNA promoter region and at least a portion of the VA RNA,

[0076] (b) a E2A Fragment comprising or consisting of at least the E2A region encoding DBP protein and a fragment of the gene encoding pVIII protein, and

[0077] (c) at least a portion of E4 region comprising or consisting of the E4 ORF6 / 7 region; and wherein the recombinant polynucleotide does not comprise any adenoviral genome sequences or fragments thereof encoding any one of:

[0078] (1) pllla protein,

[0079] (2) H EXON protein,

[0080] (3) U exon and

[0081] 4) Fiber protein.

[0082] Preferably, in the context of the invention, each viral elements are kept in the order and direction they exist in the corresponding wild type adenovirus: i.e. E2B or a fragment thereof, VA RNA I, VA RNA II or a fragment thereof, 52K or a fragment thereof (when present), E2A or a fragment thereof, pVIII or a fragment thereof, E3 12.5K or a fragment thereof (when present), Fiber fragment (when present), E4 or a fragment thereof (such as E4 Orf 6 / 7) (See Figure 1).

[0083] In the context of the invention as a whole, the VA Fragment comprises or consists of at least an E2B region comprising the VA RNA promoter region and at least a portion of the VA RNA.

[0084] Preferably, the at least a portion of E2B comprises or consists of at least the VA RNA promoter region or at least the VA RNA promoter region and also its TATA box sequence. Preferably, the at least a portion of E2B comprises or consists of at least 650 contiguous nucleotides of the full E2B region (SEQ ID NO: 36), or a variant thereof, more preferably between 680 and 750 contiguous nucleotides or even preferably between 703 and 750 contiguous nucleotides of the full E2B region (SEQ ID NO: 36), and wherein the contiguous nucleotides comprise at least the VA RNA promoter region or at least the VA RNA promoter region and also its TATA box sequence (represented as SEQ ID NO: 11 , which corresponds to the nucleotides 10125 to 10130 of SEQ ID NO.1). In a non-limiting example, the at least a portion of E2B region comprises or consists of SEQ ID NO: 12, which corresponds to the nucleotides 9841 to 10544 (704 contiguous nucleotides) of the adenoviral genome sequence of SEQ ID NO: 1. As variants (preferably functional variants) of the at least a portion of E2B are encompassed herein, suitably the at least a portion of E2B comprises or consists of a sequence with at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the sequence of SEQ ID NO: 12.

[0085] Preferably, the at least a portion of VA RNA comprises or consists of the full VA RNA I sequence and only a portion of VA RNA II sequence. Preferably, the at least a portion of VA RNA II does not comprise a full VA RNA II sequence. Preferably, the at least a portion of VA RNA comprises or consists of the 157 contiguous nucleotides of the VA RNA I sequence and a fragment of between 50 to 100 contiguous nucleotides of VA RNA II, more preferably between 60 and 90 contiguous nucleotides of VA RNA II or even preferably between 70 and 80 contiguous nucleotides of VA RNA II, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79 or 80 contiguous nucleotides of VA RNA II. The at least a portion of VA RNA preferable comprises or consists of the full VA RNA I sequence which corresponds to the nucleotides 10620 to 10776 (157 bp) as shown in SEQ ID NO: 8 and only a portion of VA RNA II which corresponds to the nucleotides 10876 to 10951 (76bp) as shown in SEQ ID NO: 10. In a non-limiting example, the at least a portion of DNA comprising VA RNA comprises or consists of SEQ ID NO: 6, which corresponds to the nucleotides 10620 to 10951 (332 contiguous nucleotides) of the adenoviral genome sequence of SEQ ID NO: 1. Alternatively, the at least a portion of VA RNA comprises or consists of the full VA RNA I sequence and the full VA RNA II sequence. In this case, the at least a portion ofVA RNA comprises or consists of the 157 contiguous nucleotides of the VA RNA I sequence which corresponds to the nucleotides 10620 to 10776 as shown in SEQ ID NO: 8 and of the 158 contiguous nucleotides of VA RNA II which corresponds to the nucleotides 10876 to 11033 as shown in SEQ ID NO: 9. In a non-limiting example, the at least a portion of DNA comprising VA RNA comprises or consists of SEQ ID NO: 6, which corresponds to the nucleotides 10620 to 10951 (332 contiguous nucleotides) of the adenoviral genome sequence of SEQ ID NO: 1 or comprises or consists of SEQ ID NO: 7, which corresponds to the nucleotides 10620 to 11033 (414 contiguous nucleotides) of the adenoviral genome sequence of SEQ ID NO: 1. As variants (preferably functional variants) of at least a portion of VA RNA are encompassed herein, suitably the at least a portion of VA RNA comprises or consists of a sequence with at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the sequence of SEQ ID NO: 6 or of SEQ ID NO: 7. SEQ ID NO: 38 represents the VA Fragment as used in the constructs pHP925 and corresponds to nucleotides 9842 to 11573 of the adenoviral genome sequence of SEQ ID NO: 1

[0086] In the context of the invention as a whole, the VA Fragment comprises or consists of from about 900 to about 1800 bp, preferably from about 1000 to 1750, from about 1000 to 1150 or from about 1100 to 1740, such as for example 1110 bp or 1732 bp.

[0087] In the context of the invention as a whole, the E2A Fragment comprises or consists of at least the E2A region encoding DBP protein and a fragment of the gene encoding pVIII protein. Preferably, the at least a portion of the E2A region encoding DBP protein comprises or consists of at least 1590 contiguous nucleotides of the gene encoding DBP protein, or of a variant thereof. In a non-limiting example, the at least a portion of E2A region encoding DBP protein comprises or consists of SEQ ID NO: 15, corresponding to nucleotides 22443 to 24032 of the adenoviral genome sequence of SEQ ID NO: 1. The at least a portion of the gene encoding pVIII protein comprises or consists of at least 350 contiguous nucleotides of the gene encoding pVIII, or a variant thereof. Preferably, the at least a portion of pVIII comprises or consists of between 350 and 684 contiguous nucleotides of the gene encoding pVIII protein or even preferably between 363 and 684 contiguous nucleotides of the gene encoding pVIII protein. In a non-limiting example, the at least a portion of the gene encoding pVIII protein comprises or consists of SEQ ID NO: 17, corresponding to nucleotides 27174 to 27536 of the adenoviral genome sequence of SEQ ID NO: 1. Such sequence can be named “minimal pVIII region”. In a further non-limiting example, the at least a portion of the gene encoding pVIII protein comprises or consists of SEQ ID NO: 16, corresponding to nucleotides 27174 to 27857 of the adenoviral genome sequence of SEQ ID NO: 1. The E2A Fragment preferably further comprises a portion of the E2A region encoding the 100K protein (also named L4 100K; corresponding to nucleotides 24061 to 26484 of SEQ ID NO: 1) as well as the 22K / 33K (also named L4 22K / 33K; both located in the region of nucleotides 26195 to 27086 of SEQ ID NO: 1 or part thereof) proteins.

[0088] In a non-limiting examples, the at least a portion of E2A region comprises or consists of SEQ ID NO: 13 or 14, corresponding respectively to nucleotides 22443 to 27536 or to nucleotides 22443 to 27857 of the adenoviral genome sequence of SEQ ID NO: 1. As variants (preferably functional variants) of at least a portion of E2A region are encompassed herein, suitably the at least a portion of E2A region comprises or consists of a sequence with at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the sequences of SEQ ID NO: 13, 14 or 15, 16 or 17. SEQ ID NO: 14 represents the E2A region as used in the construct pHP925 and corresponds to nucleotides 22443 to 27857 of the adenoviral genome sequence of SEQ ID NO: 1. SEQ ID NO: 13 represents an alternative E2A region and corresponds to nucleotides 22443 to 27536 of the adenoviral genome sequence of SEQ ID NO: 1. Alternatively, the entire E2A region is encoded in the recombinant polynucleotide of the invention.

[0089] In the context of the invention as a whole, the E2A Fragment comprises or consists of from about 2000 to about 5415 bp of the entire natural E2A region, preferably from about 3000 to 5415, from about 4000 to 5415 or from about 5000 to 5415, including size such as 5093 or 5415 bp of the entire natural E2A region, wherein the entire natural E2A region corresponds to nucleotides 18842 to 27857 of the adenoviral genome sequence of SEQ ID NO: 1 .

[0090] In the context of the invention as a whole, the at least a portion of E4 region comprising the E4ORF6 / 7 region, which encodes the protein E4ORF6 / 7, comprises or consists of at least 1000 contiguous nucleotides of the region encoding E4ORF6 / 7, or a variant thereof. Preferably, the at least a portion of E4 region comprising the E4ORF6 / 7 region comprises or consists of between 1000 and 1300 contiguous nucleotides of said region, more preferably between 1100 and 1200 contiguous nucleotides of said region or even preferably between 1140 and 1180, such as 1143, 1146, 1149, 1152, 1155, 1158, 1161 , 1164, 1167, 1170, 1173, 1176 or 1179 contiguous nucleotides of said region. In a non-limiting example, the at least a portion of E4 region comprising the E4ORF6 / 7 region consists or comprises of SEQ ID NO: 19, corresponding to nucleotides 32914 to 34077 of the adenoviral genome sequence of SEQ ID NO: 1. Such sequence can be named “minimal E4 region”. Alternatively, the at least a portion of E4 region comprising the E4ORF6 / 7 region comprises solely the E4ORF6 / 7 region (i.e. no other E4 ORFs are included), consisting of nucleotides 32914 to 34077 of the adenoviral genome sequence of SEQ ID NO: 1. As variants (preferably functional variants) of at least a portion of E4 region comprising the E4ORF6 / 7 region are encompassed herein, the at least a portion of E4 region comprising the E4ORF6 / 7 region comprises or consists of an adenoviral genome sequence having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to the sequence of SEQ ID NO: 19. SEQ ID NO: 39 represents the E4 fragment (i.e. E4 region) as used in the constructs pHP925 and corresponds to nucleotides 32914 to 35835 of the adenoviral genome sequence of SEQ ID NO: 1.

[0091] In the context of the invention as a whole, the recombinant polynucleotide preferably does not comprise any adenoviral genome sequence encoding any portion (or all) of pl I la protein, or a variant thereof. Alternatively, the recombinant polynucleotide of the invention does not comprise more than 10% of adenoviral genome sequence encoding pllla protein, or a variant thereof, wherein pllla protein is encoded by SEQ ID NO: 20 (corresponding to nucleotides 12318 to 14075 of SEQ ID NO: 1), such as not more than 9 %, not more than 8%, not more than 7%, not more than 6%, not more than 5%. In other words, the recombinant polynucleotide of the invention does comprise between 0 and 10% of adenoviral genome sequence encoding pllla protein, ora variant thereof.- Preferably, the recombinant polynucleotide of the invention does not comprise a DNA sequence encoding more than 50, more than 40, more than 30, more than 20, more than 10 or more than 5 contiguous residues from the sequence of pllla protein, wherein pllla protein is encoded by SEQ ID NO: 20.

[0092] In the context of the invention as a whole, the recombinant polynucleotide preferably does not comprise any adenoviral genome sequence encoding any portion (or all) of Hexon protein, or a variant thereof. Alternatively, the recombinant polynucleotide of the invention does not comprise more than 10% of adenoviral genome sequence encoding the Hexon protein, or a variant thereof, wherein Hexon protein is encoded by SEQ ID NO: 21 (corresponding to nucleotides 18842 to 21700 of SEQ ID NO: 1), such as not more than 9 %, not more than 8%, not more than 7%, not more than 6%, not more than 5%. In other words, the recombinant polynucleotide of the invention does comprise between 0 and 10% of adenoviral genome sequence encoding Hexon protein, or a variant thereof. Preferably, the recombinant polynucleotide of the invention does not comprise a DNA sequence encoding more than 50, more than 40, more than 30, more than 20, more than 10 or more than 5 contiguous residues from the sequence of Hexon protein, wherein Hexon protein is encoded by SEQ ID NO: 21.

[0093] In the context of the invention as a whole, the recombinant polynucleotide of the invention preferably does not comprise any adenoviral genome sequence comprising any portion (or all) of U exon, or a variant thereof. Alternatively, the recombinant polynucleotide of the invention does not comprise more than 10% of the adenoviral genome sequence corresponding to U exon, or a variant thereof, wherein the U exon sequence corresponds to SEQ ID NO: 22 (corresponding to nucleotides 30864 to 31031 of SEQ ID NO: 1), such as not more than 9 %, not more than 8%, not more than 7%, not more than 6%, not more than 5%. In otherwords, the recombinant polynucleotide of the invention does comprise between 0 and 10% of adenoviral genome sequence encoding U exon, ora variant thereof. Preferably, the recombinant polynucleotide of the invention does not comprise a DNA sequence encoding more than 5 contiguous residues from the sequence of U exon, wherein the U exon sequence corresponds to SEQ ID NO: 22.

[0094] In an embodiment of the invention, the recombinant polynucleotide does not comprise adenoviral genome sequence encoding any portion (or all) of Fiber protein, or a variant thereof. The full Fiber sequence corresponds to nucleotides 31042 to 32787 SEQ ID NO: 23. For example, the recombinant polynucleotide does not comprise any recombinant polynucleotides as shown in SEQ ID NO: 23 to 25.

[0095] Although it has been shown by the inventors that a recombinant polynucleotide comprising adenoviral sequences comprising or consisting of 1) a VA Fragment comprising or consisting of at least a E2B region comprising the VA RNA promoter region and at least a portion of the VA RNA, 2) an E2A Fragment comprising or consisting of at least the E2A region encoding DBP protein and a fragment of the gene encoding pVIII protein and 3) at least a portion of E4 region comprising or consisting of the E4 ORF6 / 7 region; and that does not comprise any region encoding pllla protein, Hexon protein, U exon and Fiber protein, is sufficient to get a helper activity (see examples section), said recombinant polynucleotide may optionally comprise one or more additional regions.

[0096] Therefore, in an embodiment of the invention, the at least a portion of E4 region consists solely of the E4ORF6 / 7 region.. Alternatively, the at least a portion of E4 region not only comprises the E4ORF6 / 7 region but can also further comprise one or more of the regions encoding a functional fragment of any one of E4ORF1 , E4ORFB, E4ORF3 and / or E4ORF4 proteins. SEQ ID NO: 30 is an example of a region encoding functional E4ORF1 , E4ORFB, E4ORF3 and E4ORF4 corresponding to nucleotides 34079 to 35526. In non-limiting examples, the at least a portion of E4 region comprises or consists of the entire E4 region, SEQ ID NO: 18, corresponding to nucleotides 32914 to 35835 of SEQ ID NO: 1. Suitably the at least a portion of E4 region comprises or consists of an adenoviral genome sequence having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to any one of SEQ ID NO: 18 or 19. Example of recombinant polynucleotide further comprising one or more of the regions encoding a functional fragment of any one of E4ORF1 , E4ORFB, E4ORF3 and / or E4ORF4 is shown in SEQ ID NO: 2 or 4. In the context of the invention as a whole, the at least a portion of E4 region comprises from about 1200 to about 3000 bp of the entire natural E4 region, preferably from about 1300 to 2950, from about 1400 to 2950, including size such as 1473 or 1922 bp of the entire natural E4 region, wherein the entire natural E4 region corresponds to nucleotides 32914 to 35835 of the adenoviral genome sequence of SEQ ID NO: 1 .

[0097] In an embodiment of the invention, the recombinant polynucleotide does not comprise an adenoviral genome sequence encoding any portion (or all) of 23K protease, or a variant thereof, wherein the full 23K encoding sequence is shown as SEQ ID NO: 26 corresponding to nucleotides 21733 to 22347 of SEQ ID NO: 1. An example of such a recombinant polynucleotide that does not comprise any sequence encoding for 23K is shown in SEQ ID NO: 2 or 4.

[0098] In an embodiment of the invention, the recombinant polynucleotide further comprises an adenoviral genome sequence encoding at least a portion of 52K protein. Preferably, when present, the at least a portion encoding 52K protein comprises or consists of between 100 and 1248 contiguous nucleotides of said region, such as between 100 and 1000 contiguous nucleotides of said region or 100 and 600 contiguous nucleotides of said region. 52K protein complete sequence, as shown as SEQ ID NO: 27, corresponds to nucleotides 11050 to 12297 of SEQ ID NO: 1. In a non-limiting example, the adenoviral genome sequence encoding at least a portion of 52K protein comprises or consists of SEQ ID NO: 28 corresponding to nucleotides 11050 to 11573 of SEQ ID NO: 1 (524bp). Suitably the adenoviral genome sequence encoding a portion of 52K protein comprises a sequence having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 27 or 28. An example of recombinant polynucleotides comprising an adenoviral genome sequence encoding at least a portion of 52K protease is shown in SEQ ID NO: 2 or 4. In another non-limiting example, the adenoviral genome sequence encoding at least a portion of 52K protein comprises or consists of nucleotides 11050 to 12297 of SEQ ID NO: 1 , or a sequence having at least 90, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereof.

[0099] In an embodiment of the invention, the recombinant polynucleotide does not comprise an adenoviral genome sequence encoding any portion (or all) of the E3 region, wherein the full E3 region corresponds to nucleotides 27858 to 32787 of SEQ ID NO: 1. In an alternative embodiment of the invention, the recombinant polynucleotide does not comprise an adenoviral genome sequence encoding any portion (or all) of the E3 region, except for at least a fragment of E3 12.5K protein. Preferably such a fragment of E3 12.5K protein is equal to or shorter than 300bp, such as equal to or shorter than 290bp or such as equal to or shorter than 280bp.

[0100] In an embodiment of the invention, the recombinant polynucleotide does not comprise an adenoviral genome sequence encoding any portion (or all) of E3 14.7K protein, wherein the full E3 14.7K encoding sequence is shown as SEQ ID NO: 41 corresponding to nucleotides 30453 to 30839 of SEQ ID NO: 1. Example of recombinant polynucleotides that do not comprise an adenoviral genome sequence encoding E3 14.7K protease is shown in SEQ ID NO: 2 or 4.

[0101] In the context of the invention as a whole, the recombinant polynucleotide of the invention does not comprise at least one of the adenoviral ITRs. Preferably, the recombinant polynucleotide does not comprise either a 3’ adenoviral ITR or a 5’ adenoviral ITR. Alternatively, the recombinant polynucleotide does not comprise any one of the 3’ adenoviral ITR and the 5’ adenoviral ITR.

[0102] In the context of the invention as a whole, also provided is a recombinant polynucleotide comprising adenoviral genome sequences, assembled in the order of wild type adenovirus genome, comprising or consisting of: (a) an VA Fragment comprising or consisting of E2B region comprising a the VA RNA promoter region and at least VA RNA I,

[0103] (b) an E2A fragment comprising or consisting of at least the E2A region coding DBP, 100K, 33K, 22K and at least a fragment of pVIII,

[0104] (c) at least a portion of E4 region comprising or consisting of the E4 ORF6 / 7 region, and wherein the polynucleotide does comprise between 0 and 10% of adenoviral genome sequences encoding any of:

[0105] (1) pllla protein,

[0106] (2) HEXON protein,

[0107] (3) U exon, and

[0108] (4) Fiber protein.

[0109] Also herein provided, in the context of the invention as a whole, is a recombinant polynucleotide comprising adenoviral genome sequences, assembled in the order of wild type adenovirus genome, comprising:

[0110] (a) a VA Fragment comprising or consisting of at least an E2B region comprising the VA RNA promoter region and at least a portion of the VA RNA,

[0111] (b) an E2A fragment comprising or consisting of at least the E2A region coding DBP, 100K, 33K, 22K and a fragment of the gene encoding pVIII protein,

[0112] (c) at least a portion of E4 region comprising or consisting of the E4 ORF6 / 7 region, wherein the polynucleotide does not comprise any of pllla protein, HEXON protein, U exon, 23K and Fiber protein.

[0113] Additional elements

[0114] The skilled person will understand that the recombinant polynucleotides according to the invention can comprise additional elements such as: regulatory elements, short sequences resulting from the cloning, which can also be called junction sequences, plasmid backbone elements, such as ori sequence (SEQ ID NO: 34) and / or antibiotic resistance sequence (SEQ ID NO: 35), and / or additional elements that are not part of the wild type Adenovirus sequence such as new / synthetic promoters other than the endogenous promoters.

[0115] In a preferred aspect of the invention, the recombinant polynucleotide comprises native regulatory elements, such as the native p4 promoter and / or E2A natural promoter and any other native promoters that are needed in order the recombinant polynucleotide according to the invention can be used as a helper sequence for instance. In an embodiment, the recombinant polynucleotide of the invention comprises a native p4 promoter which consists of or comprises SEQ ID NO: 33, corresponding to nucleotides 35527 to 35835 of SEQ ID NO: 1 . In a further embodiment, the recombinant polynucleotide of the invention comprises a native E2A promoter. The invention further provides a recombinant polynucleotide which comprises or consists of SEQ ID NO: 2 or 4 or a sequence having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 or 4.

[0116] The invention further provides a recombinant polynucleotide of the invention wherein the recombinant polynucleotide is isolated.

[0117] Plasmid

[0118] In an aspect of the invention there is provided an engineered plasmid comprising a recombinant polynucleotide of the invention.

[0119] Suitably the plasmid comprises the recombinant polynucleotide of the invention and a bacterial replication origin capable of propagating the plasmid in a bacterial host cell.

[0120] Suitably the plasmid further comprises a selectable marker gene in addition to the recombinant polynucleotide of the invention and a bacterial replication origin capable of propagating the plasmid in a bacterial host cell. In some embodiments, the selectable marker gene is a drug resistance gene. In some embodiments, the selectable marker gene is a kanamycin resistance gene. In some embodiments, the selectable marker gene is an ampicillin resistance gene.

[0121] Suitably the plasmid comprising the recombinant polynucleotide of the invention (such as the engineered recombinant polynucleotide of the invention) is a helper plasmid as described hereinbefore. Helper plasmids, their use and benefits are well known in the art. In particular, the recombinant polynucleotide of the invention provides flexibility in production methods and sites for a plasmid in which it is comprised. For example, production of a helper plasmid comprising the recombinant polynucleotide of the invention may be manufactured at low standard laboratory biological safety levels due to the production only of plasmid DNA and resulting separation of plasmid DNA production from virus (e.g. viral vector) production, and enables the safe production of rAAV particles comprising a polynucleotide to be delivered into a host cell in a controlled system. However, the resulting rAAV do not comprise the machinery required fortranscription, translation and replication of the rAAV and thus are safe vectors for the delivery of e.g. therapeutic transgenes for genetic therapies.

[0122] Suitable methods of plasmid design and production which may be used for the plasmid and helper plasmid described herein are well known in the art. In one embodiment, the plasmid or other polynucleotide comprising the recombinant polynucleotide of the invention is produced using a doggybone DNA as described herein.

[0123] In non-limiting examples, the helper plasmid according to the invention comprises or consists of a nucleic acid sequence according to SEQ ID NO: 2 or 4 or a sequence having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 or 4. In a further non-limiting example, the helper plasmid according to the invention comprises or consists of (i) a nucleic acid sequence according to SEQ ID NO: 2 or 4 or a sequence having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 or4 and (ii) a backbone sequence comprising or consisting of a bacterial replication origin capable of propagating the plasmid in a bacterial host cell and a selectable marker gene as well as any needed regulatory elements.

[0124] It was shown in the present invention that plasmid shorter than 15Kbp (such as between 10 kbp and 13kbp) enable increase of successful transfection and rAAV production titres. Therefore, in the context of the invention as a whole, such a plasmid (e.g. a helper plasmid) has preferably a total size below 15kbp. More preferably, the plasmid has a size of between 10000 and 13000 nucleotides, or even more preferably between 10500 and 12800 nucleotides (including both adenoviral sequences such as those of the recombinant polynucleotide and backbone sequences). As a non-limiting example, herein exemplified is pHP925 having a size of 12541 bp and leading to rAAV yield well above 1e11. The benefit of having a shorter plasmid is also from a toxicity viewpoint. An additional benefit is a commercial benefit because having a shorter plasmid lowers the cost of good (as typically, especially when production is externalised, one pays per gram and when one produces itself, the cells will make more copies of it).

[0125] Adenoviruses

[0126] Another aspect of the invention describes an engineered adenovirus comprising the recombinant polynucleotide of the invention (such as the engineered recombinant polynucleotide of the invention). In particular, such recombinant adenovirus is considered as a ’’helper virus” or alternatively a “helper vector” as described hereinbefore, and provides the machinery required by the rAAV but not comprised therein fortranscription, translation and replication. Helper adenoviruses and their uses are known in the art. The provision of a helper virus with an rAAV enables the safe production of rAAV particles comprising a polynucleotide to be delivered into a host cell in a controlled system. However, the resulting rAAV do not comprise the machinery required fortranscription, translation and replication of the rAAV and thus are safe vectors for the delivery of e.g. therapeutic transgenes for genetic therapies. Such rAAV particles are termed replication deficient.

[0127] As described hereinbefore, a helper adenovirus comprising the recombinant polynucleotide of the invention (such as the engineered recombinant polynucleotide of the invention), by virtue of said sequence, provides for safer manufacturing due to the removal of unnecessary sequences, increases rAAV production titres, and boosts rAAV product quality by reducing unwanted packaging of plasmid backbone and host cell DNA.

[0128] Dogaybone™ DNA

[0129] In an embodiment of the invention there is provided a doggybone DNA molecule which comprises the recombinant polynucleotide of the invention (such as the engineered recombinant polynucleotide of the invention). In an embodiment of the invention there is provided a linear double stranded, covalently closed DNA construct which comprises the recombinant polynucleotide of the invention.

[0130] Doggybone™ DNA (dbDNA™) is a synthetic DNA vector comprising sequences of interest (e.g. the recombinant polynucleotide of the invention) which is used in an enzymatic manufacturing process to synthesise DNA at high scale and purity. The dbDNA™ is a linear double stranded, covalently closed DNA construct which can encode long and complex sequences and allows in vitro enzymatic synthesis by rolling circle amplification. Long, linear double stranded concatemers are produced and then cleaved at specified sites with a cleavage-joining reaction to covalently close the dbDNA™. The dbDNA™ can then be purified using standard techniques such as column chromatography, filtration and restriction enzymatic digestion of sequences other than those of interest. Thus, in one embodiment there is provided a linear double stranded, covalently closed DNA construct comprising the recombinant polynucleotide of the invention.

[0131] Transient transfection plasmid system

[0132] In another aspect of the invention, herein provided is a plasmid system, such as a transient transfection plasmid system, comprising or consisting of: a) a cis-plasmid comprising a heterologous nucleic acid and 5’ and 3’AAV-ITRs, b) an AAV replication and capsid gene (rep / cap) comprised in one or more trans-plasmid(s), and c) a helper plasmid comprising a recombinant polynucleotide according to the invention.

[0133] In non-limiting examples, the plasmid system, such as a transient transfection plasmid system, comprising: a) a cis-plasmid comprising a heterologous nucleic acid sequence and 5’ and 3’AAV-ITRs, b) an AAV replication and capsid gene (rep / cap) comprised in one or more trans-plasmid(s), and c) a helper plasmid comprising or consisting of a recombinant polynucleotide selected from the group consisting of (i) SEQ ID NO: 2 or 4, and (ii) a sequence having at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 2 or 4.

[0134] Typically, the cis-plasmid comprises a backbone and an expression cassette comprising 5’ ITR, a promoter, the heterologous nucleic acid to be expressed, a polyA signal and a 3’ ITR The heterologous nucleic acid can encode a peptide or a protein. Alternatively, it can express an RNA such as a siRNA, a miRNA, an shRNA, etc.

[0135] Typically, the trans-plasmid comprises a backbone together with the AAV rep gene and AAV cap gene under the control of one or more promoters. The most commonly used promoter is the natural promoter p5. The AAV Rep gene sequence and the AAV Cap gene can be of the same origin (i.e. same serotype) or from different origins (i.e. different serotypes). Non-limiting examples of AAV serotypes, including any modification, derivative or pseudotype thereof, are AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or AAV-TT. Methods and processes

[0136] As will be readily appreciated, the recombinant polynucleotide of the invention finds utility in the production of recombinant adeno-associated virus (rAAV) vectors (useful in the field of gene therapy), such as when such method / process of production comprises a helper plasmid and / or helper adenovirus. Thus, in one aspect there is provided a method or a process of production of recombinant adeno associated virus (rAAV) vectors, the method or the process comprising the steps of: (a) transfection of cells with i) the recombinant polynucleotide of the invention (which can be used for instance as a helper sequence or as a helper plasmid), ii) a plasmid comprising AAV-ITRs and the target transfer gene (i.e. the cis-plasmid) and iii) one or more plasmid(s) comprising AAV capsid and non-structural replication genes (i.e. the trans plasmid(s)), (b) allowing cells sufficient time to produce rAAV vectors, and (c) producing clarified lysate comprising rAAV vectors. The method or the process of the invention can further comprise the steps of (d) further purifying the rAAV vectors and / or I further formulating the rAAV vectors. Typically, a transfection reagent is used for step b). Such transfection reagents are well known from the skilled person. Well-known protocols can be used for the different steps.

[0137] In another aspect of the invention, herein provided is a method or a process for the production of recombinant adeno-associated virus (rAAV) vectors comprising the steps of: a) providing a cell culture comprising mammalian cells (such as HEK293 cells or derivatives thereof), wherein the cells are in suspension or adherent, b) transfecting the cells with the plasmid system (such as the transient transfection plasmid system) according to the invention, c) cultivating the cells under conditions so that they produce rAAV vectors, d) lysing the cells and harvesting the rAAV vectors, e) purifying the rAAV vectors, and f) optionally formulating the rAAV vectors to obtain a pharmaceutical composition comprising the rAAV vectors and acceptable carriers and / or excipients.

[0138] Typically, a transfection reagent is used for step b). Such transfection reagents are well known from the skilled person. Well-known protocols can be used for the different steps.

[0139] The invention further provides an rAAV vector produced by a process or a method of the invention. Any recombinant adeno-associated virus (rAAV) vector can be produced according to any one of the methods according to the invention. Such rAAV vector may be of any AAV serotype, including any modification, derivative or pseudotype (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or derivatives / modifications / pseudotypes thereof such as AAV-TT). Such AAV serotypes and derivatives / modifications / pseudotypes, and methods of producing such serotypes / derivatives / modifications / pseudotypes are known in the art (see, e.g., Asokan et al., Mol. Ther. 20(4):699-708 (2012).

[0140] Uses

[0141] As will be readily appreciated, the recombinant polynucleotides of the claimed invention can be used as helper plasmids, as their reduced size can be an advantage when it comes to rAAV vectors manufacturing process. Further, the recombinant polynucleotide of the claimed invention can be used to improve the yield and quality of the rAAV vectors produced through one of the methods or processes of production herein described.

[0142] The rAAV vectors obtained through one of the methods or processes of production herein described, can be used as a medicament, for instance as a gene therapy-based medicament.

[0143] The present invention will now be further described by means of the following non-limiting examples. The application of which this description and claims form part forms an invention as a whole and may be used as a basis for any combination of features described herein.

[0144] All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

[0145] Information Related To Sequences

[0146] SEQ ID NO: 1 - Nucleic acid sequence of the native Human Ad 5 genome, accession AC_000008.1

[0147] SEQ ID NO: 2 - Nucleic acid sequence of the plasmid pHP925

[0148] SEQ ID NO: 3 - Nucleic acid sequence of the backbone for plasmid pHP925

[0149] SEQ ID NO: 4 - Nucleic acid sequence of the adenovirus part of plasmid pHP925

[0150] SEQ ID NO: 5 - Nucleic acid sequence of the plasmid PHP1138

[0151] SEQ ID NO: 6 - Nucleic acid sequence of VA RNA fragment (332bp)

[0152] SEQ ID NO: 7 - Nucleic acid sequence of VA RNA minimal fragment (414bp)

[0153] - Nucleic acid sequence of VA RNA I full length (157bp)

[0154] SEQ ID NO: 9 - Nucleic acid sequence of VA RNA II full length (158bp)

[0155] SEQ ID NO: 10 - Nucleic acid sequence of VA RNA II partial (76 bp)

[0156] SEQ ID NO: 11 - Nucleic acid sequence of VA RNA related TATA box (6 bp; TAATAT)

[0157] SEQ ID NO: 12 - Nucleic acid sequence of E2B minimal fragment (704 bp)

[0158] SEQ ID NO: 13 - Nucleic acid sequence of E2A fragment in pHP922, 923 and 926 (5094bp)

[0159] SEQ ID NO: 14 - Nucleic acid sequence of complete E2A region as is in pHP925 (5415bp)

[0160] SEQ ID NO: 15 - Nucleic acid sequence of DBP gene (1590bp)

[0161] SEQ ID NO: 16 - Nucleic acid sequence of pVIII full size (684bp)

[0162] SEQ ID NO: 17 - Nucleic acid sequence of pVIII minimal fragment (363bp)

[0163] SEQ ID NO: 18 - Nucleic acid sequence of complete E4 region (2922bp)

[0164] SEQ ID NO: 19 - Nucleic acid sequence of E4 minimal fragment comprising E4ORF6 / 7 (1164bp)

[0165] SEQ ID NO: 20- Nucleic acid sequence of pllla full size (1758bp)

[0166] SEQ ID NO: 21 - Nucleic acid sequence of HEXON full size (2859bp)

[0167] SEQ ID NO: 22 - Nucleic acid sequence of U Exon full size (168bp)

[0168] SEQ ID NO: 23 - Nucleic acid sequence of Fiber full size (1746bp)

[0169] SEQ ID NO: 24 - Nucleic acid sequence of Fiber fragment (37bp)

[0170] SEQ ID NO: 25 - Nucleic acid sequence of Fiber fragment (103bp)

[0171] SEQ ID NO: 26 - Nucleic acid sequence of complete 23K endopeptidase (615bp) SEQ ID NO: 27 - Nucleic acid sequence of complete 52K endopeptidase (1248bp)

[0172] SEQ ID NO: 28 - Nucleic acid sequence of 52K endopeptidase fragment (524bp)

[0173] SEQ ID NO: 29 - Nucleic acid sequence of complete 12.5 K endopeptidase fragment (324bp)

[0174] SEQ ID NO: 30 - Nucleic acid sequence of E4 ORF1-4 fragment (1448bp)

[0175] SEQ ID NO: 31 - Nucleic acid sequence of 3’ITR Ad5 (103bp)

[0176] SEQ ID NO: 32 - Nucleic acid sequence of 5’ITR Ad5 (103bp)

[0177] SEQ ID NO: 33 - Nucleic acid sequence of E4 promoter region (308bp)

[0178] SEQ ID NO: 34 - Nucleic acid sequence of high-copy-number ColE1 / pMB1 / pBR322 / pUC origin of replication (589bp)

[0179] SEQ ID NO: 35 - Nucleic acid sequence of kanamycin resistance (81 Obp)

[0180] SEQ ID NO: 36 - Nucleic acid sequence of E2B region comprising Pol, pTP genes, 52K and pl I la gene (8923bp)

[0181] SEQ ID NO: 37 - Nucleic acid sequence of pTP gene (5537bp)

[0182] SEQ ID NO: 38 - Nucleic acid sequence of the VA Fragment of pHP925 (1732bp) SEQ ID NO: 39 - Nucleic acid sequence of the E4 fragment of pHP925 (2922bp)

[0183] Summary of the figures

[0184] Figure 1 : Diagrammatic overview of the native Adenovirus type 5 genome.

[0185] Figure 2: Screening of rAAV titres (A) and relative ddPCR / ELISA ratios for AAVTT- Protein 1 (B). Figure 3: Alkaline gels showing quantify, purity and size of packaged genome after recovery. Figure 4: (A&B) Potency assay for AAVTT-Product 1.

[0186] Figure 5: Nucleic acid sequence of plasmid pH925.

[0187] EXAMPLES

[0188] Material and Methods rAAV vectors production method: rAAV vectors were produced by transfecting HEK293 cells in suspension with three plasmids (respectively for 1) the heterologous nucleic acid (e.g. the transgene), 2) rep / cap, and 3) helper sequences). Post transfection working volume was fixed at 30mL. The HEK293 cells were seeded at a density of 1.5 x 106cells / mL in shake flasks and grown for 24-hours at 37°C, 5% CO2 in a culture medium specific for AAV production (ThermoFisher). Just prior to transfection, the three plasmids required for viral production were complexed with a transfection reagent in the culture medium following the instruction of the supplier (so that to obtain a transfection reagent-DNA complex mixture). After about 24-hours (when cells reached a density of about 3 x 106cells / mL), transfection took place, by addition of the transfection reagent-DNA complex mixture directly to the flasks. Cell culture was continued for about 72 hours under the same culture conditions to allow for production of the rAAV vectors. After this time cells were lysed using a chemical lysis buffer and removed and viral vectors were quantified. For higher scale bioreactors (e.g. 3L bioreactors), similar steps and process were used. Genome titre by ddPCR method:

[0189] The quantification of packaged viral genome containing the transgene (within the rAAV produced according to the above method) was conducted by droplet digital polymerase chain reaction (ddPCR, equipment from Bio-Rad). To quantify the packaged viral genome, unpackaged DNAs were degraded by Dnase digestion. rAAV were then broken down by heat treatment (at 95°C) to release genomes. The released genomes were quantified by serial dilutions to a range that was readable within the metrics set by the ddPCR equipment. Transgene specific primers (i.e. forward and reverse) and FAM- labelled probe were mixed with Bio-Rad PCR master mixture and appropriate dilutions of Dnase- digested test samples. These Dnase-digested and appropriately diluted samples, along with a negative control containing ddPCR diluent buffer (NTC), and a positive control rAAV vector were plated in duplicate. Following droplet generation using Bio-Rad automated droplet generator, the samples and controls underwent polymerase chain reaction in a thermo cycler, and amplified droplets were read on Bio-Rad droplet reader. Samples were distributed between positive and negative droplets and calculated using the Poisson Distribution to quantify vector genome copies per millilitre of sample (VG / mL).

[0190] Capsid titre by ELISA:

[0191] Quantitation of intact rAAV capsids was conducted using Progen sandwich ELISA kit. rAAV vectors containing intact AAV capsids were diluted such that capsid concentration was estimated to be within range of quantitation for the assay (note that the range of quantitation for the assay varies by kit lots). Dilution series of the Kit Control (AAV standard), sample(s), Positive Control (PC), and Blank (1x ASSB buffer) were added to a microtitre plate pre-coated with a monoclonal antibody specific to intact AAV capsid, as per supplier’s instructions. Capsids present in the samples were captured onto the plate. Bound capsids were then detected by the same monoclonal antibody conjugated with biotin, followed by a streptavidin peroxidase conjugate. TMB substrate was added and enzymatically turned over into a coloured product by peroxidase-containing immune complexes; colour development was stopped with the addition of acid. Colorimetric signal of the microtitre plate containing diluted samples and AAV standard was measured on a spectrophotometer microplate reader at 450 nm with a reference wavelength of 650 nm. The absorbance signal obtained is proportional to the number of intact capsids present in the sample, therefore, a standard curve was created by plotting absorbance against known titre for the Kit Control and fitting with a 4-parameter logistic (4-PL) curve. Sample absorbances were interpolated off the 4-PL standard curve to determine rAAV capsid titre (capsids / mL).

[0192] Alkaline pel:

[0193] 2.50E+10 vg of AAV was incubated at 95°C for 5min and placed on ice immediately after the heat treatment. The denatured AAV was then loaded in prechilled 0.8% alkaline agarose gel. Electrophoresis occurred in the cold room for 17-19 hours. The agarose gel was then neutralized in 0.1 M Tris-HCI pH8.5 followed by staining with SYBR™ Gold nucleic acid gel stain. The gel image was captured with GelDoc Go Imaging system.

[0194] Cell based potency assay:

[0195] Permissive cells were seeded in a 96-well plate and incubated for 24 hours. On day 2, cells were transduced in triplicate with five 2-fold serially diluted AAV to reach MOI starting at 2.00E+5 to 1 .25E+4 vg / cells. 72 hours after transduction total cell RNA was extracted using KingFisher Apex system and then treated with DNase. DNAse-treated RNA was used for duplex RT-ddPCR reaction targeting CNS Proteinl and TATA-box binding protein (TBP). Droplet generation, PCR amplification and signal detection were performed using Bio-Rad QX200 system. CNS Proteinl expression is normalized by TBP. Relative potency is determined using JMP software.

[0196] Example 1 : Preparation of vectors

[0197] Cloning of the different recombinant polynucleotides / helper plasmids

[0198] The cloning of our basal working plasmid was accomplished using standard techniques of molecular biology. In short five fragments were synthetised based on the sequences of wildtype adenovirus 5 from GenBank, using Genscript service. These fragments encompassed VA-RNA, the E2A region, E4ORF, and other pertinent sequences. The Gibson assembly method was then utilized to seamlessly integrate these fragments onto a pUC-57 vector, which carries a kanamycin resistance gene. For the cloning of additional helper plasmids, individual fragments were extracted from this basal working plasmid using the Gibson assembly method, in accordance with varying designs.

[0199] It is noted that despite the teaching from the art that DBP protein was not required in the AAV helper function, some constructs were made with sequences coding DBP protein (Barrie et al., 1992).

[0200] Example 2 - new helper plasmid for expressing an AAV-TT based product

[0201] The efficiency of various helper plasmids has been assessed in a method for producing an AAV-TT based product. The transgene to be expressed was encoding Protein 1 (a CNS target). The resulting rAAV vector was called AAVTT-Protein 1 .

[0202] A helper plasmid designed according to the invention (pHP925) has been assessed, together with 1 commercially available plasmid (control, pHP052) and another in-house plasmid (pHP1138 lacking full E4 region).

[0203] HEK293 cells were cultivated in 30 mL shake flasks as described in the Material & Methods section to produce AAVTT-Protein 1 .

[0204] The y-axis in Figure 2A is viral genome copies / mL measured by ddPCR and the y-axis of Figure 2B is a relative representation of %full, as measured by ddPCR to ELISA ratio, with respect to the control plasmid (pHP052). As shown in Figure 2, helper plasmid pHP925 was promising, with yield of AAVTT- Protein 1 above the one obtained with the control helper plasmid. In comparison pHP1138 helper plasmid is not efficient at all, with vg titres well below 1 e11 vg / mL. pHP925 provided the highest quality of rAAV vectors. Example 3 - Manufacturing at bioreactor scale

[0205] The helper plasmid pHP925 was scaled up in a 3L bioreactor to evaluate its manufacturability by producing AAVTT-Protein 1. A titre of 4.12E+11 vg / mL was obtained at scale, showing good manufacturability of the results obtained at small scale.

[0206] Example 4 - Process recovery

[0207] Further to the very good results obtained from the previous examples, it was important to assess the percentage of process recovery from clarification step to final fill (including affinity chromatography step, polishing step, and concentration steps). For that purpose, the batch of AAVTT-Protein 1 , produced with helper plasmid pHP925 in example 3, was assessed.

[0208] Starting from samples having an average titre of 4.12x1011vg / mL, the overall recovery was at about 41% with a standard purification process, not optimised (the step during which most loss happened being the polishing step). Alkaline gels were run to quantify purity and size of the packaged genome in the final formulated purified rAAV vector. Major band was observed at the intended size with no other impurities (Figure 3).

[0209] Example 5 - Potency assay

[0210] Relative potency was tested against a reference AAV vector produced with commercially available helper. Relative potency of AAVTT-Protein 1 produced at 3L scale using pHP925 was 79% compared to the same AAVTT-Protein 1 vector produced with a commercially available helper (“reference vector”). (See Figures 4A and 4B). These results demonstrate potent vector is produced at scale even without an optimized process.

[0211] References

[0212] 1) Gao Z, Herrera-Carrillo E, Berkhout B. RNA Polymerase II Activity of Type 3 Pol III Promoters.

[0213] Mol Ther Nucleic Acids. 2018 Sep 7; 12: 135-145

[0214] 2) Asokan et al., 2012, Mol. Ther. 20(4):699-708.

[0215] 3) Asubel et al., 2003, Current Protocols in Molecular Biology. Ed. John Wily & Sons.

[0216] 4) Saha and Parks, 2017, PLOSone, 12(7): e0181012

[0217] 5) Barrie et al., 1992, Virology, 191 :473-476

Claims

Claims1 . A recombinant polynucleotide comprising adenoviral genome sequences comprising or consisting of:(a) a VA Fragment comprising or consisting of at least an E2B region comprising the VA RNA promoter region and at least a portion of the VA RNA,(b) a E2A Fragment comprising or consisting of at least the E2A region encoding DBP protein and a fragment of the gene encoding pVIII protein, and(c) at least a portion of E4 region comprising or consisting of the E4 ORF6 / 7 region, and wherein the recombinant polynucleotide does not comprise any adenoviral genome sequences or fragments thereof encoding any one of:(1 ) pl Ila protein,(2) HEXON protein,(3) U exon, and(4) Fiber protein.

2. The recombinant polynucleotide according to claim 1 wherein the recombinant polynucleotide comprises:(a) a VA Fragment comprising or consisting of (i) between 650 and 750 contiguous nucleotides of the E2B region comprising the VA RNA promoter region, (ii) the full VA RNA I sequence and (iii) a fragment of between 50 to 100 XX contiguous nucleotides of VA RNA II;(b) a E2A Fragment comprising or consisting of at least 1590 contiguous nucleotides of the E2A region encoding DBP protein and a fragment of 300 to 500 contiguous nucleotides of the gene encoding pVIII protein, and(c) at least 1000 contiguous nucleotides of the E4 ORF6 / 7 region.

3. The recombinant polynucleotide according to any one of claims 1 to 2, wherein the at least a portion of the E2B region comprises or consists of SEQ ID NO: 12, or a sequence with at least 90% identity thereof.

4. The recombinant polynucleotide according to any one of claims 1 to 3 wherein the at least a portion ofVA RNA comprises or consists of SEQ ID NO: 7 ora sequence with at least 90% identity thereof.

5. The recombinant polynucleotide according to any one of claims 1 to 4 wherein the E2A region comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 13 or 14, or a sequence with at least 90% identity thereof.

6. The recombinant polynucleotide according to any one of claims 1 to 5 wherein the fragment of the gene encoding pVIII comprises or consists of SEQ ID NO: 16 or SEQ ID NO: 17, or a sequence with at least 90% identity thereof.

7. The recombinant polynucleotide according to any one of claims 1 to 6 wherein the plasmid comprises a region of E4 which consists solely of E4 ORF6 / 7.

8. The recombinant polynucleotide according to claim 7 wherein the region of E4 comprises or consists of SEQ ID NO: 19, or a sequence with at least 90% identity thereof.

9. The recombinant polynucleotide according to any one of claims 1 to 6 wherein the plasmid comprises a region of E4 which further comprises E4 ORF4, E4 ORF3, E4 ORFB and / or E4 ORF1 in their native sequences or any active portion thereof.

10. The recombinant polynucleotide according to claim 9 wherein the region of E4 comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 18 or 19, or a sequence with at least 75% identity thereof.11 . The recombinant polynucleotide according to any one of claims 1 to 11 wherein the recombinant polynucleotide further does not comprise an adenoviral genome sequence encoding (i) 23K protease or any fragment thereof and / or (ii) E3 14.7K or any fragment thereof.

12. The recombinant polynucleotide according to any one of claims 1 to 11 wherein the recombinant polynucleotide further comprises an adenoviral genome sequence encoding at least a portion of 52K protein, wherein the at least a portion of 52K protein comprises or consists of SEQ ID NO: 27 or SEQ ID NO: 28, or a sequence with at least 90% identity thereof.

13. The recombinant polynucleotide according to any one of claims 1 to 12, wherein the recombinant polynucleotide does not comprise adenoviral Fiber sequence.

14. The recombinant polynucleotide according to any one of claims 1 to 13 wherein the recombinant polynucleotide does not comprise a 3’ ITR.

15. The recombinant polynucleotide according to any one of claims 1 to 14 wherein the recombinant polynucleotide comprises or consists of the sequence of SEQ I D NO: 2 or 4, or a sequence having at least 90% identity to SEQ ID NO: 2 or 4.

16. A plasmid comprising or consist of: (i) the recombinant polynucleotide of any one of claims 1 to 15 or (ii) the recombinant polynucleotide of any one of claims 1 to 15 and a backbone sequence comprising or consisting of a bacterial replication origin capable of propagating the plasmid in a bacterial host cell and a selectable marker gene as well as any needed regulatory elements, and, wherein the plasmid is a helper plasmid.

17. A linear double stranded, covalently closed DNA construct comprising or consist of: (i) the recombinant polynucleotide of any one of claims 1 to 15 or (ii) the recombinant polynucleotide of any one of claims 1 to 15 and a backbone sequence comprising or consisting of a bacterial replication origin capable of propagating the plasmid in a bacterial host cell and a selectable marker gene as well as any needed regulatory elements.

18. The recombinant polynucleotide of any one of claims 1 to 15 wherein the recombinant polynucleotide is isolated.

19. A plasmid system comprising: a) a cis-plasmid comprising a heterologous nucleic acid and 5’ and 3’AAV-ITRs, b) an AAV replication and capsid gene (rep / cap) comprised in one or more trans-plasmid(s), and c) (i) a helper plasmid comprising a recombinant polynucleotide according to any one of claims1 to 15 or (ii) the helper plasmid according to claim 16 or (iii) the linear double stranded, covalently closed DNA construct according to claim 17.

0. A process for the production of recombinant adeno-associated virus (rAAV) vectors comprising the steps of: a) providing a cell culture comprising HEK293 cells or derivatives thereof, wherein the cells are in suspension or adherent, b) transfecting the cells with the plasmid system according to claim 19, c) cultivating the cells under conditions so that they produce rAAV vectors, d) lysing the cells and harvesting the rAAV vectors, e) purifying the rAAV vectors, and f) optionally formulating the rAAV vectors to obtain a pharmaceutical composition comprising the rAAV vectors and acceptable carriers and / or excipients.

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