Adenovirus vector circular RNA expression system

Abstract

The present invention relates to an adenovirus genome comprising a heterologous expression cassette encoding a circular RNA and uses thereof.

Description

[0001] This invention relates to an adenoviral genome containing a heterologous expression cassette encoding circular RNA. Background Technology

[0002] Adenoviruses (AdVs) are non-enveloped, double-stranded DNA vectors. AdVs can infect a wide range of cell types, and unlike retroviruses, these vectors are non-integrating. Therefore, AdVs are attractive vectors for gene transfer, applied in vaccination, cancer treatment, and gene therapy. Adenoviruses possess many characteristics that make them attractive vectors for transgene delivery: adenovirus biology has been extensively studied; the adenovirus genome is relatively easy to manipulate, and they are capable of carrying relatively large amounts of exogenous DNA. They have the ability to infect both dividing and non-dividing cells, thus facilitating their use for a variety of purposes. Furthermore, AdVs can induce humoral and cellular immune responses, which is beneficial for some aspects of vaccine development and immuno-oncology therapy.

[0003] Most AdVs are genetically modified forms of human mammalian adenoviruses, particularly adenovirus serotype 5. Different forms of AdV exist, but based on their replication capacity, they are generally classified into two categories: conditionally replicating (CR) and replication-deficient (RD). Conditionally replicating adenoviruses (crAdVs) have been used, for example, in cancer therapy to specifically target and replicate within cancer cells; they are also known as oncolytic adenoviruses. The lytic nature of adenovirus replication directly kills infected cells, releasing relevant antigens recognized by the host's immune system. Furthermore, after lysis, progeny viruses can subsequently spread throughout the affected tissue, such as a tumor, further infecting and destroying other affected cells.

[0004] The construction of crAdV is based on the deletion or modification of viral gene functions that are essential for viral replication in normal cells but unnecessary in the targeted cells. This includes insertion mutations in the E1A region or deletion of E1B from the wild-type genome to target retinoblastoma (Rb) and p53 pathway-deficient cancer cells, respectively, as these pathways are defective in most human tumors. Another strategy for targeting crAdV replication in cancer cells involves controlling E1 transcription by introducing tumor- or tissue-specific promoters, such as prostate-specific enhancers / promoters for prostate cancer or E2F-I for cancer cells with defective Rb pathways. Furthermore, many crAdVs contain partial or complete deletions of the E3 region. The E3 region encodes several genes involved in preventing infected cells from being eliminated by the immune system. Deletion experiments have determined that this region is not essential for Ad replication in cell culture or in vivo. Therefore, removing these genes does not interfere with the replication capacity of the adenovirus and increases the transgene insertion capacity of the adenoviral vector.

[0005] Several regions within the adenovirus genome where desired transgenes can be inserted have been identified. Depending on the insertion site, the transgene can be transcribed under the control of an adenovirus promoter. Alternatively, transgene expression cassettes can be incorporated into adenovirus vectors and controlled by exogenous promoters such as highly active CMV promoters or tissue-specific promoters. Typically, in advs with partial / full E3 deletions, the transgene expression cassette is incorporated into the site of the E3-deleted gene / region. Numerous studies have demonstrated that transgene cassettes inserted under the control of adenovirus E3 promoters or exogenous promoters produce high-yield transgene expression and functional viruses.

[0006] While AdVs have proven to be powerful vectors for transgene delivery, a limitation of using AdVs for transgene delivery is that transgene expression is coupled to the presence of the virus. These vectors can be cleared from the host relatively rapidly and efficiently due to the presence of pre-existing neutralizing antibodies and the induction of a strong antiviral innate immune response after treatment with AdVs, thus limiting the duration of transgene expression. Therefore, to improve the duration of transgene expression from AdVs without further interfering with the adenoviral genome, this invention provides AdVs that, in contrast to conventional linear mRNA transcripts, are capable of encoding circular RNA transcripts to deliver transgene payloads.

[0007] Circular RNAs (circRNAs) constitute a recently recognized class of RNAs. In contrast to conventional linear splicing, circRNAs are characterized by covalently closed molecules typically generated via a nonlinear "backsplicing" event using a downstream splice donor (SD) and an upstream splice acceptor (SA). Endogenous circular RNAs have been extensively studied over the past decade, and despite some controversies regarding their functional relevance, circRNAs are widely recognized as resistant to exonuclease lysis and decay due to their circular nature, thus constituting a highly stable class of RNAs with half-lives significantly exceeding those of conventional linear mRNAs. Furthermore, knowledge of in vitro and in vivo circRNA production has significantly improved, leading to the recent emergence of therapeutic potential for engineered, persistent circular RNAs. Broadly speaking, circRNA production can be achieved through two distinct mechanisms: 1) circylation using type I intron-derived ribozymes, which has shown effectiveness for in vitro production, or 2) spliceosome-based backsplicing, similar to the biogenesis of endogenous circRNAs, which can be used for in vivo production. In the latter setting, it is known that inserting flanking inverse elements significantly stimulates backsplicing by positioning the splicing sites involved in very close proximity. Although circRNAs lack a 5' cap and a 3' poly-A tail and are therefore not substrates for translation themselves, insertion at the internal ribosome entry site (IRES) efficiently converts non-coding circRNAs into highly efficient and persistent protein-coding molecules.

[0008] Circular RNAs (circRNAs) are attractive payloads for introduction into adenoviruses due to their inherent stability and their ability to be modified to express proteins. Engineering circRNAs to express therapeutic proteins, as well as expressing them from AdvV, has the potential to further enhance adenovirus-associated antitumor responses and improve their therapeutic potential in cancer treatment and other therapeutic applications. Furthermore, several immunotherapeutic agents have shown toxicity upon systemic delivery. In these cases, the use of conditionally oncolytic adenoviruses that specifically replicate in the tumor microenvironment limits the expression of the therapeutic proteins in question with high efficacy and improved safety.

[0009] However, surprisingly, expression cassettes encoding circular RNA cannot be inserted in the same way as expression cassettes encoding, for example, linear mRNA. The structural uniqueness of the circular RNA coding sequence and other elements, such as those required for circRNA formation and translation, necessitates insertion into specific regions of the adenovirus to produce viable viruses. This invention teaches integration sites that can be used for circular RNA coding constructs. Furthermore, the inventors have unexpectedly identified integration sites that lead to particularly high levels of circRNA expression. Summary of the Invention

[0010] In a first aspect, the present invention provides an adenovirus genome encoding an adenovirus, the adenovirus genome comprising at least one heterologous expression cassette, the at least one heterologous expression cassette comprising or consisting of a nucleic acid sequence encoding a circular RNA (circRNA), wherein the heterologous expression cassette is inserted outside a functional transcription unit of the adenovirus genome, the functional transcription unit having the same reading orientation as the inserted heterologous expression cassette. Attached Figure Description

[0011] The contents of the accompanying drawings included in this specification are described below. In this context, please also refer to the specific embodiments described above and / or below.

[0012] Figure 1: Insertion of the missing gene into the region of reference Example 1. A) Elements of the circRNA cassette, promoter, inverted repeat (IR), and splice acceptor (SA) are depicted. The open reading frame (ORF) used is a split ORF flanking the CVB3 IRES element. B) The mRNA expression cassette used as a control is depicted. It differs from the circRNA cassette in that it lacks the inverted repeat element and IRES sequence. The mRNA ORF is arranged as a continuous ORF, while the circRNA is arranged as a split ORF. C) A schematic diagram of the Ad5 genome used is depicted, indicating the major transcriptional units of the Ad5 genome and their reading directions. The elements at both ends of the genome are ITRs. V1 indicates the insertion site and orientation of the inserted circRNA or mRNA expression cassette.

[0013] Figure 2: Insertion between transcription units refers to the experiment disclosed in Example 2. A) A schematic diagram of the Ad5 genome is depicted, indicating transcription units E1A, E1B, E2B, L1-5, E3A / B, and E4. Additionally, the ITRs are shown at both ends of the adenovirus genome. The insertion sites of the expression cassettes (V1 to V9) are shown below and indicated by drawn boxes. Arrows indicating the orientation of the elements are shown for both transcription units and expression cassettes. B) and C) depict immunoblotting analysis of test constructs producing live viruses (i.e., V2, V3, V4, V5, V8, and V9), showing the detection of the encoded circRNA at 24 hours (B) and 48 hours (C) post-infection of host cells (A549 cells) using equal numbers of viral particles.

[0014] Figure 3: Schematic diagram of the adenovirus 5 genome. Major transcription units and their orientations are indicated by broad arrows. Gene names appear above each transcription unit. In the adenovirus genome used in this example, the italicized gene names in the E3 region (i.e., 6.7k, gp19k, ADP, RID⍺ / β, and 14.7k) have been deleted. The smaller arrows below the schematic diagram (i.e., V1 to V9) indicate the insertion sites and orientations of heterologous expression cassettes. Solid arrows (i.e., V2, V3, V4, V5, V8, and V9) depict insertion sites where live virus was recovered, while hollow arrows (i.e., V1, V6, and V7) depict insertion sites where live virus was not recovered.

[0015] Figure 4: Alignment of human adenoviruses. Alignment of mammalian adenoviridae genomes showing the indicated transcription units / genes is depicted. Available adenovirus genomes for the family have been grouped by species, and only one genome is shown for each species. The general genome organization throughout the family is highly conserved. The relative positions of transcription units to each other are identical throughout the family. Therefore, the insertion sites identified in Ad5 (the adenovirus genome used is depicted as ONCOS-d2.7kb) are conserved within this adenoviridae family, and insertions at the same relative positions will also result in insertions outside the transcription units. The insertion sites disclosed in the Examples section are represented in the ONCOS-d2.7kb genome as black triangles ▲ (V5), addition operators + (V6 / V7), and hollow rectangles. (V1 / V2), cross-shaped box (V3 / V4) or solid box (V8 / V9). Detailed Implementation

[0016] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these can be modified. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be defined only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0017] Preferably, the terms used herein are as defined in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, edited by Leuenberger, HGW, Nagel, B. and Klbl, H. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0018] Throughout this specification and the claims thereafter, unless the context otherwise requires, the word “comprise” and its variations such as “comprises” and “comprising” shall be understood to imply inclusion of the stated whole or step or group of wholes or steps, but not to exclude any other whole or step or group of wholes or steps. Different aspects of the invention are defined in more detail in the following paragraphs. Each aspect so defined may be combined with any other aspect or group of aspects unless explicitly indicated otherwise. In particular, any feature indicated as optional, preferred, or advantageous may be combined with any other feature or group of features indicated as optional, preferred, or advantageous.

[0019] Numerous references are cited throughout this specification. Every reference cited herein, whether above or below (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to any prior disclosure. Some documents cited herein are marked as " By incorporating references In the event of a conflict between the definitions or teachings of such incorporated references and those listed in this specification, the text of this specification shall prevail.

[0020] The elements of the invention will be described below. These elements are listed using specific embodiments; however, it should be understood that these specific embodiments can be combined in any manner and in any number to produce other embodiments. The differently described embodiments and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This specification should be understood to support and cover embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, any arrangement and combination of the elements described herein should be considered.

[0021] definition The following provides definitions for some terms that are frequently used in this specification. These terms will have their respective defined and preferred meanings in the remainder of this specification in each instance of their use.

[0022] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural indicators unless the context clearly indicates otherwise.

[0023] When used in conjunction with numerical values, the term “about” is intended to cover values ​​within a range having a lower limit of 5% less than the indicated value and an upper limit of 5% greater than the indicated value.

[0024] As used herein, the term "adenovirus genome" refers to the genetic material of an adenovirus. Adenoviruses are double-stranded DNA viruses, and compared to many other viruses, their genomes are relatively large, containing approximately 30-40 kilobase pairs of DNA. The adenovirus genome contains several genes responsible for viral replication, assembly, and release. These genes encode proteins essential for the virus to complete its life cycle, such as polymerases that replicate viral DNA, capsid proteins that form a protective shell around the virus, and fibrin proteins that help the virus attach and enter host cells. The adenovirus genome also contains non-coding regions that regulate gene expression and replication, including promoters, enhancers, and origins of replication. These regulatory regions are important for controlling when and how adenovirus genes are expressed and for ensuring efficient viral replication.

[0025] In the adenovirus genome, genes can be broadly classified into early, mid, and late genes based on their expression time during the viral replication cycle. Early genes (E) are expressed early in the infection process and are primarily involved in regulating viral replication and evading the host's immune response. Mid-term genes are typically transcribed after early genes, while late genes (L) are expressed late in the infection process and primarily encode structural proteins that make up the viral capsid.

[0026] The adenovirus genome contains the following genes, which can be further subdivided into subgroups: early genes E1 to E4, mid-stage genes pIX and pIVa2, and late genes L1 to L5. These genes can be further subdivided into E1A, E1B, E2A, E2B, E3A / B, and E4. Late (L) genes are controlled by MLP promoter sequences and are spliced ​​to produce specific proteins. The E2 gene is thought to be regulated by multiple promoter / enhancer sequences. Different genes in the adenovirus genome can encode on different strands of the double-stranded genome, or even overlap if they encode on the same strand.

[0027] As used herein, the term "conditionally replicating adenovirus" (crAdV) refers to a type of adenovirus that is genetically modified to selectively replicate only under specific conditions, such as those present in a particular cell type or tissue (e.g., cancer cells). crAdV is engineered to have mutations or deletions in one or more genes essential for viral replication. However, these mutations or deletions are compensated for by the presence or absence of certain cytokines or genetic alterations specific to the cells in which crAdV can replicate (e.g., cancer cells). This allows crAdV to selectively replicate and lyse in specific cells (e.g., cancer cells) while excluding cells that do not provide these additional factors.

[0028] As used herein, the term "mammalian adenovirus" refers to a genus within the family Adenoviridae that infects a wide range of vertebrate hosts, including humans, other mammals, birds, and reptiles. Based on their genetic and antigenic characteristics, as well as their host range and tissue tropism, mammalian adenoviruses are further classified into seven species (A through G). Humans are known to be susceptible to several mammalian adenovirus species, including human mammalian adenoviruses A through G, which can cause a variety of clinical manifestations, such as respiratory illnesses, gastroenteritis, conjunctivitis, and hemorrhagic cystitis. In a preferred embodiment of the invention, the adenovirus genome is a mammalian adenovirus genome. In a preferred embodiment of the invention, the adenovirus genome is human mammalian adenovirus C, more preferably adenovirus serotype 5 (Ad5).

[0029] As used herein, the term "transcription unit" refers to a segment of DNA containing all the genetic elements necessary to transcribe one or more genes into RNA. Therefore, in the context of this invention, a transcription unit typically comprises or consists of a TATA box, one or more exons (coding genes), a polyadenylation signal, and optionally one or more introns. In some cases, the transcription unit is defined by elements other than the TATA box, but depends on other elements that initiate transcription. Preferably, the transcription unit comprises i) a TATA box or another promoter element, preferably having a sequence selected from CTTAAG or CCAAT, ii) one or more exons, iii) a polyadenylation signal, and iv) optionally one or more introns.

[0030] In a preferred embodiment, the transcription unit is defined by a TATA box and a PAS sequence.

[0031] Non-limiting examples of transcription units in an adenovirus genome are early genes E1 to E4, mid-stage genes pIX and pIVa2, and late genes L1 to L5. Table 2 provides a list of transcription units present in human adenovirus 5 used in the examples. In a preferred embodiment, the adenovirus genome comprises transcription units E1A, E1B, pIX, pIVa2, E2A, E2B, L1-L5, E3, and E4.

[0032] As used herein, the term "TATA box" refers to a DNA sequence that plays a crucial role in initiating the transcription of protein-coding genes in eukaryotes. It is typically located upstream of the transcription start site and is recognized by the transcription factor IID (TFIID) complex, a component of the RNA polymerase II pre-initiation complex. The TATA box is characterized by the presence of conserved nucleotide sequences. The common sequence of the TATA box comprises 5'-TAT(A / T)(A / T)-3' or 5'-TAT(A / T)(A / T)A-3'. Preferably, the sequence of the TATA box is selected from TATTTATA, TATATAA, TATATAAA, TATAA, and TATATA.

[0033] The term "polyadenylation signal" is used interchangeably with the terms "poly(A) signal" or "PAS" and, herein, refers to a DNA sequence located downstream of the coding sequence in a gene that emits a signal during transcription to add a poly(A) tail to the 3' end of the mRNA. The sequence of the polyadenylation signal is typically 5'-AATAAA-3' or 5'-ATTAAA-3'. In a preferred embodiment, the PAS has the sequence 5'-AATAAA-3'. For example, transcription units E1A, pIX, pIVa2, E3, L5, and E4 contain a PAS having the sequence 5'-AATAAA-3'.

[0034] As used herein, the terms “5’” and “3’” identify one end of a single-stranded nucleic acid molecule. The 5’ end is the end of the molecule that terminates at the 5’ phosphate group, and the 5’ direction is the direction toward the 5’ end. Similarly, the 3’ end is the end of the molecule that terminates at the 3’ hydroxyl group, and the 3’ direction is the direction toward the 3’ end. In the context of this invention, referring to the orientation of DNA synthesis during replication (from 5’ to 3’), RNA synthesis during transcription (from 5’ to 3’), and the reading of the mRNA sequence during translation (from 5’ to 3’), nucleic acid sequences are written with the 5’ end on the left and the 3’ end on the right (unless otherwise stated).

[0035] As used herein, the term "reading direction" refers to the direction of transcription. Transcription occurs in the 5' to 3' direction, meaning that RNA polymerase reads the DNA template strand from the 3' end to the 5' end and synthesizes complementary RNA molecules in the 5' to 3' direction. Therefore, the reading direction of the adenoviral genome of this invention refers to the 5' to 3' direction in which a specific gene is transcribed. This applies to both the forward and reverse strands of DNA, where in both cases, transcription occurs in the 5' to 3' direction with respect to the coding DNA strand.

[0036] As used in this article, the terms “upstream” and “downstream” refer to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence on a particular nucleic acid molecule.

[0037] Specifically, upstream refers to the nucleotide sequence located in the direction towards the 5' end of the DNA strand. This means that upstream is located "upstream" of the target gene or nucleotide sequence and in the opposite direction of transcription.

[0038] Similarly, downstream refers to the nucleotide sequence located in the direction towards the 3' end of the DNA strand. This means that downstream is located "downstream" of the target gene or nucleotide sequence and is in the same direction as transcription.

[0039] As used herein, the term "distance" refers to the number of nucleotides (in base pairs (bp) between two nucleic acid sequences (or specific points, such as insertion sites) located within the same nucleic acid molecule. For example, the two nucleic acid sequences X1 and X2 in sequence X1ATGCCAATGCX2 have a distance of 10 base pairs because 10 nucleotides lie between these sequences.

[0040] As used in this article, the term "inverted terminal repeat" (ITR) refers to specific DNA sequences found at both ends of the linear double-stranded DNA genome of many viruses, including adenoviruses. ITRs are short sequences repeated in opposite orientations, meaning that the sequence at one end is complementary to the sequence at the other end, and the two ends can anneal together to form a hairpin structure.

[0041] As used herein (especially in the context of expression cassettes), the term "heterogeneous" refers to genetic elements that originate from a different species or source compared to the object being compared or used together. For example, a heterologous expression cassette is an expression cassette containing or composed of such elements that do not originate from the same species in which they are inserted.

[0042] As used herein, the term "open reading frame" refers to a portion of a nucleic acid sequence between the start codon and the stop codon, excluding the stop codon (which acts as a termination signal). A codon is a three-nucleotide sequence of DNA or RNA that forms a unit of genomic information that encodes a specific amino acid or issues a termination signal (stop codon) for protein synthesis. In the standard genetic code, three different stop codons are known: TAG, TAA, or TGA at the DNA level, or UAG, UAA, and UGA at the RNA level. Other stop codons may exist in variations of the standard genetic code. The start codon is the first codon of an RNA transcript (e.g., linear mRNA, circRNA) translated by ribosomes. Typically, the start codon has the sequence AUG. In rare cases, the start codon may also have the sequence NUG (where N is any nucleotide).

[0043] As used in this article, the term "reverse splicing" refers to reverse splicing (i.e., reverse splicing), in which the upstream 3' splice site is connected to the downstream 5' splice site.

[0044] As used herein, the term "backsplicing site" refers to the nucleotide sequence at the intron-exon boundary (i.e., the splicing site). A backsplicing site typically consists of two halves, one located on an intron and the other on an exon. The spliced ​​nucleotide sequence splits between the two halves of the backsplicing site.

[0045] The term "internal ribosome entry site (IRES)" refers to a region in RNA that allows translation to be initiated internally in a cap-independent manner. IRES elements typically comprise a highly structured RNA segment containing several stem-loop structures. IRES were initially identified in picornaviruses, but they are present in a wide variety of viruses. IRES sequences have also recently been identified in many cellular mRNAs. Both types of IRES sequences—viral and cellular IRES sequences—are generally available for practice in this invention.

[0046] The term "pharmaceutical acceptable" means that a substance approved by a federal or state regulatory agency or listed in the United States Pharmacopeia, the European Pharmacopeia (Ph. Eur.), or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0047] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as saline solutions in water, and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions are preferred carriers when the drug composition is administered intravenously. Saline solutions, as well as aqueous dextran and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated as a suppository using conventional binders and carriers (such as triglycerides). The compounds of the present invention can be formulated in a neutral or salt form. Pharmaceutically acceptable salts include salts formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; and salts formed with free carboxyl groups, such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in EW Martin's "Remington's Pharmaceutical Sciences". Such compositions contain a therapeutically effective amount of the compound, preferably in a purified form, along with a suitable amount of carrier to provide a form suitable for appropriate administration to the patient. The formulation should conform to the administration method.

[0048] Implementation Plan The various aspects of the invention are defined in more detail below. Unless explicitly indicated otherwise, each aspect so defined may be combined with any other aspect or combination thereof. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or combination thereof indicated as preferred or advantageous.

[0049] In a first aspect, the present invention provides an adenovirus genome encoding an adenovirus, the adenovirus genome comprising at least one (preferably heterologous) expression cassette, the at least one (preferably heterologous) expression cassette comprising or consisting of a nucleic acid sequence encoding a circular RNA (circRNA), wherein the (preferably heterologous) expression cassette is inserted outside a functional transcription unit of the adenovirus genome, the functional transcription unit having the same reading orientation as the inserted (preferably heterologous) expression cassette.

[0050] The inventors unexpectedly discovered that, depending on the insertion site, inserting an expression cassette encoding circRNA into the adenovirus genome may not allow adenovirus formation. This differs from inserting an expression cassette that does not encode circRNA but rather encodes elements such as mRNA. Not wanting to be bound by theory, the inventors believe that this incompatibility of expression cassettes encoding circRNA may be related to the specific structure of circRNA and related elements (such as splice sites (i.e., splice acceptors and splice donor elements) and IRES elements) that can prevent viral particle formation. In particular, the splice site of the circRNA expression cassette required for in vivo circRNA circularization during backsplicing events may interfere with the splice acceptor / donor sites of the adenovirus genome, thus leading to unfavorable splicing events. It was found that inserting the expression cassette outside the functional transcription unit of adenoviruses as disclosed herein produces functional adenoviruses. Inserting a heterologous expression cassette outside a functional adenovirus transcription unit having the same reading direction as the inserted heterologous expression cassette... For example, as illustrated in the embodiments, a heterologous expression cassette inserted with orientation V1 will be inserted into a transcription unit (i.e., E3A / B) having the same orientation. A heterologous expression cassette inserted with orientation V2 (i.e., at the same point as V1 but in the opposite direction) will be inserted outside a functional adenovirus transcription unit having the same reading direction.

[0051] In a preferred embodiment, the heterologous expression cassette is not inserted between the 5' terminal ITR and E1A, between E2A and E3, or between pIX and pIVa2.

[0052] In a preferred embodiment, the heterologous expression cassette is not inserted between the 5' terminal ITR and E1A.

[0053] In a preferred embodiment, the heterologous expression cassette is not inserted between E2A and E3.

[0054] In a preferred embodiment, the heterologous expression cassette is not inserted between pIX and pIVa2.

[0055] In a preferred embodiment, the expression cassette is a heterologous expression cassette, wherein the genetic elements (such as encoding circRNA and / or other elements of the expression cassette) do not originate from the adenovirus genome used.

[0056] In a preferred embodiment, the transcription units mentioned are early genes E1 to E4, mid-term genes pIX and pIVa2, and late genes L1 to L5.

[0057] In a preferred embodiment, the transcription units mentioned are those shown in Table 2 below.

[0058] In another preferred embodiment, the transcription unit is a transcription unit encoding a specific adenoviral protein. Non-limiting examples are pIX and pIVa2. Other examples can be found in Table 2.

[0059] Furthermore, the location of the insertion site within the adenovirus genome does indeed have a significant impact on the expression level of the encoded circRNA.

[0060] In a preferred embodiment, a heterologous expression cassette is inserted outside a functional transcription unit of the adenovirus genome, which is encoded on the same strand of the adenovirus genome (in which the heterologous expression cassette (specifically, the circRNA encoded therein) is encoded).

[0061] In a preferred embodiment, the heterologous expression cassette is inserted at a point outside the functional transcriptional units of the adenovirus genome (encoded on either strand of the adenovirus genome). In another preferred embodiment, the heterologous expression cassette is inserted outside the functional transcriptional units encoding the early genes E1 to E4 (encoded on either strand of the adenovirus genome).

[0062] In a preferred embodiment, the heterologous expression cassette is inserted at a point between functional transcription units (encoded on either strand of the adenoviral genome). Preferably, the functional transcription unit into which the heterologous expression cassette is inserted is the closest functional transcription unit, i.e., there are no other functional transcription units between them.

[0063] In a preferred embodiment of the first aspect of the invention, the (preferably heterologous) expression cassette is not inserted between the transcription units pIX and pIVa2.

[0064] In a preferred embodiment of the first aspect of the invention, a (preferably heterologous) expression cassette is inserted between a functional transcription unit of the adenovirus genome and the ITR, wherein there are no other functional transcription units between the functional transcription unit and the ITR.

[0065] In a preferred embodiment of the first aspect of the invention, a (preferably heterologous) expression cassette is inserted: a) Among the functional transcriptional units encoded on either strand of the adenoviral genome, the functional transcriptional unit in which a heterologous expression cassette is inserted is the closest functional transcriptional unit (i.e., there are no other functional transcriptional units between them); or b) Between the functional transcriptional units of the adenovirus genome and the ITR, where there are no other functional transcriptional units between the functional transcriptional units and the ITR.

[0066] In a preferred embodiment of the first aspect of the invention, the adenovirus is a conditionally replicating adenovirus. In replication-defective adenoviruses, not all genes in the adenovirus genome are required to produce a functional adenovirus. This provides more opportunities in selecting insertion sites within the adenovirus genome, because even if interference with some elements in the adenovirus genome occurs as described above, it is irrelevant if the affected elements are not essential in replication-defective adenoviruses. Therefore, in conditionally replicating adenoviruses, there are fewer suitable insertion sites available. However, the present invention provides insertion sites that take into account these additional requirements and are also functional in conditionally replicating adenoviruses.

[0067] In a preferred embodiment of the first aspect of the invention, the heterologous expression cassette is inserted into the adenovirus genome at a point at least 24 bp (at least 25 bp, at least 30 bp, at least 35 bp) away from any transcriptional unit encoding a mid-term gene (preferably encoded on any strand of the adenovirus genome). Preferred examples of transcriptional units encoding mid-term genes are pIX and pIVa2.

[0068] In a preferred embodiment of the first aspect of the invention, the heterologous expression cassette is located at a distance of at least 24 bp upstream (i.e., in the 5' direction) or at least 24 bp downstream (i.e., in the 3' direction) from any functional transcriptional unit of the adenovirus genome.

[0069] In a preferred embodiment of the first aspect of the invention, the distance from the heterologous expression cassette to the inverted terminal repeat (ITR) of the adenovirus genome is shorter upstream of the heterologous expression cassette than downstream of it.

[0070] The inventors further observed that the orientation of the expression cassette relative to the nearest ITR of the adenovirus affects the expression intensity of the circRNA encoded within the expression cassette. Expression cassettes with a reading direction opposite to the nearest ITR exhibited lower expression levels of the encoded circRNA compared to those with a reading direction away from the nearest ITR. Not wishing to be bound by theory, the inventors hypothesized that, if inserted with the correct orientation, regulatory elements located within the ITR could increase the transcription of the circRNA encoded in the expression cassette.

[0071] In a preferred embodiment of the first aspect of the invention, the inverted terminal repeat (ITR) of the adenovirus genome is located within 6000 nucleotides upstream of the heterologous expression cassette. In other words, the 5' end of the heterologous expression cassette is 6000 nucleotides or less from the ITR in the 5' direction. In a preferred embodiment, the distance from the ITR in the 5' direction is less than 5000, less than 4000, less than 3000, or less than 2000 nucleotides.

[0072] In a preferred embodiment of the first aspect of the invention, a heterologous expression cassette is inserted between transcription units E1A / E1B, E4 / E2A, L5 / E4, or between E4 and the 3' ITR of the adenovirus genome (i.e., upstream of E4). These transcription units can encode on different strands of the adenovirus genome.

[0073] In a preferred embodiment, the termination of transcription unit E1A is characterized by the PAS of E1A, and the initiation of transcription unit E1B is characterized by the TATA box of E1B.

[0074] In a preferred embodiment, the termination of transcription unit E4 is characterized by the PAS of E4, and the initiation of transcription unit E2A is characterized by the promoter of E2A.

[0075] In a preferred embodiment, the termination of transcription unit L5 is characterized by the PAS of L5, and the termination of transcription unit E4 is characterized by the PAS of E4.

[0076] In a preferred embodiment, the initiation of transcription unit E4 is characterized by the TATA box of E4 and the 3' terminal ITR of the adenoviral genome located upstream of E4.

[0077] In a preferred embodiment, the (preferably heterologous) expression cassette is inserted between transcription units defined by the nucleic acid sequences shown in Table 1 below. Preferably, the heterologous expression cassette is inserted between transcription units E1A / E1B, E4 / E2A, L5 / E4, or between E4 and the 3' terminal ITR of the adenovirus genome (i.e., upstream of E4), as noted in Table 1 below. Table 1: Annotated TATA (or promoter) and PAS signals and surrounding sequences SID refers to SEQ ID NO; in the above sequence, the TATA box (or related promoter element) and PAS sequence are underlined.

[0078] In a preferred embodiment of the first aspect of the invention, the adenovirus genome is a mammalian adenovirus genome.

[0079] In a preferred embodiment of the first aspect of the invention, the adenovirus genome is a large ape mammal adenovirus genome.

[0080] In a preferred embodiment of the first aspect of the invention, the adenovirus genome is selected from: African pygmy hedgehog, alpaca, bat, cattle, dog, deer, dolphin, horse, poultry, frog, guinea pig, lemur, mandrill, rat, sheep, pigeon, proboscis monkey, polar bear, pig, reindeer, rhesus monkey, sea lion, ape, skunk, squirrel, squirrel monkey, tree shrew and turkey adenovirus genomes.

[0081] In a preferred embodiment of the first aspect of the invention, the adenovirus genome is a human mammalian adenovirus genome.

[0082] In a preferred embodiment of the first aspect of the present invention, the adenovirus genome is a human mammalian adenovirus group C genome.

[0083] In a preferred embodiment of the first aspect of the invention, the adenovirus genome is the human adenovirus 5 (i.e., serotype 5; Ad5, HADV-5) genome.

[0084] In a preferred embodiment of the first aspect of the invention, the adenovirus genome is selected from the following human mammalian adenovirus genomes: HAdV-1, HAdV-105, HAdV-106, HAdV-11, HAdV-11+34, HAdV-11a, HAdV-12, HAdV-14, HAdV-16, HAdV-17, HAdV-18, HAdV-19, HAdV-2, HAdV-20, HAdV-1 ...3, HAdV-14, HAdV-15, HAdV-16, HAdV-17, HAdV-18, HAdV- AdV-21, HAdV-21a, HAdV-22, HAdV-23, HAdV-24, HAdV-25, HAdV-27, HAdV-29, HAdV-3+7, HAdV-3 0. HAdV-31, HAdV-32, HAdV-33, HAdV-35, HAdV-36, HAdV-38, HAdV-39, HAdV-40, HAdV-41, HAdV-4 2. HAdV-43, HAdV-44, HAdV-45, HAdV-47, HAdV-4a, HAdV-4p, HAdV-5, HAdV-51, HAdV-53, HAdV-5 4. HAdV-55, HAdV-56, HAdV-58, HAdV-6, HAdV-60a, HAdV-61, HAdV-62, HAdV-63, HAdV-64, HAdV-6 6. HAdV-68, HAdV-69, HAdV-7, HAdV-71, HAdV-7d, HAdV-7d2, HAdV-86, HAdV-89, HAdV-B3, HAdV- D10, HAdV-D13, HAdV-D37, HAdV-D8, HAdV-E4, HAdV-A, HAdV-B, HAdV-C, HAdV-D, HAdV-E, HAdV-F.

[0085] In a preferred embodiment of the first aspect of the invention, the adenovirus genome is selected from the following human mammalian adenovirus genomes: African pygmy hedgehog adenovirus 1, bat mammalian adenovirus, bat mammalian adenovirus A, bat mammalian adenovirus B, bat mammalian adenovirus C, bat mammalian adenovirus D, bat mammalian adenovirus E, bat mammalian adenovirus F, bat mammalian adenovirus G, bovine mammalian adenovirus B, bovine mammalian adenovirus C, canine mammalian adenovirus A, deer mammalian adenovirus B, dolphin mammalian adenovirus B, yellow-haired fruit bat adenovirus, equine mammalian adenovirus A, equine mammalian adenovirus B, guinea pig adenovirus, human adenovirus 86, species of the genus *Adenovirus*, human mammalian adenovirus A, human mammalian adenovirus B, human mammalian adenovirus C, human mammalian adenovirus D, human mammalian adenovirus E, human mammalian adenovirus F, human mammalian adenovirus G, species of the genus *Adenovirus*, mouse mammalian adenovirus A, mouse mammalian adenovirus B, mouse mammalian adenovirus C, ONCOS-d2.7kb, sheep mammalian adenovirus A, sheep... Mammalian adenovirus C, Proboscis monkey mammalian adenovirus A, Polar bear mammalian adenovirus A, Swine mammalian adenovirus A, Swine mammalian adenovirus B, Swine mammalian adenovirus C, Rhesus monkey adenovirus 54, Rhesus monkey adenovirus 55, Rhesus monkey adenovirus 56, Rhesus monkey adenovirus 57, Rhesus monkey adenovirus 58, Rhesus monkey adenovirus 59, Rhesus monkey adenovirus 60, Rhesus monkey adenovirus 61, Rhesus monkey adenovirus 62, Rhesus monkey adenovirus 63, Rhesus monkey adenovirus 64, Rhesus monkey adenovirus 65, Rhesus monkey adenovirus 66, Rhesus monkey adenovirus 67 Egyptian fruit bat adenovirus, sea lion mammal adenovirus A, simian adenovirus B, simian adenovirus ER, simian mammal adenovirus A, simian mammal adenovirus B, simian mammal adenovirus C, simian mammal adenovirus D, simian mammal adenovirus E, simian mammal adenovirus F, simian mammal adenovirus G, simian mammal adenovirus H, simian mammal adenovirus I, skunk adenovirus HUN / 2009, skunk mammal adenovirus A, squirrel mammal adenovirus A, squirrel monkey adenovirus, and tree shrew mammal adenovirus A.

[0086] In a preferred embodiment of the first aspect of the invention, the (preferably heterologous) expression cassette includes an open reading frame (ORF) encoding a target circRNA and an internal ribosome entry site (IRES) operatively linked to the ORF.

[0087] In a preferred embodiment of the first aspect of the invention, the (preferably heterologous) expression cassette includes an open reading frame (ORF) encoding a target circular RNA and an internal ribosome entry site (IRES) operatively linked to the ORF, a first backsplicing site located on the 5' side of the IRES, and a second backsplicing site located on the 3' side of the IRES.

[0088] In a preferred embodiment of the first aspect of the invention, the (preferably heterologous) expression cassette further includes a first inverse repeat (IR) element located on the 5' side of the first inverse splice site and a second IR element located on the 3' side of the second inverse splice site.

[0089] In a preferred embodiment of the first aspect of the invention, the (preferably heterogeneous) expression cassette further includes a promoter operatively connected to the expression cassette to guide expression of the expression cassette.

[0090] In a preferred embodiment of the first aspect of the invention, the (preferably heterogeneous) expression cassette further includes a first inverse repeat (IR) element located on the 5' side of the first inverse splice site and a second IR element located on the 3' side of the second inverse splice site, as well as a promoter operatively connected to the expression cassette to guide expression of the expression cassette.

[0091] In a preferred embodiment of the first aspect of the invention, the (preferably heterologous) expression cassette further includes a polyadenylation signal.

[0092] In a preferred embodiment of the first aspect of the invention, the ORF is a split ORF, wherein the flanks of the IRES are two parts of the split ORF.

[0093] In a preferred embodiment of the first aspect of the invention, the IRES is contained in a (preferably heterologous) expression cassette, and the IRES is positioned within the (preferably heterologous) expression cassette, wherein: (I) The 5' end of IRES and the 3' end of the first backsplicing site are separated by at least 50 nucleotides, preferably by at least 200 nucleotides, more preferably by at least 300 nucleotides, and (II) The 3' end of the IRES and the 5' end of the second back splice site are separated by at least 300 nucleotides, preferably at least 350 nucleotides.

[0094] In a preferred embodiment of the first aspect of the invention, the target circular RNA in the heterologous expression cassette is a transgene.

[0095] In a preferred embodiment of the first aspect of the invention, the expression cassette has a sequence as shown in SEQ ID NO: 001.

[0096] In a second aspect of the invention, an infectious adenovirus particle comprising the adenovirus genome of the first aspect of the invention is provided.

[0097] In a preferred embodiment of the second aspect of the invention, the adenovirus particles are conditionally replicating adenoviruses.

[0098] In a preferred embodiment of the second aspect of the invention, the adenovirus particles are replication-defective adenoviruses.

[0099] In a third aspect of the invention, a pharmaceutical composition comprising the adenovirus genome of the first aspect of the invention or the infectious adenovirus particles of the second aspect of the invention further comprises a pharmaceutically acceptable carrier and / or excipient.

[0100] In a fourth aspect, the present invention also provides medical uses of the adenovirus genome according to the first aspect of the present invention, the infectious adenovirus particles according to the second aspect of the present invention, and the pharmaceutical composition according to the third aspect of the present invention. Medical uses include uses in therapy and prevention. Preferably, it is used in the therapy and prevention of proliferative diseases.

[0101] Example The inventors have observed that insertion of circular RNA expression cassettes into the adenovirus genome has a significant impact on both transgene expression and adenovirus fitness. The following examples provide the identification of insertion sites in the adenovirus genome suitable for expressing expression cassettes encoding circRNAs. Specifically, sites leading to superior expression intensity of the encoded circRNA are provided.

[0102] Example 1: Insertion at the missing adenovirus gene site In the first set of experiments, circRNA and mRNA expression cassettes (Figures 1A-1B) were inserted into the E3 region of the adenovirus serotype 5 backbone (Figure 1C). The circRNA cassette consists of an exogenous CMV promoter, an inverted repeat element, a splitting ORF, an IRES element, and a polyadenylation signal (the sequence in SEQ ID NO: 001). The mRNA expression cassette consists of a CMV promoter, an ORF, and a polyadenylation signal.

[0103] The adenovirus vector used contained a 2.7 kb deletion in the E3 region to allow sufficient space for insertion of the payload cassette into the adenovirus 5 genome. The expression cassette was inserted with the same orientation as the E3 encoding region in the adenovirus genome. Table 2 below shows the elements encoded in the adenovirus genome.

[0104] The inventors observed that insertion of circular RNA expression cassettes into the E3 region inhibited adenovirus vector rescue, while insertion of linear mRNA expression cassettes did not impede viral rescue or adaptation. This indicates that insertion of circular RNA expression cassettes has a significant impact on viral adaptation. Not wanting to be bound by theory, the inventors believe that circRNA expression cassettes may contain actual splice acceptor and splice donor sequences that could potentially interfere with adenovirus gene splicing. Therefore, insertion into the E3 region could potentially interfere with L5 fiber gene splicing. Table 2: Elements encoded in the Ad5 genome used in the examples Example 2: Insertion outside the transcription unit of the adenovirus genome To identify suitable integration sites within the adenovirus genome, the inventors inserted circRNA expression cassettes into different regions of the adenovirus 5 genome. Based on the hypothesis that inserting circRNA cassettes might interfere with adenovirus gene splicing, the expression cassettes were inserted between transcription units within the crAdV5 vector. circRNA expression cassettes were incorporated into most integration sites in both forward and reverse orientations (Figure 2A and Table 3). Table 3: Insertion site of circRNA cassette within adenovirus 5 backbone The insertion point for the expression cassette is chosen to ideally avoid overlap with transcription units on both strands, but at least avoid overlap with transcription units encoded on the same strand. The distance from the insertion point to the nearest transcription unit on either strand is described in Table 4 below. Table 4: Relative positions of insertion sites * indicates the opposite reading direction to the inserted expression cassette (i.e., encoding on the opposite strand of the adenovirus genome); # inserts V1 into the transcription unit, so the above distance indicates the beginning and end of the transcription unit where V1 is inserted.

[0105] These distances are calculated based on the start and end of the nearest transcription unit, defined as starting with a TATA box and ending with a polyadenylation signal (PAS). The relevant elements used to calculate the distances in Table 4 are placed next to the distances expressed in base pairs (bp).

[0106] The choice of insertion site has a significant impact on viral viability (Table 3). For insertion sites V6 and V7, placing the expression cassette between pIX and IVa ORF does not produce viable virus (Figures 2B-2C).

[0107] Beyond the general applicability of the insertion sites to the recovery of live viral particles, the inventors unexpectedly discovered that these sites differed significantly in the expression intensity of the circRNA constructs. Western blotting was used to assess the expression of circRNA-derived proteins 24 h and 48 h after infecting A549 cells with equal numbers of viral particles.

[0108] circRNA-derived protein expression differed between insertion sites, with the highest yield coming from circRNA cassettes (V4) inserted in reverse orientation between the L5 and E4 regions (Fig. 2B-2C). Comparable payload expression was observed among viral V2, V5, and V9 (Fig. 2B-2C). Low levels of protein expression were observed for viral V3 and V8 (Fig. 2B-2C).

[0109] The inventors further observed that expression cassettes inserted according to the transcriptional direction (i.e., 5' to 3') do not face the nearest ITR. Example 3: The insertion site is conserved in mammalian adenoviridae families. The inventors further investigated whether the identified insertion sites were specific to adenovirus Ad5, or whether these findings could be extrapolated to at least the mammalian adenoviridae family. Therefore, comparisons were performed with available mammalian adenovirus genomes to test whether these sites also exist in other mammalian adenovirus genomes.

[0110] The alignment of annotations, including those for relevant transcription units and genes, is depicted in Figure 4. The adenovirus 5 genome used in previous experiments is labeled “ONCOS-d2.7kb”, and different insertion sites are depicted within this genome. As can be readily seen, the transcription units present in Ad5 are also present in other mammalian adenoviruses with relatively similar positions to each other. Therefore, the same sites between the transcription units tested and validated in Ad5 are present and conserved in other members of the mammalian adenoviridae family. Thus, the insertion strategy used for adenovirus 5 (Ad5) in the above embodiments can be transferred to other mammalian adenoviridae members using conserved genomic structures.

[0111] Summarize The published experimental data indicate that the expression of circular RNA coding cassettes from the adenovirus genome is complex. Unlike linear mRNA expression cassettes, inserting circRNA expression cassettes into coding regions of the genome, such as the E3 region, significantly impacts viral adaptability.

[0112] Furthermore, the inventors observed that placing the circRNA expression cassette between these coding regions and transcription units (and thus outside of them) is insufficient to ensure circRNA expression. As observed with insertion sites V6 and V7, inserting circRNA between the pIX and IVa genes does not produce live viruses.

[0113] In summary, this invention has identified specific regions in the adenovirus genome that allow for the insertion of circRNA expression cassettes. Furthermore, the inventors have also identified specific regions that allow for high-yield circRNA expression.

Claims

1. An adenoviral genome encoding an adenovirus, comprising at least one heterologous expression cassette comprising or consisting of a nucleic acid sequence encoding a circular RNA (circRNA), wherein the heterologous expression cassette is inserted outside of a functional transcription unit of the adenoviral genome having the same reading direction as the inserted heterologous expression cassette.

2. The adenoviral genome according to claim 1, wherein the adenovirus is a conditionally replicating adenovirus.

3. The adenoviral genome according to any one of the preceding claims, wherein the heterologous expression cassette is inserted at a point in the adenoviral genome at least 24 bp distance from a transcription unit encoding a middle gene.

4. The adenoviral genome according to any one of the preceding claims, wherein the distance of the heterologous expression cassette to the inverted terminal repeat (ITR) of the adenoviral genome is shorter upstream of the heterologous expression cassette compared to downstream of the heterologous expression cassette.

5. The adenoviral genome according to any one of the preceding claims, wherein the inverted terminal repeat (ITR) of the adenoviral genome is within 6000 nucleotides upstream of the heterologous expression cassette.

6. The adenoviral genome according to any one of the preceding claims, wherein the heterologous expression cassette is inserted between the transcription units E1A / E1B, E4 / E2A, L5 / E4 or between E4 and the 3’ terminal ITR.

7. The adenoviral genome according to any one of the preceding claims, wherein the adenoviral genome is a mammalian adenoviral genome, preferably a human or great ape adenoviral genome, more preferably a group C adenoviral genome.

8. The adenoviral genome according to claim 7, wherein the adenoviral genome is the genome of human adenovirus 5 (hAd5).

9. The adenoviral genome according to any one of the preceding claims, wherein the heterologous expression cassette comprises an open reading frame (ORF) encoding a circular RNA of interest and an internal ribosome entry site (IRES) operably linked to the ORF.

10. The adenoviral genome according to any one of the preceding claims, wherein the circular RNA of interest in the heterologous expression cassette is a transgene.

11. The adenoviral genome according to any one of the preceding claims, wherein the heterologous expression cassette further comprises: - a first and a second inverted splice site allowing circularization of the circRNA; - a promoter operably linked to the ORF; - a first and a second inverted repeat (IR) element; and / or - a polyadenylation signal.

12. An infectious, preferably conditionally replicating adenoviral particle comprising the adenoviral genome of any one of the preceding claims.

13. A pharmaceutical composition comprising the adenoviral genome according to claims 1 to 11 or the infectious, preferably conditionally replicating adenoviral particle according to claim 12 and a pharmaceutically acceptable carrier and / or excipient.

14. An adenoviral genome according to claims 1 to 11 or an infectious, preferably conditionally replicating adenoviral particle according to claim 12 or a pharmaceutical composition according to claim 13 for use in medicine.

15. An adenoviral genome according to claims 1 to 11 or an infectious, preferably conditionally replicating adenoviral particle according to claim 12 or a pharmaceutical composition according to claim 13 for use in therapy and prophylaxis of proliferative diseases.

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