Patent Application
20240076689
This invention generally relates to the field of somatic gene therapy. The invention provides a nucleic acid construct comprising a transgene encoding a therapeutic protein, a tetracycline (Tet)-responsive aptazyme sequence, and inverted terminal repeats (ITRs). The nucleic acid construct can be transferred to a subject in need of the encoded therapeutic protein in the form of a viral vector, particularly an adeno-associated virus (AAV) vector. The Tet-responsive aptazyme sequence allows for a tightly controlled expression of the transgene in the subject, thereby avoiding toxic side effects of the therapeutic protein. The nucleic acid construct and the viral vectors comprising same are particularly useful in the treatment of proliferative diseases like cancer.
Clinical trials using recombinant first-generation adeno-associated virus (AAV) vectors have contributed significantly to the further advancement of gene therapy by achieving important milestones, such as the first market approved AAV-based therapies (Russell et al. 2017; Jiang et al. 2018; Kumar et al. 2016). At the same time, these trials identified vector elements, whose optimization has the potential to further improve efficacy, tissue specificity and safety, e.g., by engineering vector capsids and promoter/enhancer elements (Grimm & Büning, 2017; Sarcar et al. 2019). Respective approaches are further strengthened by the aspiration to extend next-generation gene therapies beyond the field of inherited rare diseases towards acquired diseases and larger patient populations. To address potential safety issues and account for the natural variability in patients' disease biology and therapeutic response, a particularly desirable feature of gene therapy vectors would be a system that allows to control and precisely induce gene expression.
Specifically, it is envisioned that patients are able to switch on the expression of an AAV-delivered therapeutic by the temporal intake of a small-molecule drug. Such a therapeutic AAV-based system is depicted in
Achieving controllable gene expression is highly desirable, as evident from different protein-based systems that have been investigated recently, including destabilizing domains and inducible promoters as the most advanced approaches (Santiago et al. 2018; Barrett et al. 2018). These transcriptional control systems, including the Rheoswitch system, Mifepristone, and classic Tet-ON/OFF promoter systems, suffer from a common drawback: The require the expression of DNA-binding proteins that enable transcription after being activated by their cognate ligands (Chiocca et al., 2019; Wang et al., 2004; Gonzalez-Aparicio et al., 2011; Vanrell et al., 2011; Das et al., 2016). These DNA-binding proteins bear an immunogenic risk by representing T-cell epitopes. Attempts to address this for the Tet-promoter control system by engineering versions without certain T cell epitopes for HLA0201 (the most common human HLA serotype), revealed that if that is even possible—as the protein still has to retain specific binding to both the Tet repressor AND to tetracycline—there will be other humans with other serotypes that may still present epitopes from the resulting protein since there will be no immune tolerance to this foreign protein (Ginhoux et al., 2004). These data show that in order to achieve the full potential of gene therapy technologies, genetic switches with wide dynamic ranges that control transgene expression without the requirement of additional protein components are required.
In this context, so-called artificial riboswitches have been described as attractive building blocks for gene expression control systems that function independently of co-expressed regulatory proteins or fused destabilizing protein domains. Artificial riboswitches (or aptazymes) are DNA-encodable fusions of a ligand-binding RNA aptamer and a ribozyme, which enable to control messenger RNA (mRNA) integrity by conditional mRNA self-cleavage. As shown in
Using Theophylline, tetracycline (Tet), Guanine or protein-responsive hammerhead or Hepatitis Delta Virus (HDV)-based riboswitches, principal functionality in cell culture has been demonstrated, yet mostly using OFF-switches (Kumar et al. 2009; Ketzer et al. 2012; Ketzer et al. 2014; Nomura et al (2013); Wei & Smolke, 2015; Bloom et al. 2015; Kennedy et al. 2014). In contrast, exploration of riboswitch function in animals has been scarce. One early study in mice showed direct (i.e. non-allosteric) inhibition of a ribozyme by an RNA-binding compound (Yen et al. 2004). Another demonstrated riboswitch-mediated transgene regulation in ex vivo manipulated cells after transplantation into mice (Chen et al. 2010). In the context of viral vectors, it was previously shown that a recombinant AAV vector equipped with a guanine-HDV switch that enabled conditional shutdown of various genes' expression in vitro allowed for robust gene expression in mice, in the absence of exogenous guanine (Strobel et al. 2015b). In addition, one recent study demonstrated approximately seven-fold Tet-riboswitch-mediated downregulation of AAV-mediated reporter gene expression in the gastrocnemius muscle of mice (Zhong et al. 2016). Tight regulation of self-cleaving activity in the context of an engineered AAV-delivered aptazyme was also reported by employing steric-blocking antisense oligonucleotide resulting in an ON-fashion of transgene expression (Zhong et al., 2020). However, versatility of this approach is hampered by the poor bioavailability of antisense oligonucleotides compared to small molecule ligands.
Whereas ligand-induced suppression of gene expression might in principle find applications as a safety switch, e.g. in oncolytic therapy, a far more attractive option for many therapeutic applications would be the ability to induce therapeutic gene expression only in response to a ligand. However, the only viral vector and ON-riboswitch-based study available to date achieved very modest effects of at best two-fold induction of GFP expression in the eye of mice (Reid et al. 2018), but only the 3×-L2Bulgel8tc riboswitch demonstrated a significant increase (2-fold) in GFP expression compared to baseline levels in vivo (p<0.05, paired test). WO2018/165536 describes the effects of K19 in cell culture, see
In summary, there is a need for a clinically applicable inducible system fulfilling the desired criteria of being efficient, non-immunogenic, small and transgene-independent. Preferably, this system should provide an expression induction which covers a broad range of therapeutic protein expression, i.e. ideally 0-100% of a conventional, riboswitch-free construct. In addition, it should render possible the fine-tuning of expression levels by ligand dose adjustment. Finally, it should allow for repeated ON and OFF switching.
The present invention provides nucleic acid constructs and viral vectors that comprise a transgene and a tetracycline-responsive aptazyme which allows for a controlled expression of the transgene. The tetracycline-responsive aptazyme preferably is the aptazyme “K19” previously described Beilstein et al. 2015. The aptazyme comprises the tetracycline aptamer (Berens et al. 2001) and the full-length hammerhead ribozyme N79 from Schistosoma mansoni (Yen et al. 2004). In has been found herein that when used in an expression cassette and delivered by a viral vector, such as an AAV vector, the K19 aptazyme effectively controls and dose-dependently induces AAV-mediated transgene expression by providing or retracting tetracycline in an animal in vivo.
Prior to the present invention, therapeutic applicability of a Tet-responsive aptazyme in eukaryotes has been hampered by (a) lack of sufficient expression induction to cover a broad range of therapeutic protein expression, i.e. ideally 0-100% of a conventional, riboswitch-free construct; (b) lack of ligand dose dependency to fine-tune therapeutic protein expression levels in vivo and (c) uncertainty whether this approach also allows for repeated ON and OFF switching. The present invention has overcome these obstacles.
As described below, the functionality of the K19 Tet riboswitch was first established herein in the context of AAV vector expression cassettes in different cellular cultures, exploring the potency of different designs and chronological aspects of inducibility. Following an in vivo pharmacokinetic (PK) study for Tet, the riboswitch performance was investigated in liver, lung, muscle and heart of mice by simultaneous AAV-mediated secretion of a liver-restricted tool antibody of the diabody type, and a ubiquitously expressed cellular luciferase. It was surprisingly found that the K19 riboswitch construct repeatedly induced reporter antibody expression in a dose-dependent and highly dynamic manner, by administering or retracting Tet treatment. The K19 Tet RNA switch construct was subsequently tested with the therapeutically relevant single-chain IL-12 gene enabling expression of fully bioactive IL-12. Tet-induced IL-12 levels and background levels in vitro were comparable for murine and human single-chain IL-12. Surprisingly, the range of Tet-induced IL-12 levels and leakiness of the system were similar to the reporter gene data observed in vivo. Following liver targeting after systemic delivery of hepatotropic AAV, IL-12 plasma levels induced by a single Tet application increased and dropped to baseline within 24 hrs. IL-12 induction could be repeated by a Tet rechallenge nine days after the first challenge and yielded clinically meaningful cytokine levels without toxicity. In a separate set of pharmacokinetic (PK) and safety experiments in naive mice, we successfully titrated the AAV vector dose so that a twice daily Tet application over five consecutive days resulted in safe and sustained inducible IL-12 expression. In contrast, IL-12 was not detectable in vector-dose matched animals receiving no Tet. The identical treatment regimen was then applied to study the pharmacokinetic and pharmacodynamic (PK/PD) of local and inducible IL-12 immunotherapy in a model of hepatocellular carcinoma (HCC). We observed comparable PK of inducible IL-12 in tumor-bearing and naive mice and recorded near complete remission that coincided with an influx of T-cells in the tumor nodules.
The nucleic acid constructs and viral vectors of the invention therefore allow to fine-tune the expression levels of therapeutic proteins in vivo by adjusting the dose of the Tet ligand in a rib oswitch context. Moreover, the aptazyme-mediated control over the transgene expression following AAV-mediated gene delivery enables repeated, i.e. dynamic ON-OFF switching. This renders the nucleic acid constructs and viral vectors of the invention particularly suitable for use in a clinical setting, as the system could be repeatedly switched on until full tumor remission, as well as in the case of tumor relapse, even several months after delivery of the system.
In a first aspect, the present invention relates to a nucleic acid construct comprising (i) a transgene encoding one or more therapeutic proteins, (ii) at least one tetracycline-responsive aptazyme sequence, and (iii) inverted terminal repeats (ITRs). The nucleic acid construct can comprise or consist of either RNA or DNA. Preferably, the nucleic acid construct will comprise or consist of DNA. The construct may be in linear or circular form, e.g. in the form of a plasmid. In a preferred embodiment, the nucleic acid construct of the invention comprises or consists of single-stranded or double-stranded DNA. In a particularly preferred embodiment, the nucleic acid construct of the invention consists of single-stranded DNA.
The nucleic acid construct of the invention comprises a transgene which codes for one or more therapeutic proteins. As used herein, therapeutic proteins include all types of proteins that exert, upon administration, a therapeutic benefit to a patient suffering from a disease or condition. Therapeutic proteins include proteins which are active immediately after their translation, as well as proteins which are first produced as in an inactive form and are activated after cleavage by a protease or peptidase, e.g. proteins produced with a signal peptide. Preferably, the transgene codes for a mammalian protein, more preferably a human protein. The protein is therapeutically active which means that its delivery to a subject effectively reduces or inhibits the severity of a disease or pathological condition. Preferably, the therapeutically active protein is a protein whose constitutive expression in the subject would lead to toxic or other significant side effects, so that a tight control of the expression is required. Toxic and other significant side effects caused by expression of the protein may include severe conditions caused by strong and persistent activation of the immune response, including cachexia, fever, chills, fatigue, arthromyalgia and/or headache.
In a preferred embodiment of the invention, the transgene encodes one or more immunoregulatory proteins. Immunoregulatory proteins in the sense of the instant disclosure include, but are not limited to, antibodies, such as Ipilimumab or anti-PD1 antibodies, antibody fragments, cytokines, such as interleukins, interferons, lymphokines, and pro-inflammatory and pro-apoptotic members of the tumor necrosis factor (TNF)/tumor necrosis factor receptor (TNFR) superfamily. Further included are T cell engagers, immune checkpoint inhibitors, agonists such as anti-CD137, anti-CD28, or anti-CD40 combinations of any of the above. In a preferred embodiment, the immunoregulatory protein encoded by the transgene is an interleukin selected from the group consisting of IL-4, IL-6, IL-10, IL-11, IL-12, IL-13, IL-23, IL-27, and IL-33. It is particularly preferred that the immunoregulatory protein in the construct of the invention is IL-12, preferably human IL-12.
It is known that IL-12 plays an important role in regulating both the innate and adaptive immune response. Apart from inducing potent anti-cancer effects, it synergizes with several other cytokines for increased immunoregulatory activities. A gene transfer of IL-12 would circumvent the safety issues caused by exposure to bolus doses of recombinant cytokine protein. If the gene encoding IL-12 is delivered locally, it is possible to achieve a beneficial concentration in the desired tissue, with diminished systemic exposure and hence, toxicity (Berraondo et al., 2018; Chiocca et al., 2019). Vectorized IL-12 delivery using gene shuttles with tissue tropism enables systemic delivery followed by local IL-12 production and, attraction and activation of T-cells in a paracrine fashion. IFNγ conveys beneficial anti-tumoral effects of IL-12 by modifying the tumor microenvironment. However, IFNγ is also the main mediator of the toxic effects of IL-12 and, over time, turns on immunoregulatory mechanisms, such as PD-L1 and IDO-1 expression, which mediate adaptive resistance to immunotherapy (Berraondo et al., 2018). Therefore, a genetically encoded, regulatable IL-12 expression is highly beneficial, as it indirectly also controls the level of IFNγ release from natural killer cells.
Natural IL-12 is a heterodimeric protein consisting of one p35 subunit (the alpha chain) and one p40 subunit (the beta chain). The two subunits are covalently linked via a disulfide bridge and form the biologically active 70 kDa dimer. Simultaneous expression of the alpha and beta chain required for production of active IL-12, has been achieved by bicistronic expression using an internal ribosomal entry site (IRES) or by expressing both subunits individually. However, the IRES strategy results in unequal expression of the individual subunits resulting in a bias towards p40 homodimers that display inhibitory p70 signaling. Moreover, two full expression cassettes entailing all necessary cis-acting modules exceed the packaging limit of AAV. Therefore, it is preferred to express for research purposes in mice a bioactive single chain murine IL-12 fusion protein sequence identical to mIL-12.p40.Δp35 as described by Lieschke et al. (mIL-12 hereinafter). In this construct, the p40 subunit is linked by a (Gly-Ser) linker to the p35 subunit from which the first 22 amino acids were deleted. The analogous human IL-12 fusion protein, with a p40-G6S-p35 configuration, also has been reported to retain high in vitro bioactivity (Lieschke et al., 1997; Zhang et al., 2011). For use in human cells, e.g. in cell tissues or human individuals, the corresponding protein of human origin (denoted “hIL-12”, see e.g. SEQ ID No. 6) can be used and is preferred herein. Human IL-12 is not cross-reactive for mouse cells. Consequently, mIL-12 was employed in the mouse experiments described below (for the mature sequence see SEQ ID No. 12, the precursor sequence with signal peptide is encoded by Seq ID NO:11, for the region in the plasmid flanked by ITRs see SEQ ID NO:29). Human IL-12 can be used in human tissue accordingly as an example for single chain IL-12 that works in human tissue. The term single chain IL-12 shall mean that the function of the IL-12 heterodimer is realized by one fusion protein.
In a preferred embodiment, the nucleic acid construct of the invention comprises a transgene encoding single chain IL-12. It is preferred that for the single chain IL-12, one or more of the conditions (A) to (AAAAA) are met:
(A) The single chain IL-12 shows at least 80% sequence identity to
(AA) The single chain IL-12 shows at least 90% sequence identity to
(AAA) The single chain IL-12 shows at least 95% sequence identity to
(AAAA) The single chain IL-12 shows at least 98% sequence identity to
(AAAAA) The single chain IL-12 shows 100% sequence identity to
For each case (A) to (AAAAA), it is preferred that the single chain IL-12 shows the recited level of identity for the reference sequence specified under (iii) or (iv). More preferably, the single chain IL-12 shows the recited level of identity for a sequence according to SEQ ID No. 3 and to a sequence according to SEQ ID No. 4 over the full length of SEQ ID 3 and 4, respectively.
Preferably, the single chain IL-12 comprises one or more sequences selected from the group consisting of SEQ ID No. 1, 2, 3, 4, 5, and 6. More preferably, the human single chain IL-12 comprises both a sequence according to SEQ ID No. 3 and a sequence according to SEQ ID No. 4. Most preferably, the single chain IL-12 comprises both a sequence of SEQ ID No. 3 and a sequence of SEQ ID No. 4, wherein the sequence of SEQ ID No. 3 is followed by the sequence of SEQ ID No. 4, see e.g. SEQ ID No. 1, 2, 5, 6, optionally separated by a linker sequence such as (G 4 5) 3 (i.e. GGGGSGGGGSGGGGS) or G 4 5 (i.e. GGGGS) or G6s (i.e. GGGGGGS) (see e.g. SEQ ID No. 1, 2, 5, 6, 34, 35, 36, 37, 38, 39, 40, or 41). To allow or facilitate secretion, the single chain human IL-12 protein encoded by the transgene of the nucleic acid construct [which comprises a transgene encoding one or more therapeutic proteins, at least one tetracycline-responsive aptazyme sequence, and inverted terminal repeats (ITRs)] should preferably comprise an N-terminal signal sequence, such as the authentic signal sequence (e.g. SEQ ID No. 33 in SEQ ID No. 2, 6) or a signal sequence that stems from a different secreted protein (see e.g. the amino acid sequence encoded by the sequence SEQ ID No. 13) or an artificial signal sequence having the same function to allow cleavage by the signal peptidase. Such single chain IL-12 proteins are known in the art, see EP 3 211 000 B1 (sequence referred to as SEQ ID No. 6 therein) and US 10,646,549 B2 (sequence referred to as SEQ ID No. 48 therein). In another preferred embodiment, the single chain IL-12 comprises the sequence of SEQ ID No. 75.
In a particularly preferred embodiment, the nucleic acid construct of the invention comprises a transgene that encodes a single chain IL-12 comprising the sequence of SEQ ID No. 3, the sequence of SEQ ID No. 4, a linker sequence between the sequence of SEQ ID No. 3 and the sequence of SEQ ID No. 4, and an N-terminal signal sequence that provides for secretion of the single chain IL-12.
As used herein, the terms “identical” or “percent identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same in length and/or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence.
To determine the percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity =number of identical positions/total number of positions (e.g., overlapping positions)×100). In some embodiments, the two sequences that are compared are the same length after gaps are introduced within the sequences, as appropriate (e.g., excluding additional sequence extending beyond the sequences being compared).
The term “% sequence identity to the amino acid sequence of SEQ ID No. X over the length of SEQ ID No. X” means that the alignment should cover the entire length of the sequence of SEQ ID No. X (the reference sequence). In case the algorithms mentioned below do not render an alignment of the entire length of the reference sequence with the test sequence, but only over a subsequence of said reference sequence, amino acid residues within the reference sequence that do not have an identical counterpart on the test sequence are calculated as mismatch. The percent identity score given by said algorithm is then adjusted: If the algorithm yields K identical amino acids over an alignment length of L amino acids, and yields a percent identity of K/L*100, the term L is replaced by the number amino acids of the reference sequence if that number is higher than L. For instance, if the test sequence has one amino acid at the N-terminus less than the reference sequence SEQ ID No. 2 (but is otherwise identical except for this difference), the percent identity is 517/518*100%≈99.8%. The same applies vice versa to nucleic acid sequences.
The determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and)(BLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid encoding a protein of interest. BLAST protein searches can be performed with the) (BLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein of interest. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g.,) (BLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. If ktup=2, similar regions in the two sequences being compared are found by looking at pairs of aligned residues; if ktup=1, single aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences, or from 1 to 6 for DNA sequences. The default if ktup is not specified is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignment may be carried out using the CLUSTAL W algorithm, as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.
An alignment can easily be produced, e.g. by using the following link: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMS=blas tp&PAGE TYPE=BlastSearch&BLAST_SPEC=blast2seq&DATABASE=n/a&QUERY=&SUBJECTS=
For the purpose of calculating the percent identity, an alignment between test sequence and reference sequence (selected form the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively) is chosen among possible alignments produced by the mentioned algorithm that gives the highest identity score.
The single chain IL-12 protein preferably exhibits in an assay according to example (1.14) an immune-stimulating activity of at the same order of magnitude or better than the activity of commercially available bioactive human IL-12 consisting of two subunits linked via a disulfide bond with known half-maximum activity generally observed at concentrations of 100 to 400 pg/mL (Gately et al., 1995).
This table shows sequences mentioned in the text. In case of inconsistencies with the sequence listing, the sequences shown in the table are the authentic sequences. For explanations see Table 3.
The nucleic acid construct of the invention also comprises one or more tetracycline-responsive aptazyme sequences. As used herein, the term “aptazyme sequence” includes both an RNA, i.e. the aptazyme sequence itself, and the DNA encoding such RNA. An aptazyme as used herein refers to an RNA molecule that combines ribozyme and aptamer functionalities. An aptazyme normally comprises a first and a second RNA sequence which have been fused to each other. The first RNA sequence has ribozyme activity, i.e. it catalyzes the cleavage of an RNA molecule. Preferably, the first RNA sequence catalyzes a self-cleavage reaction which means that it provides for an intramolecular RNA cleavage within the ribozyme part of the aptazyme. The second RNA sequence of the aptazyme has aptamer functionality, i.e. it is capable of binding to a target molecule due to a stable three-dimensional structure. The first RNA sequence having ribozyme activity and the second RNA sequence having aptamer functionality are fused such that the ribozyme activity of the first RNA sequence is influenced by the binding of the second RNA sequence to its cognate ligand. In this way, the aptazyme can control the integrity of a messenger RNA (mRNA) by conditional mRNA self-cleavage.
According to the invention, the aptazyme is tetracycline-responsive which means that the aptamer sequence of the aptazyme specifically binds to tetracycline and reacts to such binding by a change in the three-dimensional structure. By changing the structure of the aptamer sequence, the activity of the ribozyme sequence is modulated, i.e. either increased or decreased. In a preferred embodiment, tetracycline binding by the aptazyme decreases, and preferably completely prevents, RNA cleavage by the ribozyme, thereby providing for an increased expression of the nucleic acid construct of the invention by mRNA stabilization.
Hence, the at least one tetracycline-responsive aptazyme preferably induces or enhances expression of the transgene upon tetracycline binding. In a particularly preferred embodiment, expression levels of a DNA construct of the invention are at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, or at least 14-fold, higher in the presence of an effective amount of tetracycline compared to the absence of tetracycline. More specifically, it is preferred that the nucleic acid construct of the invention, after delivery into a test subject, results in an at least 4-fold, preferably at least 6-fold, at least 8-fold, or at least 9-fold, higher expression level of the transgene compared to baseline level 8 hours after administration of 30 mg tetracycline per kg bodyweight to said subject. The test subject is a human, non-human primate or a mouse preferably a mouse.
In a preferred embodiment, the nucleic acid construct comprises a transgene encoding single chain IL-12, preferably human single chain IL-12, at least one tetracycline-responsive aptazyme sequence which comprises the sequence of SEQ ID NO:9 or SEQ ID NO:10 and ITRs derived from AAV2 such as the sequences according to Seq ID 8, 43, 44. 49. The construct preferably also comprises the liver-specific promoter LP1 such as the sequences according to SEQ ID NOs: 42 or 72. The nucleic acid construct preferably comprises any of the sequences set forth in SEQ ID NOs:29, 30, 31,46, 47, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 66, or a complement of any of these. The nucleic acid construct may be double stranded.
Within the nucleic acid construct of the invention, the at least one tetracycline-responsive aptazyme can be located either 5′ or 3′ of the transgene. It is however preferred that the at least one tetracycline-responsive aptazyme is located 3′ of the transgene, e.g. in the 3′ UTR region of the transgene. The nucleic acid construct of the invention may also comprise more than one tetracycline-responsive aptazyme, such as 2, 3, 4 or 5 of these aptazymes. If the construct comprises two tetracycline-responsive aptazymes, it is preferred that these are both located 3′ of the transgene, e.g. in the 3′ UTR region. Such arrangement is referred to herein as 3′3′ construct.
In a particularly preferred embodiment, the tetracycline-responsive aptazyme is the aptazyme “K19” previously described by Beilstein et al. 2015. The aptazyme comprises the tetracycline aptamer (Berens et al. 2001) and the full-length hammerhead ribozyme N79 from Schistosoma mansoni (Yen et al. 2004). The sequence of the aptazyme K19 is provided in SEQ ID NO:10 herein. The respective DNA sequence encoding the aptazyme K19 is provided in SEQ ID NO:9. Accordingly, in one embodiment of the invention, the tetracycline-responsive aptazyme sequence comprises or consists of the sequence set forth in any of SEQ ID NOs: 9 or 10.
The nucleic acid construct further comprises inverted terminal repeat (ITR) sequences. An ITR normally comprises of a first upstream nucleotide sequence which is followed by a second downstream nucleotide sequence which is the reverse complement of the first upstream nucleotide sequence. The intervening sequence of nucleotides (if any) between the first upstream and the second downstream nucleotide sequence can be of any length.
ITR sequences naturally occur in the genome of AAV and retroviruses where they are involved in packaging of the nucleic acid into viral capsids. Preferably, the ITR sequences of the nucleic acid construct of the invention comprises flank the transgene and the aptazyme, which means that the transgene and the aptazyme are located between the ITR sequences. It is preferred that the two ITR sequences that flank the transgene and the aptazyme are each about 140-145 bp in length. In a preferred embodiment, the ITR sequences in the nucleic acid construct of the invention are derived from an adeno-associated virus, preferably from AAV2, AAV8, or AAV9. It is particularly preferred that the ITR sequences comprise or consist of the ITR sequence set forth in SEQ ID NO: 8.
The ITR sequences usually both have a length between 130 and 145 nucleotides. At least one of which may be considerably shorter (Zhou, Tian et al., 2017). It is preferred that the two ITR sequences that flank the transgene and the aptazyme are each about 145 bp in length. In a preferred embodiment, the ITR sequences in the nucleic acid construct of the invention are derived from an adeno-associated virus, preferably from AAV2 (Wilmott et al., 2019; Samulski et al., 1983, Zhou et al., 2017). It is particularly preferred that the ITR sequences comprise or consist of the ITR sequence set forth in SEQ ID NO: 8, 43, 49, 50. It is understood that the ITRs have to be arranged in a certain way to exhibit their function: For the AAV2 wild-type ITR sequences according to Wilmott et al., 2019 the following set up is preferred:
In the scientific literature, the view shown for ITR right 3′-downstream is more common.
The construct comprising the transgene, the tetracycline-responsive aptazyme, and the ITRs can in principle have any size. Preferably, the size will be such that it can be packaged into the capsid of a viral vector. A skilled person will readily be able to select the size in consideration of the packaging capacity of the viral vector at hand. For example, if the nucleic acid construct, e.g. a single-stranded DNA, shall be used in combination with an AAV vector, the size of the construct should be below 4.7 kb which is the maximum size that is effectively packaged into an AAV vector. In a preferred embodiment of the invention, the nucleic acid construct is between 0.5 kb and 4.5 kb in size, such as between 0.75 kb and 4.0 kb, between 1.0 kb and 3.5 kb, between 1.5 kb and 3.0 kb, or between 2.0 kb and 2.5 kb. In a particularly preferred embodiment, the nucleic acid construct is a DNA molecule having a size between 0.5 kb and 4.5 kb, between 0.75 kb and 4.0 kb, between 1.0 kb and 3.5 kb, between 1.5 kb and 3.0 kb, or between 2.0 kb and 2.5 kb.
The nucleic acid construct may further comprise a promoter that drives the expression of the one or more transgenes. The promoter will be selected dependent on the intended use of the construct and the putative site of transgene expression. For example, where expression of the transgene in the liver is desired, a promoter having a high activity in liver tissue will be selected, such as the liver-specific promoter LP1. Similarly, if the transgene is to be expressed in tumor tissue, a tumor-specific promoter will be used, such as the alpha fetoprotein (AFP) promoter. Accordingly, in a preferred embodiment, the nucleic acid construct of the invention comprises a liver-specific promoter or a tumor-specific promoter.
As used herein, liver-specific promoters include the LP1 promoter, the transthyretin (TTR) promoter, A1AT promoter, and the thyroxine binding globulin (TPG) promoter (Greig et al., 2017), hybrid liver-specific promoter (HLP), human thyroxine-binding globulin (TBG), transthyretin (TTR), human alpha 1-antitrypsin (hAAT) promoter combined with liver-specific apolipoprotein E (ApoE) enhancer, synthetic liver-specific promoters (Okuyama et al., 1996; Cabrera-Pérez et al. 2019; EP2698163A1, WO2020104424). Tumor-specific promoters include the alpha fetoprotein (AFP) promoter (Shi, et al. 2004), the CEA promoter (Cao et al. 1998; Lan et al. 1997) and the Mucl promoter (Chen et al. 1995; Tai et al. 1999), and the hTERT promoter (Quante et al. 2005). Differential transcriptional regulation of human telomerase in a cellular model representing important genetic alterations in esophageal squamous carcinogenesis, Carcinogenesis vol.26 no.11 pp. 1879-1889). In an even more preferred embodiment, the nucleic acid construct of the invention comprises a promoter that is selected from the group of the human cytomegalovirus (CMV) promoter, the liver-specific promoter LP1, the tumor-specific alpha fetoprotein (AFP) promoter, and the human telomerase reverse transcriptase (hTERT) promoter.
As a further component, the nucleic acid construct of the invention may comprise a poly(A) signal. A poly(A) signal is a sequence motif which is recognized by the RNA cleavage complex, a multi-protein complex that cleaves the mRNA at the end of the transcription process. In a subsequent step, a tail of adenosine monophosphate residues is added to the 3′ end of a mRNA in a reaction catalyzed by the enzyme polyadenylate polymerase. The resulting poly(A) tail is in involved in nuclear export, translation, and stability of mRNA. Poly(A) signals are well known to a skilled person. Most human polyadenylation signals contain the sequence AAUAAA. Thus, in a preferred embodiment the nucleic acid construct of the invention comprises the sequence AAUAAA. In yet another preferred embodiment the nucleic acid construct comprises a synthetic poly(A) signal as described (Levitt et al., 1989). In yet another preferred embodiment the nucleic acid construct comprises the SV40 poly(A) signal depicted in SEQ ID NO: 7.
Preferably, the nucleic acid construct of the invention is a DNA construct comprising a transgene expression cassette. The expression cassette comprises a promoter which is operably linked to a transgene encoding a therapeutic protein, a sequence encoding an aptazyme upstream or downstream of the transgene, a polyadenylation signal, and ITR sequences at the 3′ and 5′ end.
In another aspect, the present invention relates to a transgene expression cassette comprising a promoter, a transgene encoding one or more therapeutic proteins, and at least one tetracycline-responsive aptazyme sequence. The promoter preferably is a liverspecific promoter, such as the liver-specific LP1 promoter, or a tumor-specific promoter as described hereinabove. The liver specific LP1 promotor may comprise an intron, see Seq ID 42. An example without SV40 intron is shown in Seq ID 72,
Preferably, the transgene expression cassette will comprise or consist of DNA. The expression cassette may be in linear or circular form, e.g. in the form of a plasmid. In a preferred embodiment, the transgene expression cassette of the invention comprises or consists of single-stranded or double-stranded DNA. In a particularly preferred embodiment, transgene expression cassette of the invention consists of single-stranded DNA.
The transgene expression cassette may further comprise a poly(A) signal, such as a SV40 poly(A) signal as described hereinabove. Thus, in a preferred embodiment the transgene expression cassette of the invention comprises the sequence AAUAAA. In yet another preferred embodiment the transgene expression cassette comprises a synthetic poly(A) signal. In yet another preferred embodiment the transgene expression cassette comprises the SV40 poly(A) signal depicted in SEQ ID NO: 7.
The transgene expression cassette comprises a transgene encoding one or more immunoregulatory proteins. As described hereinabove, immunoregulatory proteins include, but are not limited to, antibodies, such as Ipilimumab or anti-PD1 antibodies, antibody fragments, cytokines, such as interleukins, interferons, lymphokines, and pro-inflammatory and pro-apoptotic members of the tumor necrosis factor (TNF)/tumor necrosis factor receptor (TNFR) superfamily. Immunoregulatory proteins further include, but are not limited to, T cell engagers, immune checkpoint inhibitors, agonists such as anti-CD137, anti-CD28, or anti-CD40 combinations of any of the above. In a preferred embodiment, the immunoregulatory protein encoded by the transgene is an interleukin selected from the group consisting of IL-4, IL-6, IL-10, IL-11, IL-12, IL-13, IL-23, IL-27, and IL-33. It is particularly preferred that the immunoregulatory protein in the transgene expression cassette of the invention is IL-12, preferably human IL-12. Preferably, the single chain IL-12 comprises one or more of the sequences selected from the group consisting of SEQ ID Nos. 1-6 or 34 to 41. Further examples can be found in Table 4 below.
The transgene expression cassette of the invention comprises at least one tetracycline-responsive aptazyme sequence. As described herein above, the at least one tetracycline-responsive aptazyme can be located either 5′ or 3′ of the transgene. It is however preferred that the at least one tetracycline-responsive aptazyme is located 3′ of the transgene, e.g. in the 3′ UTR region of the transgene. The transgene expression cassette of the invention may also comprise more than one tetracycline-responsive aptazyme, such as 2, 3, 4 or 5 of these aptazymes. It is particularly preferred that the tetracycline-responsive aptazyme comprises or consists of the sequence set forth in any of SEQ ID NOs: 9 or 10.
The at least one tetracycline-responsive aptazyme sequence preferably induces or enhances expression of the transgene upon tetracycline binding. In a particularly preferred embodiment, expression levels of a DNA construct of the invention are at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, or at least 14-fold, higher in the presence of an effective amount of tetracycline compared to the absence of tetracycline. More specifically, it is preferred that the transgene expression cassette of the invention, after delivery into a subject, results in an at least 4-fold, preferably at least 6-fold, at least 8-fold, or at least 9-fold, higher expression level of the transgene compared to baseline level 8 hours after administration of 30 mg tetracycline per kg bodyweight to said subject. The subject is preferably a mouse.
In a preferred embodiment, the present invention provides a transgene expression cassette comprising a transgene encoding single chain IL-12, preferably human single chain IL-12, at least one tetracycline-responsive aptazyme sequence which comprises the sequence of SEQ ID NO:9 or SEQ ID NO:10 and ITRs derived from AAV2. The expression cassette preferably also comprises the liver-specific promoter LP1. The expression cassette preferably comprises any of the sequences set forth in SEQ ID NOs: SEQ ID NOs:29, 30, 31, 46, 47, 50, 51, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 66, without the flanking ITRs, preferably any of sequences set forth in SEQ ID NO: 73 or 74.
In another aspect, the invention relates to a viral vector comprising a capsid and a packaged nucleic acid, wherein the packaged nucleic acid comprises a nucleic acid construct or a transgene expression cassette as defined herein above, preferably a DNA construct. The viral vector can be selected dependent on the tissue to be transduced. Non-limiting examples of viral vectors that can be used in accordance with the invention include lentivirus vectors, adenovirus vectors, adeno-associated virus vectors (AAV vectors), and paramyxovirus vectors. Among these, the AAV vectors are particularly preferred, especially those with an AAV-2, AAV-8 or AAV-9 serotype. The viral vectors may comprise capsid proteins that have been modulated to include an amino acid sequence that provides for selective binding to a target tissue, such as liver tissue or lung tissue (see for example WO 2015/018860).
The nucleic acid constructs or transgene expression cassettes of the invention are particularly useful for the treatment of cancer diseases, in particular liver cancer. After systemic injection, the nucleic acid constructs or transgene expression cassettes of the invention locally deliver a regulatable transgene expression cassette, e.g. by use of a tissue-tropic AAV that targets towards a chosen cancerous organ (e.g. liver), induce local expression of the therapeutic protein in the cancerous organ, subsequent activation of T cells and other immune cells, and tumor elimination. The nucleic acid constructs or transgene expression cassettes can be applied to eliminate primary tumors, as well as secondary tumors (i.e. metastases) which are located in the cancerous organ provided with the regulated expression cassette. T cells and other immune cells primed locally through the described system can migrate with the blood stream to distant sites of the body, and induce abscopal anti-tumor responses towards cancer lesions that are located outside the cancerous organ that had been provided with the nucleic acid constructs or transgene expression cassettes.
It is known that many cancer patients die not as a result of their primary tumors, but rather of metastases resulting from those primary tumors (Dillekas et al., 2019). The formation of metastasis is a complex process that depends on both the circulation from the primary tumor and the properties of the target organ, such as its propensity to suppress the immune system. Several tumor types frequently metastasize to the liver, including colorectal cancer (Valderrama-Treviño et al., 2017), lung cancer and melanoma. For example, hepatic metastasis formation correlates with diminished immunotherapy efficacy in patients with cancer (Yu et al., 2021). Due to the location of the metastases in liver, their size, the amounts of liver metastases, residual normal liver, and additional hepatic disease, 85% of these patients are not eligible for surgery (Jemal et al., 2002), representing a very high medical need. The nucleic acid constructs or transgene expression cassettes of the invention therefore represent an important contribution by providing a treatment option for these patients.
For the treatment of cancers residing in the liver, hepatocytes represent an ideal target cell population in order to transduce them for release of IL-12 in the proximity of the tumor. AAV vectors have an excellent safety and efficacy profile documented in over 180 clinical trials (Paulk, 2020) and have been used widely for systemic liver gene delivery due to their natural hepatotropism (Wang et al., 2019). As such, AAV vectors encapsidating the IL-12 gene combined with a riboswitch cassette for toggleable control, would represent an ideal platform for the regulatable IL-12 gene therapy of liver cancers.
In another aspect, the invention relates to a nucleic acid construct or transgene expression cassette as defined herein above or a viral vector according as defined herein above for use in medicine. Specifically, the nucleic acid constructs, transgene expression cassettes and viral vectors are contemplated for use in a method of treating a proliferative disease, such as fibrosis or a cancer disease. Cancer diseases that can be treated by the nucleic acid constructs, transgene expression cassettes and viral vectors of the invention comprise liver cancer, brain cancer, pancreatic cancer, colorectal cancer, esophageal cancer, gastric cancer, hepatocellular cancer, anal cancer, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, testicular cancer, vulvar cancer, skin cancer, urogenital cancer, renal cancer, bladder cancer, head and neck cancer, oropharyngeal cancer, laryngeal cancer, non-small cell lung cancer, small cell lung cancer. In a particularly preferred embodiment of the invention, the nucleic acid constructs, transgene expression cassettes or viral vectors are for use in a method of treating or preventing liver cancer, such as hepatocellular carcinoma (HCC) or cholangiocarcinoma. In another preferred embodiment, the nucleic acid construct, transgene expression cassette or viral vector of the invention is used for treating colorectal cancer.
It is particularly preferred according to the invention that the nucleic acid construct, the transgene expression cassette or the viral vector of the invention is used to treat a patient that has one or more cancer lesions located in the liver. The lesions may result from a primary liver cancer or from a secondary liver cancer. As used herein, secondary liver cancer is understood to refer to metastasis in the liver that result from a primary tumor other than a liver tumor.
If the nucleic acid construct or the transgene expression cassette of the invention is administered to a subject in the form of a viral vector, it is preferred that the viral vector is administered in an amount corresponding to a dose of virus in the range of 1.0×1010 to 1.0×1014 vg/kg (virus genomes per kg body weight), although a range of 1.0×1011 to 1.0×1012 vg/kg is more preferred, and a range of 5.0×1011 to 5.0×1012 vg/kg is still more preferred, and a range of 1.0×1012 to 5.0×1011 is still more preferred. A virus dose of approximately 2.5×1012 vg/kg is most preferred. The amount of the viral vector to be administered, such as the AAV vector according to the invention, for example, can be adjusted according to the strength of the expression of the one or more transgenes.
In another aspect, the invention provides a cell which comprises a nucleic acid construct, a transgene expression cassette or a viral vector as defined herein above.
In yet another aspect, the invention provides a pharmaceutical composition comprising a nucleic acid construct, a transgene expression cassette or a viral vector as defined herein above in combination with a pharmaceutical-acceptable carrier or diluent.
In yet another aspect, the invention provides a method of treating a proliferative disease comprising administering to a patient in need thereof a therapeutically effective amount of a nucleic acid construct, transgene expression cassette or a viral vector as defined herein above. Preferably, the proliferative disease to be treated is a fibrosis or cancer disease. Cancer diseases that can be treated by the nucleic acid constructs, transgene expression cassettes and viral vectors of the invention have been discussed elsewhere herein. The treatment of liver cancer is particularly preferred.
In yet another aspect, the invention relates to the use of a nucleic acid construct, transgene expression cassette or viral vector of the invention for the manufacture of a medicament for treating a proliferative disease, such as a cancer disease.
Riboswitch or control plasmid constructs were cloned using available constructs based on CMV or LP1 promoters and an enhanced GFP (eGFP) transgene as wells as a SV40 poly(A) signal. For packaging into recombinant AAV vectors, all plasmids were equipped with AAV2 ITRs. All AAVs applied in in vivo experiments had an 11nt deletion in the left ITR (see SEQ ID NO: 46 and analogous constructs). Cellular and secreted NanoLuciferase genes were derived from pNL1.1 and pNL1.3 vectors purchased from Promega. The anti-FITC tandem scFv construct was constructed based on published sequences (Vaughan et al. 1996) and synthesized at Life technologies. The K19 riboswitch sequence was derived from Beilstein (Beilstein et al. 2015) and cloned into the reporter constructs flanked by (CAAA) 3 spacer sequences. The sequences encoding murine or human single chain IL-12 were derived from the published sequence mIL-12.p40.L.Ap35 (Lieschke et al., 1997). The human IgG signal peptide was then introduced by PCR and cloned into pCR-TOPOP3.3. Restriction enzyme mediated subcloning of the continuous sequence encoding the signal peptide and single chain IL-12 replaced the reporter genes in the respective AAV plasmids.
HEK293H and HepG2 cells were cultured in DMEM+GlutaMAX+10% FCS at 37° C. 30,000 HEK293 cells per 96-well were seeded 24 h prior to transfection using the Lipofectamine-3000 kit with 35 ng DNA, 0.07 μL P3000, 0.15 μL Lipofectamine-3000 and 10 μL Opti-MEM per well. Master mixes were prepared and up-scaled according to growth area for bigger culture formats. Transfection optimization for HepG2 resulted in 50,000 cells being seeded and transfected using 70 ng DNA, 0.14 μL P3000 and 0.15 μL Lipofectamine-3000 per 96-well. 10,000. Unless stated differently, tetracycline (Tet-HCl, Sigma-Aldrich) was added to cells 1-2 h after transfection and simultaneously to AAV addition in case of transduction. Tet was stored as frozen 2 mM stock solution in water in light-protected single-use aliquots and serially diluted in water prior to its addition (10 μL per 96-well) to the cells.
AAVs were produced in transiently transfected HEK293 cells and quantified by qPCR as described (Strobel et al. 2015a). Briefly, HEK293H cells were cultivated in DMEM+GlutaMAX media supplemented with 10% fetal calf serum. Three days before transfection, the cells were seeded in 15 cm tissue culture plates to reach 70-80% confluency on the day of transfection. For transfection, 0.5 μg total DNA per cm 2 of culture area were mixed with 1/10 culture volume of 300 mM CaCl2 as well as all plasmids required for AAV production in an equimolar ratio. The plasmid constructs were as follows: One plasmid encoding the AAV cap gene (Strobel et al., 2015a); the AAV cis-plasmid containing the expression cassette flanked by ITRs; a pHelper plasmid (AAV Helper-free system, Agilent). The plasmid CaCl2 mix was then added dropwise to an equal volume of 2× HBS buffer (50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4), incubated for 2 min at room temperature and added to the cells. After 5-6 h of incubation, the culture medium was replaced by fresh medium. The transfected cells were grown at 37° C. for a total of 72 h. Cells were detached by addition of EDTA to a final concentration of 6.25 mM and pelleted by centrifugation at room temperature and 1000× g for 10 min. The cells were then resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM MgCl2, pH 8.5).
AAV vectors were purified essentially as previously described (Strobel et al., 2015a): For iodixanol gradient based purification, cells harvested from up to 40 plates were dissolved in 8 mL lysis buffer. Cells were then lysed by three freeze/thaw cycles using liquid nitrogen and a 37° C. water bath. For each initially transfected plate, 100 units Benzonase nuclease (Merck) were added to the mix and incubated for 1 h at 37° C. After pelleting cell debris for 15 min at 2500× g, the supernatant was transferred to a 39 mL Beckman Coulter Quick-Seal tube and an iodixanol (OptiPrep, Sigma Aldrich) step gradient was prepared by layering 8 mL of 15%, 6 mL of 25%, 8 mL of 4% and 5 mL of 58% iodixanol solution diluted in PBS-MK (lx PBS, 1 mM MgCl2, 2.5 mM KCl) below the cell lysate. NaCl had previously been added to the 15% phase at 1 M final concentration. 1.5 μL of 0.5% phenol red had been added per mL to the 15% and 25% iodixanol solutions and 0.5 μL had been added to the 58% phase to facilitate easier distinguishing of the phase boundaries within the gradient. After centrifugation in a 70Ti rotor for 2 h at 63000 rpm and 18° C., the tube was punctured at the bottom. The first five milliliters (corresponding to the 58% phase) were then discarded, and the following 3.5 mL, containing AAV vector particles, were collected. PBS was added to the AAV fraction to reach a total volume of 15 mL and ultrafiltered/concentrated using Merck Millipore Amicon Ultra-15 centrifugal filter units with a MWCO of 100 kDa. After concentration to ˜1 mL, the retentate was filled up to 15 mL and concentrated again. This process was repeated three times in total. Glycerol was added to the preparation at a final concentration of 10%. After sterile filtration using the Merck Millipore Ultrafree-CL filter tubes, the AAV product was aliquoted and stored at −80° C.
9-12 weeks old C57BL/6 mice with a body weight of 19-21 g were purchased from Charles River laboratories. AAVs were diluted to the desired concentration in PBS and administered into the tail vein in a volume of 100 μL per mouse, under light isoflurane anesthesia. For preparation of a 100 mg/kg tetracycline solution, 20 mg tetracycline-HCl were dissolved in 400 μL 25% 2-hydroxypropyl beta-cyclodextrin solution (HP-β-CD, Sigma Aldrich) +600 μL PBS and adjusted to about pH 6 by addition of 35 μL 1M NaOH. For lower doses, this solution was serially diluted in PBS+HP-β-CD. Tet solutions were prepared immediately before i.p. administration (200 μL per mouse). At the respective time points, 20 μL blood were sampled by puncturing the vena saphena and collected using K3-EDTA Microvette POCT 20 μL capillary microtubes (Sarstedt) followed by centrifugation. Blood plasma was used for quantitative anti-FITC and tetracycline measurements. At the final blood draw, additional serum samples were prepared for the assessment of liver enzymes. Organs of interest were dissected and snap-frozen in liquid nitrogen for DNA/RNA extraction or the preparation of protein tissue lysates. For experiments in tumor-bearing mice, luciferase-expressing Hepal-6 tumor cells (1.0×106 cells in 50 μL PBS) were injected into the spleen of each mouse under anesthesia and allowed to migrate into the liver for 5 min via vena lienalis. Thereafter the spleen was resected. Mice received the analgeticum Meloxicam (1.0 mg/kg in 10.0 ml/kg) subcutaneously 1-2 h before surgery and 24 h later. Body weight and tumor growth were monitored. Tumor volumes were determined by in vivo bioluminescence using an IVIS® Lumina III bioluminescence imaging system (Perkin Elmer) with a CCD-camera. For this purpose, 150 mg/kg (7.5 mL/kg) D-Luciferin in aqua dest. was injected i.p. 8 min before anesthetization. Light emission was measured 10 min post injection. Tumor-bearing mice were block-randomized according to tumor sizes measured by the in vivo biolumines-cence imaging of the same day. For block-randomization, a robust automated random number generation within individual blocks was used (MS-Excel 2016).
eGFP expression was assessed by fluorescence microscopy or direct fluorescence detection using the Molecular Devices SpextraMax i3x with a MiniMax 300 imaging unit. Nano-Luciferase measurements were performed using the Promega Nano-Glo Luciferase assay as per manufacturer's instructions. If required, appropriate sample dilutions were identified prior to assessment. A detailed description of the anti-FITC ELISA setup and measurement is provided further below.
HEK293 cells were transfected using the calcium phosphate method as described for AAV production, using 30 μg of CMV-aFITC expression plasmid per 15-cm culture dish. 48 h after transfection, the culture supernatant was harvested and centrifuged at 400×g for 5 min. 45 mL supernatant were then mixed with 60 μL of anti-VS beads and purified using the “VS-tagged protein purification kit Ver.2” (3317, MBL) as per instructions. Following protein elution, V5 elution peptide was removed by ultrafiltration. Therefore, 40 μL protein eluate were added to a PBS-pre-equilibrated Vivaspin 500 column (VS0101, Sartorius) and filled up to 500 μL using PBS. Following centrifugation at 15000×g for approximately 2 min to reach a retentate volume of 50 μL, PBS was again added to 500 μL and centrifuged again. This process was repeated three times in total. After retentate recovery, anti-FITC protein was aliquoted and frozen at −20° C. Protein concentration was determined using NanoDrop-One measurements at 280 nm and calculations based on a protein size of 57 kDa and a molar extinction coefficient of 116,240 M−1 cm−1.
A standard MSD plate (L15XA-1) was coated with 30 μL of BSA-FITC (A23015, Molecular Probes) and diluted to 0.25 μg/mL in PBS under shaking for 5 min at 750 rpm. After incubation at 4° C. overnight (or 1 h at room temperature (RT)), the plate was washed three times using 300 μL/well wash buffer (PBS+0.05% Tween-20). 150 μL of blocking solution (3% Blocker A (R93BA-2, MSD) in PBS) were then added and incubated for 1 h at 750 rpm and RT. After washing three times, 25 μL of each sample, stand- and (diluted in 1% Blocker A in PBS) or blank were added per well and incubated for 1 h at 750 rpm and RT. Detection antibody (biotinylated rabbit anti-V5, ab18617, Abcam) was diluted to 1 μg/mL in 1% Blocker A in PBS and SULFO-tag labeled streptavidin (R32AD-5, MSD) was diluted to 0.5 μg/mL in 1% Blocker A in PBS. After washing the plate three times, 25 μL of antibody and streptavidin dilutions were added simultaneously to each well and incubated for 1 h at 750 rpm and RT. Following three washing steps, 150 μL/well of 2× Read Buffer T (R92TC-2, MSD) diluted in water were added per well. The plate was then read using an MSD Sector Imager 600.
Expression of IL-12 and IFNγ were analyzed using the Mouse IL-12p70 or the Proinflammatory Panel 1 Mouse Kit (K152ARB, K15048D, MSD) according to the manufacturer's instructions. Lowest standard of provided IL12p70 was taken as lower limit of detection (LLOD).
Flash frozen tissue samples were homogenized in 100 μL MSD lysis buffer (R6OTX-2), using a Precellys-24 homogenizer and ceramic (KT03961-1-009.2, VWR) or metal bead tubes (KT03961-1-001.2) at 6000 rpm for 30 sec. Homogenates were immediately placed on ice, followed by addition of additional 900 μL lysis buffer. A second round of homogenization was then carried out. Samples were again cooled on ice and centrifuged for 10 min at 20,000×g. 700 μL of supernatant were recovered and protein concentration was determined using a BCA assay (Promega). Homogenates were stored at −80° C.
Tissue samples were flash frozen immediately after dissection. For DNA and RNA isolation, samples were homogenized in 900 μL RLT buffer (79216, Qiagen), using a Precellys-24 homogenizer and ceramic bead tubes (KT03961-1-009.2, VWR) at 6000 rpm for 30 sec. Afterwards, samples were immediately placed on ice. 350 μL Phenolchloroform-isoamyl alcohol (77617, Sigma Aldrich) were then added to 700 μL homogenate in a Phase Lock gel tube and mixed by shaking. Following centrifugation for 5 min at 16000×g, 350 μL Chloroform-isoamyl alcohol (25666, Sigma-Aldrich) were added and the mixture was shaken again. After 3 min of incubation at RT and centrifugation for 5 min at 12000×g, the upper (aqueous) phase was collected and pipetted into a deep well plate placed on dry ice. After processing of all samples, DNA and RNA were purified, using the AllPrep DNA/RNA 96 kit (80311, Qiagen) as per instructions, including the optional “on-column DNase digestion” step. RNA from cell cultures was isolated by pelleting cells, followed by lysis in 350 μL RLT buffer and purification using the RNeasy mini kit (74104, Qiagen).
1.11 Analysis of Gene Expression and AAV Vector Genomes (qPCR and ddPCR)
For gene expression analysis, equal amounts of RNA were reverse transcribed to cDNA using the High-capacity cDNA RT kit (4368814, Thermo Fisher) as per instructions. qRT-PCR reactions were set up using the QuantiFast Probe RT-PCR kit (204456, Qiagen) and primers specifically binding the K19 riboswitch sequence or the anti-FITC gene. Expression was normalized to RNA polymerase II housekeeper expression. AAV vector genomes were detected using extracted DNA either for ddPCR or qPCR. For qPCR a standard curve was generated by serial dilutions of the respective expression plasmid. qPCR runs were performed on an Applied Biosystems ViiA 7 Real-Time PCR System. For ddPCR, Automated Droplet Generator, QX200 Droplet Digital PCR System, and QX200 Droplet Reader (all Bio-rad) were applied.
Pharmacokinetic of tetracycline was investigated in 12 weeks old (approximately 30 g body weight) male C57BL/6 mice purchased from Janvier Labs. A Tet solution was administered i.p. at an administration volume of 10 mL/kg and a dose of 54 mg/kg. The Tet solution contained 10% 2-hydroxypropyl beta-cyclodextrin and was adjusted to pH 6. Serial blood sampling was performed via puncture of the saphenous vein into K3-EDTA coated vials. A maximum volume of 20 μL blood was collected per sampling time point. Plasma samples were prepared by centrifugation. For tissue distribution, intraperitoneal dosing was performed as described above in the same animals at day 3. The mice were sacrificed two hours after Tet administration and subsequently the brain, liver, kidney, heart, lungs, both eyes, a piece of leg muscle and a blood sample were collected. Tissue weights were recorded and all samples were stored at −20° C. prior to bioanalysis. Plasma protein was precipitated with acetonitrile. Tissue samples were transferred to Precellys vials and three parts of acetonitrile/methanol (1:1) and one part of water was added for the homogenization step. All samples were centrifuged prior to bioanalysis. Compound concentrations were determined by high performance liquid chromatography coupled with tandem mass spectrometry.
All measurements were performed using the Konelab PRIME 60 and test kits from Thermo Scientific (following the Konelab Chemistry Information Manual 12A/2003, March 2003) and spectrophotometrical assessment at 340 nm. Aspartate aminotransferase (AST) activity was measured by an enzymatic rate method (Schumann et al. 2002a) without pyridoxal-5′-phosphate for AST activation. Alanine aminotransferase (ALT) activity was measured by an enzymatic rate method based on the IFCC method (Schumann et al. 2002b). without adding pyridoxal-5′-phosphate. The removal of NADH was measured spectrophotometrically at 340 nm. Glutamate dehydrogenase (GLDH) activity was measured by an enzymatic rate method, using a kit supplied by Roche Diagnostics.
A bioassay is employed to measure human or mouse IL-12 bioactivity as a function of the proliferation of phytohaemagglutinin (PHA)-activated human lymphoblasts, as described by Gately et al., 1995.
Briefly, this functional assay is based on the ability of IL-12 to stimulate proliferation of PHA-activated T lymphoblasts (“PHA blasts”). In this assay, IL-12 that has been bound to immobilized anti-IL-12 antibody stimulates proliferation of PHA-activated human lymphoblasts. Human or mouse IL-12 is captured from IL-12-containing culture fluid or serum by anti—human IL-12 or anti—mouse IL-12 antibody adsorbed to the wells of an EIA (enzyme immunoassay) plate. The test fluid is then washed from the wells and replaced with a PHA-activated human lymphoblast suspension. The lymphoblasts proliferation in response to the captured IL-12 is measured. Commercially available bioactive human IL-12 recombinant protein consisting of two subunits linked via a disulphide bond (for example Thermo Fisher Scientific; Cat. #PHC1124) is used as a standard.
As an alternative, a commercial IL-12 Bioassay (Promega GmbH; Cat.#J3042) can also be employed. This is a bioluminescent cell-based assay designed to measure IL-12 stimulation or inhibition and is performed according to manufacturer's instructions. Briefly, the IL-12 Bioassay consists of a genetically engineered human cell line that expresses a luciferase reporter driven by a response element (RE). When IL-12 binds to IL-12R it transduces intracellular signals resulting in luminescence. The bioluminescent signal is detected and quantified using Bio-Glo™ Luciferase Assay System (Cat.#G7940, G7941) and a standard luminometer.
As another alternative, a HEK-Blue™ assay can be used for showing IL-12 bioactivity in vitro. HEK-Blue™ IL-12 cells (InvivoGen, #hkb-i112) are designed to detect bioactive human and murine IL-12. The human embryonic kidney HEK293-based cell line expresses the human genes for the IL-12 receptor and the genes of the IL-12 signaling pathway into line, and a STAT4-inducible SEAP reporter gene. Cell surface ligand binding triggers a signaling cascade activating STAT-4 and production of the reporter protein secreted alkaline phosphatase (SEAP). SEAP can be detected in the supernatant using QUANTI-Blue™ Solution according to manufacturer's instructions. To show in vitro bioactivity of IL-12 expressed from expression plasmids, AAV plasmids or AAV vectors, cells are cultured, transfected with plasmids or transduced with AAV vectors, and a reporter assay carried out according to manufacturer information.
Statistical calculations were performed using GraphPad Prism V7.03.
Tissue samples of rat liver were fixed in 4% PFA and paraffin embedded (formalin fixed and paraffin embedded, FFPE). 3 μm thick sections of FFPE tissue on super frost plus slides were deparaffinised and rehydrated by serial passage through changes of xylene and graded ethanol for H&E and immunohistochemistry staining.
H&E staining was performed according to standard protocols (Romeis, Mikroskopische Technik; Hrsg. P. Böck; Urban and Schwarzenberg; München, Wien, Baltimore; 19. Auflage; 2015; pp 201; ISBN: 978-3-642-55189-5)
For Immunohistochemistry, antigen retrieval was performed by incubating the sections in Leica Bond Enzyme solution (Bond Enzyme Pre-treatment Kit, Cat#35607) for 5 minutes. Sections were incubated with an anti-CD45 antibody (abcam, ab10558, rabbit polyclonal). The antibody was diluted (1:400) with Leica Primary Antibody Diluent (AR9352; Leica Biosystems, Nussloch, Germany) and incubated for 30 min at room temperature. Bond Polymer Refine Detection, (Cat#37072) was used for detection (3,3′ Diaminobenzidine as chromogen, DAB) and counterstaining (hematoxylin). Staining was performed on the automated Leica IHC Bond-III platform (Leica Biosystems, Nussloch, Germany). Microscopic assessment of samples was conducted with a Zeiss Axiolmager M2 microscope and ZEN slidescan software (Zeiss, Oberkochen, Germany).
Tumor size was calculated using the image processing software HALO 3.1. A classifier based on DenseNET (Huang et al., 2017) was trained with 16 sample regions from background, healthy and cancerous tissue.
For quantitative analysis anti-CD45 stained sections of the liver were scanned with an Axio Scan.Z1 whole slide scanner (Carl Zeiss Microscopy GmbH, Jena, Germany) using an 20× objective (0.22 μm/px) in bright field illumination. The percentage of the anti-CD45-positive cells was calculated using the image processing software HALO 3.1 with CytoNuclear v2.0.9 module (Indica Labs, Corrales, NM, USA). Cell count analysis was then restricted to ‘normal’ tissue, segmented in a pre-processing step using a built-in classifier (QC Slide). The analysis module uses color deconvolution to split signals of hematoxylin and DAB. Parameters for cell detection in the hematoxylin image and thresholds for positive DAB staining intensities in the cytoplasm were optimized manually. The summed percentage of strongly and moderately stained DAB positive cells were used in the quantitative analysis.
To evaluate functionality of the K19 aptazyme in the AAV vector context, K19 was cloned into a plasmid containing AAV2 inverted terminal repeats (ITRs) and a CMV promoter-driven eGFP gene. K19 was either positioned 5′ upstream, 3′ downstream or at both positions relative to the eGFP gene (
To deepen the insight into temporal gene expression regulation, an mRNA-focused kinetics experiment was conducted. Therefore, a qPCR probe spanning the aptazyme auto-cleavage site was designed, allowing direct assessment of eGFP mRNA cleavage. Twenty-four hours after HEK293 cell transfection with plasmids containing either the active or inactive K19 switch, media were changed, baseline samples were taken and Tet was added to all remaining cultures, before being lysed at several time points to obtain RNA and protein for gene expression analysis. Slight but steadily increasing eGFP expression induction was seen from 15 min after Tet addition and full induction was observed after four hours on the mRNA level (
Because the tetracycline aptamer domain of the K19 aptazyme specifically binds Tet but not Doxycycline, preparation for the evaluation of the riboswitch system in mice included Tet pharmacokinetic (PK) studies, for which preclinical in vivo data is scarce. Following administration of 54 mg/kg i.p., peak plasma concentrations of 42 μM at 30 mins and residual levels of 3.3 μM at 8 h were measured, corresponding to a half-life of approx. 2.8 h (
Next, alternative reporter proteins were tested that would allow measuring switching performance and kinetics in vivo, ideally in a multiplexed fashion. We decided for a secreted anti-Fluorescein isothiocyanate (aFITC) tandem single chain variable fragment (scFv) antibody (Vaughan et al. 1996; Honegger et al. 2005) under the control of the liver-specific LP1 promoter (Nathwani 2006) and a CMV promoter expressed, non-secreted Nano-Luciferase (cNLuc) and cloned appropriate expression constructs and controls (
Previous studies have tested aptazyme designs in the muscle and eye of mice, however, either OFF-designs were used (Zhong et al. 2016) or the ON-designs led to very modest effects (Reid et al. 2018). Moreover, aptazyme functionality across different organs has not been studied so far. Therefore, an experiment was designed herein to explore ONswitch potency and functionality across organs in a simultaneous fashion. Specifically, advantage was taken of the broad transduction pattern of recombinant AAV9 following i.v. administration (Zincarelli et al. 2008) to simultaneously express and study regulation of 1) intracellularly expressed cNLuc in the liver, lung, heart and muscle tissue and 2) the transcriptionally liver-targeted, secreted aFITC antibody, by measuring its levels in plasma. Therefore, mixtures of AAV9-CMV-cNLuc-K19 (mediating ubiquitous expression) and AAV9-LP1-aFITC-K19 (mediating liver specific expression) were administered to mice (1x10 11 vg in total per animal, N=8 animals per group). Two weeks after AAV administration, a total of four 100 mg/kg doses of Tet was administered in 8 h intervals to induce expression from the aptazyme constructs (see scheme in
In addition to the potent induction of aFITC, successful regulation was also observed for the intracellular expression of cNLuc driven by a CMV promoter. In the liver, a 3.3-fold increase upon Tet treatment was observed by measuring luciferase activity in tissue lysates (
Although impeded by the fact that by the time of induction, basal cNLuc expression had already proceeded for two weeks, leading to intracellular accumulation and less pronounced induction, our results clearly demonstrate that gene expression induction by a riboswitch is feasible in different organs in mice. Another interesting finding in this regard was that total Tet exposure in the liver was approximately 18-fold higher than in the heart and muscle, but nevertheless, CMV promoter-driven intracellular cNLuc expression was similarly induced by the switch (3.3-, 4.1- and 2-fold in liver, heart and muscle, respectively). Given that AAV9 transduction efficiency is similar in liver and heart (Zincarelli et al. 2008), these results might suggest that cardiac expression could be particularly well regulated by a riboswitch, possibly due to higher transcriptional and/or mRNA degradation activity. While systematic follow-up studies are required to prove this particular hypothesis, our data support the general assumption that the potency of riboswitch-controlled gene regulation might be partly dependent on the cellular context.
Having demonstrated functionality of the Tet switch in an in vivo setting, we examined how Tet-induced expression levels compare to those of a conventional riboswitch-free construct, mediating constitutive expression. We therefore re-assessed expression induction in a simple follow-up experiment, including an AAV9-LP1-aFITC control vector and a single Tet trigger (100 mg/kg) to induce expression in mice (
One expected feature of riboswitch vectors, which, however, has not been proven so far, is the potential to fine-tune expression levels in vivo by adjusting the dose of ligand. Moreover, aptazymes should in principle allow for repeated, i.e. dynamic ON-OFF switching, yet also this aspect has not been experimentally proven in animals so far. Therefore, we finally investigated the degree and kinetics of reporter expression induction by four different (3, 10, 30, 90 mg/kg) single-dose Tet administrations (N=8 animals per group) and further explored the possibility to re-stimulate expression one week after the first induction. Pharmacokinetic (PK) measurements further enabled to investigate associated PK/PD relationships. For this experiment, we again made use of the AAV9LP1-aFITC vectors (
To assure complete Tet clearance and to simulate a treatment-free phase with desired expression shutdown, proofing persistent riboswitch activity (compare
In contrast to the previous experiment, using multiple dosing (
Pharmacokinetic and -dynamic (PD) measurements finally allowed for correlation assessment between Tet plasma levels (
Following successful proof of concept in the context of reporter genes, the reporter gene was next replaced by the IL-12 gene encoding murine single chain IL-12 and packaged as AAV9. Transduction of HepG2 cells with the active K19-IL-12 vector carrying the liver-specific LP1 promoter revealed 3% background levels of IL-12 in the supernatant and a 6.4-fold induction at the highest Tet dose (
The following
The dose-finding study using i.v. delivery of the AAV9 harboring the constitutive mIL-12-K19 inactive construct to naïve C57BL/6 mice at three different doses (5×109, 5×1010, 5×1011 vg) is shown in
The low vector dose was selected to be used for all vectors nominated to be assessed for a PD study on Tet-induced IL-12 expression in naive mice (N=5 per group). The study design (
In addition, we showed in HEK293 cells that the K19 riboswitch responds to Tet by inducing human IL-12 (5.6-fold) in a drug-dose-dependent manner (
Finally, using HEK-Blue™ IL-12 cells, we showed bioactivity of human single chain IL-12 contained in supernatants of HEK293 cell that had been transfected with plasmids encoding human single chainIL-12 (
In summary, these data suggest that IL-12 gene therapy can be tightly controlled in a spatio-temporal manner for a safe and efficient immunomodulatory effect using a rational combination of AAV serotype, vector dose, Tet dosing regimen and target organ. The IL-12 data confirm the potential to fine tune expression levels of a therapeutic protein in vivo by adjusting the dose of ligand in a riboswitch context. Moreover, the aptazyme-mediated control over IL-12 expression enables for repeated, i.e. dynamic ON-OFF switching.
Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402
Ausländer, S., and Fussenegger, M. (2013) From gene switches to mammalian designer cells: present and future prospects, Trends Biotechnol. 31, 155-168
Barrett, J. A., Cai, H., Miao, J., Khare, P. D., Gonzalez, P., Dalsing-Hernandez, J., Sharma, G., Chan, T., Cooper, L. J. N., and Lebel, F. (2018) Regulated intratumoral expression of IL-12 using a RheoSwitch Therapeutic System((R)) (RTS((R))) gene switch as gene therapy for the treatment of glioma, Cancer Gene Ther 25, 106-116
Beilstein, K., Wittmann, A., Grez, M., and Suess, B. (2015) Conditional Control of Mammalian Gene Expression by Tetracycline-Dependent Hammerhead Ribozymes, ACS synthetic biology, 4, 526-534
Berens, C., Thain, A., and Schroeder, R. (2001) A tetracycline-binding RNA aptamer, Bioorg Med Chem 9, 2549-2556
Berens, C., Groher, F., and Suess, B. (2015) RNA aptamers as genetic control devices: the potential of riboswitches as synthetic elements for regulating gene expression, Biotechnology Journal 10, 246-257
Berraondo, P., Etxeberria, I., Ponz-Sarvise, M., and Melero, I. (2018) Revisiting Interleukin-12 as a Cancer Immunotherapy Agent, Clin Cancer Res 24, 2716-2718
Bloom, R. J., Winkler, S. M., and Smolke, C. D. (2015) Synthetic feedback control using an RNAi-based gene-regulatory device, J Biol Eng 9, 5
Cao G. et al. 1998. Comparison of carcinoembryonic antigen promoter regions isolated from human colorectal carcinoma and normal adjacent mucosa to induce strong tumor specific gene expression. Int J Cancer 78:242-247
Chen L. et al. 1995. Breast cancer selective gene expression and therapy mediated by recombinant adenoviruses containing the DF3/MUC1 promoter. J. Clin. Invest. 96:2775-2782
Chen, Y. Y., Jensen, M. C., and Smolke, C. D. (2010) Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems, Proc. Natl. Acad. Sci. U.S.A 107, 8531-8536
Chiocca E A, Yu J S, Lukas R V, Solomon I H, Ligon K L, Nakashima H, Triggs D A, Reardon D A, Wen P, Stopa B M, Naik A, Rudnick J, Hu J L, Kumthekar P, Yamini B, Buck J Y, Demars N, Barrett J A, Gelb A B, Zhou J, Lebel F, Cooper L J N. (2019). Regulatable interleukin-12 gene therapy in patients with recurrent high-grade glioma: Results of a phase 1 trial. Sci Transl Med.11(505).
Choi, Y. J., Lee, C. H., Lee, K. Y., Jung, S. H., and Lee, B. H. (2015) Increased hepatic Fatty Acid uptake and esterification contribute to tetracycline-induced steatosis in mice, Toxicol Sci 145, 273-282
Cohen J. (1995). IL-12 Deaths: Explanation and a Puzzle. Science 270:908.
Das A T, Tenenbaum L, Berkhout B. (2016). Tet-On Systems For Doxycycline-inducible Gene Expression. Curr Gene Ther. 16(3):156-67.
Dillekas H, Rogers M S, Straume O. Are 90% of deaths from cancer caused by metastases? (2019). Cancer Med. 8(12):5574-5576.
Gately, M. K, Chizzonite R., Presky D. H. (1995). Measurement of human and mouse interleukin-12. Curr Protoc Immunol 1995;6(16):1-15.
Ginhoux F, Turbant S, Gross DA, Poupiot J, Marais T, Lone Y, Lemonnier FA, Firat H, Perez N, Danos 0, Davoust J. (2004). HLA-A*0201-restricted cytolytic responses to the rtTA transactivator dominant and cryptic epitopes compromise transgene expression induced by the tetracycline on system. Mol Ther. 10(2):279-89.
Gonzalez-Aparicio M, Alzuguren P, Mauleon I, Medina-Echeverz J, Hervas-Stubbs S, Mancheno U, Berraondo P, Crettaz J, Gonzalez-Aseguinolaza G, Prieto J, Hernandez-Alcoceba R. (2011). Oxaliplatin in combination with liver-specific expression of interleukin 12 reduces the immunosuppressive microenvironment of tumours and eradicates metastatic colorectal cancer in mice. Gut 60(3):341-9.
Greig J A, Wang Q, Reicherter A L, Chen S J, Hanlon A L, Tipper C H, Clark K R, Wadsworth S, Wang L, Wilson J M. (2017). Characterization of Adeno-Associated Viral Vector-Mediated Human Factor VIII Gene Therapy in Hemophilia A Mice. Hum Gene Ther. 2017 28:392-402
Grimm, D., and Büning, H. (2017) Small But Increasingly Mighty: Latest Advances in AAV Vector Research, Design, and Evolution, Human Gene Therapy 28, 1075-1086
Higgins et al., 1996, Methods Enzymol. 266:383-402
Honegger, A., Spinelli, S., Cambillau, C., and Plückthun, A. (2005) A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex, Protein Sci. 14, 2537-2549
Huang, G.; Liu, Z.; van der Maaten, L.; Weinberger, K. Q. (2017). Densely Connected Convolutional Networks. In Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, Honolulu, HI, USA, 21-26 Jul. 2017.
Jemal, A., Thomas, A., Murray, T., Thun, M. (2002). Cancer statistics, 2002. CA Cancer J Clin. 52(1):23-47.
Jiang, D. J., Xu, C. L., and Tsang, S. H. (2018) Revolution in Gene Medicine Therapy and Genome Surgery, Genes (Basel) 9, 575
Kamimura K, Yokoo T, Abe H, Terai S. (2019). Gene Therapy for Liver Cancers: Current Status from Basic to Clinics. Cancers (Basel). 11(12):1865.
Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268
Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877
Kennedy, A. B., Vowles, J. V., d'Espaux, L., and Smolke, C. D. (2014) Proteinresponsive ribozyme switches in eukaryotic cells, Nucleic Acids Research. 19, 12306-12321
Ketzer, P., Haas, S. F., Engelhardt, S., Hartig, J. S., and Nettelbeck, D. M. (2012) Synthetic riboswitches for external regulation of genes transferred by replication-deficient and oncolytic adenoviruses, Nucleic Acids Research 40, e167
Ketzer, P., Kaufmann, J. K., Engelhardt, S., Bossow, S., von Kalle, C., Hartig, J. S., Ungerechts, G., and Nettelbeck, D. M. (2014) Artificial riboswitches for gene expression and replication control of DNA and RNA viruses, Proc. Natl. Acad. Sci. U.S.A 111, E554-562
Kitada, T., DiAndreth, B., Teague, B., and Weiss, R. (2018) Programming gene and engineered-cell therapies with synthetic biology, Science 359
Kumar, D., An, C.-I., and Yokobayashi, Y. (2009) Conditional RNA interference mediated by allosteric ribozyme, J. Am. Chem. Soc. 131, 13906-13907
Kumar, S. R., Markusic, D. M., Biswas, M., High, K. A., and Herzog, R. W. (2016) Clinical development of gene therapy: results and lessons from recent successes, Mol Ther Methods Clin Dev 3, 16034
Lan K H. et al. 1997. In vivo selective gene expression and therapy mediated by adenoviral vectors for human carcinoembryonic antigen-producing gastric carcinoma. Cancer Res. 57(19):4279-84
Leonard, J. P., Sherman, M. L., Fisher, G. L., Buchanan, L. J., Larsen, G., Atkins, M. B., Sosman, J. A., Dutcher, J. P., Vogelzang, N. J., and Ryan, J. L. (1997) Effects of singledose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production, Blood 90, 2541-2548
Levitt, N., Briggs, D., Gil, A., Proudfoot, N. J. (1989). Definition of an efficient synthetic poly(A) site. Genes Dev. 3(7):1019-25.
Lieschke G J I, Rao P K, Gately M K, Mulligan R C. Bioactive murine and human interleukin-12 fusion proteins which retain antitumor activity in vivo. Nat Biotechnol. 1997 January;15(1):35-40.
Lo C H, Chang C M, Tang S W, Pan W Y, Fang C C, Chen Y, Wu P Y, Chen K Y, Ma H I, Xiao X, Tao M H. (2010). Differential antitumor effect of interleukin-12 family cytokines on orthotopic hepatocellular carcinoma. J Gene Med. 12(5):423-34.
Manno, C. S., Pierce, G. F., Arruda, V. R., Glader, B., Ragni, M., Rasko, J. J., Rasko, J., Ozelo, M. C., Hoots, K., Blatt, P., Konkle, B., Dake, M., Kaye, R., Razavi, M., Zajko, A., Zehnder, J., Rustagi, P. K., Nakai, H., Chew, A., Leonard, D., Wright, J. F., Lessard, R. R., Sommer, J. M., Tigges, M., Sabatino, D., Luk, A., Jiang, H., Mingozzi, F., Couto, L., Ertl, H. C., High, K. A., and Kay, M. A. (2006) Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response, Nature Medicine 12, 342-347.
Nathwani A C, Gray J T, Ng C Y, Zhou J, Spence Y, Waddington S N, Tuddenham E G, Kemball-Cook G, McIntosh J, Boon-Spijker M, Mertens K, Davidoff A M. (2006) Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver, Blood 107, 2653-2661
Nathwani, A. C., Tuddenham, E. G. D., Rangarajan, S., Rosales, C., McIntosh, J., Linch, D. C., Chowdary, P., Riddell, A., Pie, A. J., Harrington, C., O'Beirne, J., Smith, K., Pasi, J., Glader, B., Rustagi, P., Ng, C. Y. C., Kay, M. A., Zhou, J., Spence, Y., Morton, C. L., Allay, J., Coleman, J., Sleep, S., Cunningham, J. M., Srivastava, D., Basner-Tschakarjan, E., Mingozzi, F., High, K. A., Gray, J. T., Reiss, U. M., Nienhuis, A. W., and Davidoff, A. M. (2011) Adenovirus-associated virus vector-mediated gene transfer in hemophilia B, N. Engl. J. Med. 365, 2357-2365
Nathwani, A. C., Reiss, U. M., Tuddenham, E. G. D., Rosales, C., Chowdary, P., McIntosh, J., Della Peruta, M., Lheriteau, E., Patel, N., Raj, D., Riddell, A., Pie, J., Rangarajan, S., Bevan, D., Recht, M., Shen, Y.-M., Halka, K. G., Basner-Tschakarj an, E., Mingozzi, F., High, K. A., Allay, J., Kay, M. A., Ng, C. Y. C., Zhou, J., Cancio, M., Morton, C. L., Gray, J. T., Srivastava, D., Nienhuis, A. W., and Davidoff, A. M. (2014) Long-term safety and efficacy of factor IX gene therapy in hemophilia B, N. Engl. J. Med. 371, 1994-2004
Nomura, Y., Zhou, L., Miu, A., and Yokobayashi, Y. (2013) Controlling mammalian gene expression by allosteric hepatitis delta virus ribozymes, ACS synthetic biology 2, 684-689
Okuyama T, Huber RM, Bowling W, et al. Liver-directed gene therapy: a retroviral vector with a complete LTR and the ApoE enhancer-alpha 1-antitrypsin promoter dramatically increases expression of human alpha 1-antitrypsin in vivo. Human Gene Therapy. 1996 March;7(5):637-645. DOI: 10.1089/hum.1996.7.5-637.
Paulk, N. (2020). Gene Therapy: It's Time to Talk about High-Dose AAV. The deaths of two children with X-linked myotubular myopathy in the ASPIRO trial prompts a reexamination of vector safety. Genetic Engineering & Biotechnology News 40(9):14-16.
Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8
Cabrera-Pérez R, Vila-Julia F, Hirano M, Mingozzi F, Torres-Torronteras J, Marti R. Alpha-1-Antitrypsin Promoter Improves the Efficacy of an Adeno-Associated Virus Vector for the Treatment of Mitochondrial Neurogastrointestinal Encephalomyopathy. Hum Gene Ther. 2019 August;30(8):985-998. doi: 10.1089/hum.2018.217. Epub 2019 Apr 24. PMID: 30900470; PMCID: PMC7647930.
Qian C, Liu X Y, Prieto J. (2006). Therapy of cancer by cytokines mediated by gene therapy approach. Cell Res. 16(2):182-8.
Michael Quante, Steffen Heeg, Alexander von Werder, Gitta Goessel, Christine Fulda, Michaela Doebele, Hiroshi Nakagawa, Roderick Beijersbergen, Hubert E Blum, Oliver G Opitz (2005). Differential transcriptional regulation of human telomerase in a cellular model representing important genetic alterations in esophageal squamous carcinogenesis, Carcinogenesis vol.26 no.11 pp. 1879-1889
Reid, C. A., Nettesheim, E. R., Connor, T. B., and Lipinski, D. M. (2018) Development of an inducible anti-VEGF rAAV gene therapy strategy for the treatment of wet AMD, Sci Rep 8, 11763
Russell, S., Bennett, J., Wellman, J. A., Chung, D. C., Yu, Z.-F., Tillman, A., Wittes, J., Pappas, J., Elci, O., McCague, S., Cross, D., Marshall, K. A., Walshire, J., Kehoe, T. L., Reichert, H., Davis, M., Raffini, L., George, L. A., Hudson, F. P., Dingfield, L., Zhu, X., Haller, J. A., Sohn, E. H., Mahaj an, V. B., Pfeifer, W., Weckmann, M., Johnson, C., Gewaily, D., Drack, A., Stone, E., Wachtel, K., Simonelli, F., Leroy, B. P., Wright, J. F., High, K. A., and Maguire, A. M. (2017) Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial, Lancet 390, 849-860
Samulski R. J., Srivastava A., Berns K. I., Muzyczka, N. (1983). Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV. Cell 33(1):135-43.
Santiago, C. P., Keuthan, C. J., Boye, S. L., Boye, S. E., Imam, A. A., and Ash, J. D. (2018) A Drug-Tunable Gene Therapy for Broad-Spectrum Protection against Retinal Degeneration, Mol Ther 26, 2407-2417.
Sarcar, S., Tulalamba, W., Rincon, M. Y., Tipanee, J., Pham, H. Q., Evens, H., Boon, D., Samara-Kuko, E., Keyaerts, M., Loperfido, M., Berardi, E., Jarmin, S., In't Veld, P., Dickson, G., Lahoutte, T., Sampaolesi, M., De Bleser, P., VandenDriessche, T., and Chuah, M. K. (2019) Next-generation muscle-directed gene therapy by in silico vector design, Nat Commun 10, 492
Schumann et al. 2002a IFCC Primary Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes at 37° C., Clin Chem Lab Med 2002; 40(7):718-724© 2002 by Walter de Gruyter·Berlin·New York
Schumann et al. 2002b, IFCC Primary Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes at 37° C. Clin Chem Lab Med 2002; 40(6):643-648© 2002 by Walter de Gruyter·Berlin·New York
Sheridan, C. (2015). Mayo clinician reports on patient death in oncolytic virus trial. https://www.bioworld.com/articles/396511-mayo-clinician-reports-on-patient-death-in-oncolytic-virus-trial?v=preview.
Yu-Jun Shi, Jian-Ping Gong, Chang-An Liu, Xu-Hong Li, Ying Mei, Can Mi, and YanYing Huo (2004). Construction of a targeting adenoviral vector carrying AFP promoter for expressing EGFP gene in AFP producing hepatocarcinoma cell, World J Gastroenterol. 2004 Jan. 15; 10(2): 186-189.
Strobel, B., Miller, F. D., Rist, W., and Lamla, T. (2015a) Comparative analysis of cesium chloride- and iodixanol-based purification of recombinant Adeno-associated virus (AAV) vectors for preclinical applications, Hum Gene Ther Methods 26, 147-57
Strobel, B., Klauser, B., Hartig, J. S., Lamla, T., Gantner, F., and Kreuz, S. (2015b) Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells, Molecular Therapy 23, 1582-1591
Tang, A., Harding, F. (2019). The challenges and molecular approaches surrounding interleukin-2-based therapeutics in cancer. Cytokine: X1. 1:100001
Tai YT. et al. 1999. In vivo cytotoxicity of ovarian cancer cells through tumor-selective expression of the BAX gene. Cancer Res. 59(9):2121-6.
Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5
Valderrama-Treviño, A. I., Barrera-Mera, B., Ceballos-Villalva, J. C., and Montalvo-Javé, E. E. (2017). Hepatic Metastasis from Colorectal Cancer. Euroasian J Hepatogastroenterol. 7: 166-175.
Vanrell L, Di Scala M, Blanco L, Otano I, Gil-Farina I, Baldim V, Paneda A, Berraondo P, Beattie S G, Chtarto A, Tenenbaum L, Prieto J, Gonzalez-Aseguinolaza G. (2011). Development of a liver-specific Tet-on inducible system for AAV vectors and its application in the treatment of liver cancer. Mol Ther. 19(7):1245-53.
Vaughan, T. J., Williams, A. J., Pritchard, K., Osbourn, J. K., Pope, A. R., Earnshaw, J. C., McCafferty, J., Hodits, R. A., Wilton, J., and Johnson, K. S. (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library, Nature Biotechnology 14, 309-314
Wang, L., Hernandez-Alcoceba, R., Shankar, V., Zabala, M., Kochanek, S., Sangro, B., Kramer, M. G., Prieto, J., Qian, C. (2004). Prolonged and inducible transgene expression in the liver using gutless adenovirus: a potential therapy for liver cancer. Gastroenterology. 126(1):278-89.
Wang, D., Tai, P. W. L., Gao, G. (2019). Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug. Disc.18:358-378.
Wei, K. Y., and Smolke, C. D. (2015) Engineering dynamic cell cycle control with synthetic small molecule-responsive RNA devices, J Biol Eng 9, 21.
Wilmott P., Lisowski L., Alexander I. E., Logan G. J. (2019). A User's Guide to the Inverted Terminal Repeats of Adeno-Associated Virus Hum Gene Ther Methods. 30(6):206-213.
Yen, L., Svendsen, J., Lee, J. -S., Gray, J. T., Magnier, M., Baba, T., D'Amato, R. J., and Mulligan, R. C. (2004) Exogenous control of mammalian gene expression through modulation of RNA self-cleavage, Nature 431, 471-476
Yu J, Green M D, Li S, Sun Y, Journey S N, Choi J E, Rizvi S M, Qin A, Waninger J J, Lang X, Chopra Z, El Naqa I, Zhou J, Bian Y, Jiang L, Tezel A, Skvarce J, Achar R K, Sitto M, Rosen BS, Su F, Narayanan S P, Cao X, Wei S, Szeliga W, Vatan L, Mayo C, Morgan M A, Schonewolf C A, Cuneo K, Kryczek I, Ma V T, Lao C D, Lawrence T S, Ramnath N, Wen F, Chinnaiyan A M, Cieslik M, Alva A, Zou W. (2021). Liver metastasis restrains immunotherapy efficacy via macrophage-mediated T cell elimination. Nat Med. 27(1):152-164.
Zhang, L., Morgan, R. A., Beane J. D., Zheng Z., Dudley M. E., Kassim S. H., Nahvi A. V., Ngo L. T., Sherry R. M., Phan G. Q., Hughes M. S., Kammula U. S., Feldman S. A., Toomey M. A., Kerkar S. P., Restifo N. P., Yang J. C., Rosenberg S. A. (2011) Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Molecular Therapy 19, 751-759
Zhong, G., Wang, H., Bailey, C. C., Gao, G., and Farzan, M. (2016) Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells, Elife 5:e18858
Zhong (2020) A reversible RNA on-switch that controls gene expression of AAV-delivered therapeutics in vivo Nat Biotechnol. 2020 February;38(2):169-175. doi: 10.1038/541587-019-0357-y. Epub 2019 Dec. 23
Zhou Q, Tian W, Liu C, Lian Z, Dong X, Wu X. (2017). Deletion of the B-B′ and C-C′ regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression. Sci Rep. 7(1):5432.
Zou, W. (2005) Immunosuppressive networks in the tumour environment and their therapeutic relevance, Nat Rev Cancer 5, 263-274
Zincarelli, C., Soltys, S., Rengo, G., and Rabinowitz, J. E. (2008) Analysis of AAV Serotypes 1-9 Mediated Gene Expression and Tropism in Mice After Systemic Injection, Molecular Therapy 16, 1073-1080
Qingzhang Zhou, Wenhong Tian, Qingzhang Zhou, Wenhong Tian, Deletion of the B-B′ and C-C′ regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression, Sci Rep. 2017 Jul. 14;7(1):5432. doi: 10.1038/s41598-017-04054-4
Shin, Yue, and Duan Recombinant Adeno-Associated Viral Vector Production and Purification Methods Mol Biol. 2012; 798: 267-284.doi: 10.1007/978-1-61779-343-1_15
This table shows sequences in FASTA format mentioned in the text. In case of inconsistencies with the sequence listing, the sequences shown in the table are the authentic sequences.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20175116.1 | May 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/063034 | 5/17/2021 | WO |
