Patent Application
20250145990
The invention concerns a trans-splicing RNA (tsRNA) molecule comprising at least one or multiple unstructured target binding domains complementary to at least one or multiple precursor messenger RNA (pre-mRNA) targets and adapted to prevent off-target trans-splicing by the inclusion of a safety domain; a cell or vector or therapeutic or composition or pharmaceutical composition comprising said tsRNA; and a method for killing cells or treating a disease or for imaging or for a cosmetic application using said tsRNA. The invention has use in the medical, cosmetic, and veterinary field.
Spliceosome-meditated RNA trans-splicing (SMART) is the process by which two distinct precursor messenger RNAs (pre-mRNAs), or other spliceable RNAs, are joint in trans to generate a chimeric RNA molecule in the nucleus that, after nuclear export, triggers the formation of a chimeric protein in the cytoplasm. This technology can be used to repair defective RNA, e.g., by replacing a mutated with an intact exon, or to label an endogenous message with a functional sequence. This functional sequence can trigger the expression of a death signal to kill the targeted cell, of a fluorescent protein to image the targeted cells, of a therapeutic or cosmetic protein to treat or cure the targeted cells, or of any gene of interest to program/re-program the targeted cell.
Trans-splicing-based repair is difficult to achieve because durable repair requires the trans-splicing RNA to be delivered continuously or to be expressed endogenously after genomic integration, it also requires precise splicing towards the intended splice sites within the target, and it must be efficient enough to trigger the therapeutic phenotype despite strong competition with regular cis-splicing.
Trans-splicing-based labelling with a functional sequence concerns, for example, an RNA coding for a fluorescent protein to monitor the expression of genes in living cells or a death signal to selectively trigger death of cells expressing aberrant transcripts in a suicide gene therapy approach. An aberrant transcript can be a biomarker for diseased cells such as transcripts of oncogenes specific for cancer or viral transcripts. The death signal can be triggered by a) a direct signal such as a toxin e.g., diphtheria or the cholera toxin, b) an apoptotic or necrotic gene such as a caspase, or c) an enzyme such as the herpes simplex virus thymidine kinase (HSVtk) that triggers a death signal upon co-delivery of a drug like ganciclovir (GCV). Direct toxins a) or apoptotic signals b) can immediately trigger cell death which, unfortunately, increases the risks involved with unspecific targeting or off-targeting. This makes the regulatory approval of such technologies problematical. In contrast, the use of a combination of two components c) which, by themselves, are not toxic to the cells represents a much safer approach.
As a therapy, trans-splicing-triggered cell death is easier to achieve than trans-splicing-based repair because the trans-splicing construct needs to be delivered only once into the target cells and so long-term expression is not necessary. Moreover, alternative on-target trans-splicing, i.e., trans-splicing towards the right target but involving any splice sites of that target, is not disadvantageous but instead contributes to a target-specific death signal and trans-splicing doesn't need to be highly efficient to trigger a signal that is strong enough to kill the targeted cells.
Based on the HSVtk/GCV-system we have developed an efficient trans-splicing-based suicide gene therapy approach. We have designed new trans-splicing RNAs both for 5′ and 3′ exon replacement (ER), i.e., for attaching a suicide gene or a component of a suicide gene system such, as the HSVtk, either to the 5′ or 3′ end of the target message. We have investigated RNA structure design to improve both on-target activity and specificity of trans-splicing RNA (tsRNA).
The efficiency of methods such as gene therapy of inherited and acquired genetic diseases, genetic vaccination, stem cell programming, somatic cell reprogramming, immunotherapy and manipulation of protein expression and trans-splicing-based suicide gene therapy in vivo is dependent on the delivery of recombinant DNA into primary cells ex vivo or in vivo in order to trigger the expression of non-coding RNAs or proteins. In primary cells, the expression of recombinant foreign episomal DNA (such as plasmids) is silenced within 24 hours post-delivery, independent of the route of delivery. The mechanisms underlying this effect are poorly understood. Only integrating viral delivery vectors, such as retroviral, lentiviral, and AAV vectors have been successfully used to trigger medium and long-term expression in primary cells. These vectors, however, are costly considering current good Manufacturing Practise (CGMP) production standards. It is considered to be several orders of magnitude more expensive to produce viral vectors under cGMP standards than generating an equivalent quantity of ‘naked’ genetic material. In addition, viral vectors harbour safety risks and concerns which are associated (i) with negative interference of the integrated foreign DNA at the loci of integration (e.g., disruption of gene function and regulation), and (ii) with the involvement of components originating from pathogenic viruses. Alternatively, the direct delivery of functional RNA into primary cells results in rapid degradation and providing only short-term effects. Hence, there is a strong desire for the development of novel genetic vectors that escape transgene silencing.
The present disclosure allows for sustained and safe transgene expression in primary cells solving the problem of transgene silencing.
Novel vectors such as DNA minicircles or dumbbell-shaped vectors consisting solely of a transcription unit comprising promoter, coding genes and RNA-stabilising sequences, have several advantages such as improved cellular delivery or nuclear diffusion due to small size. Moreover, these small vectors are resistant to exonucleases due to the covalently closed structure, whereas plasmids often harbour single-strand breaks, so-called nicks, triggered by shearing forces. The lack of unnecessary bacterial sequences or resistance proteins eliminates unwanted side effects in the host, and the controlled in vitro synthesis and the option to chemically link fluorophores, cell-penetrating peptides or immune stimulatory peptides to the loop structures, allows easy manipulation of these vectors. Dumbbell-shaped DNA vectors, sometimes called dumbbells, doggybone DNA or closed-ended DNA, comprise (i) a double-stranded DNA core that includes any gene or genes of interest to be delivered as well as regulatory sequences such as promoters, enhancers, nuclear localisation signals, transcriptional termination or polyadenylation signals, and (ii) single-stranded loop structures closing the ends on both sides. As described above transgenic silencing in plasmids is frequent. DNA minicircles lacking extragenic spacers between the 5′ and 3′ ends of the transgene expression cassette were shown to allow sustained transgene expression in mice. When compared with minicircles, dumbbell-shaped vectors can be an order of magnitude smaller in molecular weight, in particular those for the expression of small non-coding RNA. WO2012/032114 discloses a DNA expression construct comprising a dumbbell-shaped circular vector which maintains expression for seven days post injection into melanomas. As opposed to any other genetic vectors, dumbbells can be featured with helper functions for targeted delivery, imaging, immune sensing etc. via the loops.
This disclosure relates to novel dumbbell vector conjugates for targeted delivery. We present dumbbell vectors that are, amongst other things, conjugated with tri-antennary N-acetylgalactosamine (GalNAc)3 for targeted delivery into asialoglycoprotein-positive cells including hepatocytes. We further present dumbbell vectors that are conjugated with aptamers, i.e., a CD137 and a prostate-specific membrane antigen (PSMA)-targeting aptamer. Both, (GalNAc)3 and the aptamers are non-covalently linked to an enlarged dumbbell loop structure via complementary base pairing.
According to a first aspect of the invention, there is provided a trans-splicing RNA (tsRNA) molecule comprising:
In a preferred embodiment of the invention said binding domain comprises a binding site comprising at least 25, more preferably 35, even more preferably 45, and most preferably 55 or more consecutive unstructured nucleotides (nt) having no internal binding and/or self-complementary sequences; and said binding domain, when of a length of 44 nt or longer, has at least one, or a plurality of, mismatch nucleotide(s) with respect to said gene.
In a preferred embodiment of the invention any one or more, including all, of parts b) i-iii may be present in said tsRNA, including any combination thereof.
Reference herein to a safety domain is reference to an antisense binding domain specific for a splice site in the splice signal of the trans-splicing RNA. The safety domain is arranged to prevent off-target trans-splicing as the binding domain has to bind to its target to release the safety domain from the splice signal in order to enable on-target trans-splicing. This is best seen with reference to
In a preferred embodiment of the invention said safety domain is either a linear sequence of nucleic acids or a folded sequence of nucleic acids comprising one or more folds, herein referred to as a continuous safety domain and a segmented safety domain, respectively.
Accordingly in other preferred embodiments of this invention are the novel safety domains, i.e., antisense sequences, with respect to the trans-splicing RNAs, that are complementary to, at least a part of the tsRNA's splice signal domain, or its splice donor (SD) and polypyrimidine tract (PPY). These safety domains prevent off-target trans-splicing as any of the target binding domains has to bind to its target first to release the safety domain from the SD and PPY in order to enable on-target trans-splicing. Two designs of safety domains were invented and tested: 1. a continuous safety domain and 2. a segmented safety domain the latter of which was disrupted by the various target binding domains.
Reference herein to a trans-splicing RNA molecule is reference to a molecule that interacts with a target precursor mRNA molecule and mediates a trans-splicing event to generate a novel chimeric mRNA that can be processed in the cell to yield a protein product.
Reference herein to a gene that associates with or is a biomarker for a target cell or a diseased cell to be treated is reference to a gene that is characteristic of said cell or said diseased cell and so is exclusively or preferentially present or expressed in/by said cell or when said disease occurs.
Reference herein to a housekeeping gene is reference to a gene that does not associate with a disease and one that is ubiquitously and abundantly expressed in any cell.
A novel aspect or embodiment of the invention is the generation of multi-targeting trans-splicing-based suicide RNAs targeting disease-specific and/or housekeeping gene derived pre-mRNAs. As opposed to the disease-specific pre-mRNA biomarkers, pre-mRNAs derived from housekeeping genes are constitutively expressed in all cell types. The housekeeping genes include but are not limited to genes involved in gene expression, metabolism, cellular structure, cellular surfaces, signaling, and others. This novel design applies to target cells in which the disease-specific biomarkers are very limited by numbers and/or the level of expression. In such a scenario, trans-splicing towards a housekeeping sequence can trigger basal expression of the death signal but without yet killing the cells. Only additional trans-splicing towards a disease or cell type-specific pre-mRNA biomarker will elevate the expression of the death signal above the threshold that finally kills the cell.
Reference herein to a protein that is a component of a suicide system is reference to a protein that interacts with, directly or indirectly, at least one other molecule to trigger or result in death of a cell in which said protein is expressed.
Those skilled in the art will appreciate that the tsRNA has nucleotides complementary to a gene with which it is to bind and because it is RNA it will include the nucleotides adenosine, guanosine, cytidine or uridine and the respective bases adenine, guanine, cytosine, and uracil or known chemical modifications of the same.
As those skilled in the art know, RNA is a chain of nucleotides, but unlike DNA, it is often found in nature as a single strand folded onto itself due to the presence of self-complementary sequences that allow parts of the RNA to fold and pair with itself to form a highly structured molecule. Thus, reference herein to an unstructured state is reference to a state within said binding sites where the sequence of RNA nucleotides exists in an unfolded chain. This chain may be curved or bent but it is not folded; thus there is no internal binding or self-complementary sequences.
As an alternative, a further way of describing an unstructured state is where said binding site comprises a sequence predicted not to fold into a stable minimum free energy secondary structure (Gibbs free energy of RNA secondary structure formation ΔG≥0 kcal/mol) or is at least less structured than the average of possible binding domains (ΔG>ΔGaverage). While RNAfold indicates such structures as open circle, mfold would not give any result for structures with ΔG≥0 kcal/mol.
In preferred embodiments of the invention, said binding domain is not fully complementary to the target gene, or pre-mRNA, and so said binding domain does not form perfect duplexes with the target gene and, usually, is not longer than 200 bp, most usually not longer than 100 bp.
In certain embodiments of the invention said binding domain, including said binding site, comprise a sequence of nucleotides selected from the list comprising or consisting of: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 7 3, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300 or more nucleotides.
In yet further certain embodiments of the invention said binding domain comprises a sequence of nucleotides that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to said part of said gene that associates with or is a biomarker for a disease to be treated, or, that alternatively is expressed in any cell. Ideally, the mismatches in said binding domain are positioned in way to avoid any stretches of 44 nt or longer that are perfectly complementary to the target, including or excluding said binding sites, and ideally at least 5 nucleotides from the 5′ or 3′ end. We think these features most likely act by suppressing antisense effects including A-to-I editing that might result from the formation of long double-stranded RNA in the nucleus. These features appear to be particularly beneficial in 3′ER.
In certain embodiments the binding domain, particularly when it is short i.e., less than 100 nucleotides including the binding site, is located adjacent a spacer sequence that helps to maintain the unstructured nature of the binding domain and so in this embodiment the tsRNA also includes a spacer sequence, known to those skilled in the art, adjacent said binding domain.
Those skilled in the art will appreciate that said binding domain can be designed to bind to any pre-mRNA including any biomarker of any disease and, providing it has the recited features, efficient trans-splicing will occur. Thus, the suicide gene therapy constructs, or sequences described herein can easily be reprogrammed to target alternative diseases or diseased tissue simply by exchanging the target binding domain(s).
In yet further certain embodiments of the invention said trans-splicing RNA molecule comprises a plurality of said binding domains which are complementary to the same or different parts of a gene that associates with or is a biomarker for a disease to be treated. Where more than one binding domain is for the same part of the gene, we have found it significantly improves specific on-target trans-splicing. Where more than one binding domain is for a different part of the gene, or even a different gene, we have found it enhances trans-splicing activity and improves the trans-splicing phenotype.
In certain further embodiments said tsRNA is either 5′ or 3′ tsRNA.
Accordingly, in one aspect or embodiment of the invention there is provided a cytotoxic tsRNA, ideally based on a suicide gene therapy approach.
In yet a further preferred embodiment said tsRNA comprises a tri-antennary N-acetylgalactosamine (GalNAc)3, for targeted delivery into asialoglycoprotein-positive cells, including hepatocytes.
In yet a further preferred embodiment said tsRNA is a dumbbell RNA. More preferably said dumbbell RNA comprises a tri-antennary N-acetylgalactosamine (GalNAc)3, for targeted delivery into asialoglycoprotein-positive cells, including hepatocytes.
In yet a further preferred embodiment said tsRNA is encoded by a dumbbell-shaped DNA vector. More preferably said tsRNA is a dumbbell RNA which comprises a tri-antennary N-acetylgalactosamine (GalNAc)3, for targeted delivery into asialoglycoprotein-positive cells, including hepatocytes.
In another embodiment of this invention are dumbbell-shaped delivery vectors featured with (GalNAc)3 residues for targeted delivery into hepatocytes, wherein they comprise at least one residue but, ideally, multiple residues. GalNAc residues are frequently used to deliver oligomeric nucleic acids including antisense oligodeoxyribonucleotides (ASOs) or small interfering RNAS (siRNAs) into hepatocytes. We have for the first-time provided gene expression vectors, i.e., dumbbell vectors, with (GalNAc)3 residues. Therefore (GalNAc)3 labelled antisense DNA or RNA oligos were hybridized to produce an extended complementary dumbbell loop via complementary base pairing (
In another embodiment of this invention are trans-splicing-based suicide RNAs targeting hepatoblastoma-derived cells harbouring target binding domains which are complementary to alpha-feto protein (AFP), Vascular endothelium growth factor (VEGF), Γ-glutamyl transferase (GGT), Hepatocellular carcinoma associated protein 2 (HCCA2), Transforming growth factor beta 1 (TGF-β1), cluster of differentiation 24 (CD24), Cyclin D1 (CCND1), Glypican 3 (GPC3) and Telomerase reverse transcriptase (TERT).
Additionally or alternatively, said tsRNA comprises at least one aptamer, such as, CD137 and/or a prostate-specific membrane antigen (PSMA)-targeting aptamer. Preferably, either or both, (GalNAc)3 and said aptamer(s) is/are non-covalently or covalently linked to the tsRNA.
Additionally or alternatively, said tsRNA is a dumbbell RNA which comprises at least one aptamer, such as, CD137 and/or a protatate-specific membrane antigen (PSMA)-targeting aptamer. Preferably, either or both, (GalNAc)3 and said aptamer(s) is/are non-covalently linked to an enlarged dumbbell loop structure via complementary base pairing.
Additionally or alternatively, said tsRNA is encoded by a dumbbell-shaped DNA vector which encodes/comprises at least one aptamer, such as, CD137 and/or a prostate-specific membrane antigen (PSMA)-targeting aptamer. Preferably, either or both, (GalNAc)3 and said aptamer(s) is/are non-covalently linked to an enlarged dumbbell loop structure via complementary base pairing.
In another embodiment of this invention are dumbbell-shaped delivery vectors featured with at least one aptamer, e.g., a CD137 binding aptamer, for targeted delivery into CD137+ cells. In another embodiment of this invention are dumbbell-shaped delivery vectors featured with at least one aptCD137-2 residue, but, ideally, multiple residues.
In another embodiment of this invention are dumbbell-shaped delivery vectors featured with at least one prostate-specific membrane antigen (PSMA) binding aptamer for targeted delivery into prostate cancer cells. These dumbbells deliver suicide RNAs comprise prostate-specific antigen (PSA) pre-mRNA targeting domains.
In yet a further embodiment of the invention said tsRNA comprises or is characterized by any one or more of the sequences herein described, including any combination thereof.
According to a second aspect of the invention, there is provided a trans-splicing RNA (tsRNA) molecule comprising:
Ideally, said binding domain comprises a binding site comprising at least 25, more preferably 35, even more preferably 45, and most preferably 55 or more consecutive unstructured nucleotides (nt) having no internal binding and/or self-complementary sequences and said binding domain, when of a length of 44 nt or longer, has at least one, or a plurality of, mismatch nucleotide(s) with respect to said gene.
In a preferred embodiment of the invention any one or more, including all, of parts c) i-iii may be present in said tsRNA.
Our work shows, where part b (i) of the first aspect of the invention or part c (i) of the second first aspect of the invention is deployed, that these optimised tsRNAs efficiently triggered the death of cells containing same, such as, hepatoblastoma-derived cells, HBV-positive cells, CD137-positive cells, nasopharyngeal cancer cells, epidermal cells, basal cells, hair follicle cells, and senescent cells.
In another embodiment of this invention are trans-splicing-based suicide RNAs targeting HBV-positive cells harbouring target binding domains which are complementary to HBV pre-genomic RNA (pgRNA), AFP and GPC3. As the expression of HBV pgRNA is lower in cell culture than the expression of other HCC-specific targets, HBV-targeting RNAs are less active at lower concentration. However, HBV RNA targets are expressed at much higher levels in HBV-infected cells in vivo.
In another embodiment of this invention are trans-splicing-based suicide RNAs targeting nasopharyngeal cancer cells harbouring target binding domains which are complementary to various oncogenic pre-mRNAs as described herein.
In another embodiment of this invention are trans-splicing-based suicide RNAs targeting EBV-positive cells harbouring target binding domains which are complementary to Epstein-Barr virus pre-mRNAs (i.e. BZLF1, EBNA-3B, LMP1 and LMP2A).
In another embodiment of this invention are trans-splicing RNAs targeting epidermal cells harbouring target binding domains which are complementary to Keratin 1 (KRT1), Keratin 2 (KRT2), Keratin 10 (KRT10), Keratin 14 (KRT14), Caspase-14 precursor (CASP14), Neuroblast differentiation-associated protein 2 (AHNAK2). Another embodiment of this invention are trans-splicing RNAs targeting basal cells harbouring target binding domains which are complementary to Keratin 15 (KRT15), Collagen 17A1 (COL17A1), Tumour protein 73 (TP73). In another embodiment of this invention are trans-splicing RNAs targeting hair follicle cells harbouring target binding domains which are complementary to Homeobox C13 (HOXC13), Fibroblast growth factor 7 (FGF-7). In another embodiment of this invention are trans-splicing RNAs targeting senescent cells harbouring target binding domains which are complementary to Forkhead Box O4 (FOXO4) and cyclin-dependent kinase inhibitor 2A (p16). All these trans-splicing RNAs were either featured with a GFP gene for imaging or the HSVtk gene to trigger cell death. The sequences were delivered using dumbbell vectors into the epidermis of domestic pigs following non-invasive topical application.
In another embodiment of this invention are trans-splicing-based suicide RNAs encoding death signals other than the HSVtk, such as CYLD Lysine 63 Deubiquitinase (CYLD), tumor necrosis factor-like weak inducer of apoptosis (TWEAK), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Tumor necrosis factor alpha (TNF-α) pre-mRNA.
Thus, spliceosome-mediated RNA trans-splicing represents a promising therapeutic strategy to trigger cell death in suicide gene therapy approaches.
In another preferred embodiment of this invention, said biomarker is any single biomarker or combination of biomarkers selected from the group comprising cancer biomarkers, including HCC biomarkers alpha-feto protein (AFP), Vascular endothelium growth factor (VEGF), γ-glutamyl transferase (GGT), Hepatocellular carcinoma associated protein 2 (HCCA2), Transforming growth factor beta 1 (TGF-β1), cluster of differentiation 24 (CD24), Cyclin D1 (CCND1), Glypican 3 (GPC3), Telomerase reverse transcriptase (TERT), α-L-fucosidase (AFU), CD19, CD34, CD44, CD49E, CD51, CD105, Collagen type XV alpha 1 (COL15A1), C-X-C motif chemokine receptor 4 (CXCR4), Denticleless E3 ubiquitin protein ligase homolog (DTL), Epithelial cell adhesion molecule (EPCAM), Golgi protein 73 (GP73), G protein signaling modulator 2 (GPSM2), Hepatocyte growth factor (HGF), Heat shock protein 70 (HSP70), Insulin like growth factor 2 (IGF2), Immunoglobulin superfamily member 3 precursor (IGSF3), Integrin Subunit Alpha 6 (ITGA6), Kell blood group glycoprotein (KEL), KIT Proto-Oncogene, Receptor Tyrosine Kinase (KIT), Minichromosome Maintenance Complex Component 3 (MCM3), Minichromosome Maintenance Complex Component 7 (MCM7), PDZ Binding Kinase (PBK), DNA Polymerase Delta 1, Catalytic Subunit (POLD1), Protein Regulator Of Cytokinesis 1 (PRC1), SRY-Box Transcription Factor 17 (SOX17), Spermatogenesis-associated serine-rich protein 2 (SPATS2), Translocon-associated protein subunit beta (SSR2), Stathmin 1 (STMN1), Thrombomodulin (THBD), ZW10 Interacting Kinetochore Protein (ZWINT), HBV-derived RNA including HBV pgRNA, Epstein-Barr virus derived RNA and pre-mRNAs including BamHI Z Epstein-Barr virus replication activator (BZLF1), Epstein-Barr virus nuclear antigen 3B (EBNA-3B), Latent membrane protein 1 (LMP1), and Latent membrane protein 2A (LMP2A), epidermal cell markers including Keratin 1 (KRT1), Keratin 2 (KRT2), Keratin 10 (KRT10), Keratin 14 (KRT14), Caspase-14 precursor (CASP14), Neuroblast differentiation-associated protein 2 (AHNAK2), basal cell markers including Keratin 15 (KRT15), Collagen 17A1 (COL17A1), Tumour protein 73 (TP73), hair follicle cell markers including Homeobox C13 (HOXC13) and Fibroblast growth factor 7 (FGF-7), senescent cell markers including Forkhead Box 04 (FOXO4) and cyclin-dependent kinase inhibitor 2A (p16), the stratum corneum markers Kallikrein related peptidase 5 (KLK5), Small proline-rich protein 4 (SPRR4), and Arachidonate 12-lipoxygenase (ALOX12B), the Stratum spinosum (Upper epidermal layers) markers HOP homeobox (HOPX) and Kallikrein 9 (KLK9), the Stratum granulosum markers Filaggrin (FLG) and Premature ovarian failure 1B protein (POF1B), the Melanocyte markers Melan-A (MLANA) and Tyrosinase (TYR), the Langerhans cell markers CD1A and CD207, the fibroblast markers Periostin (POSTN) and Phospholipase C-eta-2 protein (PLCH2), the basal cell carcinaoma markers Glioma 1 (Gll1), Glioma 2 (GII2), Forkhead box protein (FOXM1), Forkhead box protein (FOXO3A), Desmoglein 2 (DSG2) and C3b, the basal cell carcinoma recurrence markers Cyclooxygenase (COX-2), Ezrin (EZR), CD25, Maspin, Glioma 3 (GII3) and Gremlin1.
In other embodiments said disease is cancer or a viral infection or a bacterial infection or an acquired genetic disease caused by mutations triggered by transposable elements, radiation, chemicals, or unknown triggers.
In the first instance said cancer is selected from the group comprising: hepatocellular carcinoma (HCC), cervical cancer, vaginal cancer, vulvar cancer, penile cancer, skin cancers, melanoma including malignant melanoma, squamous-cell carcinoma, basal-cell carcinoma, Merkel cell carcinoma, lung cancer, cell bladder cancer, breast cancer, colon or rectal cancer, anal cancer, endometrial cancer, kidney cancer, leukemia, acute myelogenous or myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic myelogenous or myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (P-TLL), large granular lymphocytic leukemia, adult T-cell leukemia, lymphoma, myeloma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer, nasopharyngeal cancer, mouth or throat cancer, oropharyngeal cancers, stomach cancer, brain tumours, bone cancer, and stem cell cancers and, indeed other cancers that would benefit from the treatment disclosed herein.
In the second instance said viral infection is selected from the group comprising: Papillomaviruses, human papillomavirus type 16, human papillomavirus type 18, retroviruses, lentiviruses, herpes viruses, adenovirus, adeno-associated virus, Flu virus, Hepatitis virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Epstein-Barr virus (EBV), human T-cell lymphotropic virus (HTLV), human immunodeficiency virus (HIV), human immunodeficiency virus type 1 (HIV-1), and human immunodeficiency virus type 2 (HIV-2), and others.
In the third instance said bacterial infection is selected from the group comprising: Bartonella henselae, Francisella tularensis, Listeria monocytogenes, salmonella species, Salmonella typhi, Brucella species, Legionella species. Mycobacteria species, Mycobacterium tunberculosis, Nocardia species, Rhodococcus species, Yersinia species, Neisseria meningitides and others.
In the last instance said acquired genetic disease is selected from the group comprising: Neurofibromatosis 1 and 2, Mc Cune Albright, Duchenne muscular dystrophy (DMD), Epidermolysis bullosa, Fanconi A and C, Philadelphia chromosome, Hemophilia A and B, cystic fibrosis, Muckle Wells syndrome, lipoprotein lipase deficiency, B-thalassemia, Gaucher Disease types I to III—GBA gene, Ornithine transcarbamylase (OTC) deficiency—OTC, Phenylketonuria (PKU)—PAH gene, Aspartylglucosaminuria—AGA gene, Alpha-1 anti trypsin deficiency (AATD)—SERPINA1, pyruvate dehydrogenase complex deficiency, and others.
In a further preferred embodiment of the invention, tsRNA is provided with a helper function for targeted delivery by the use of a peptide or carbohydrate such as GalNAc3, ideally more than one GalNAc3 is conjugated to the dumbbell tsRNA, ideally, by attaching two GalNAc3 residues via RNA linker oligonucleotides to a HSVtk expressing dumbbell vector, preferably in the region of the circular ends.
In an alternative aspect there is provided a medicament comprising said tsRNA according to the invention and, optionally where part b (i) of the first aspect of the invention or part c (i) of the second aspect of the invention is deployed, at least one further component of said suicide system effective to trigger death of a cell expressing said trans-spliced RNA.
In an alternative aspect there is provided a pharmaceutical composition comprising said tsRNA according to the invention and, optionally where part b (i) of the first aspect of the invention or part c (i) of the second aspect of the invention is deployed, at least one further component of said suicide system effective to trigger death of a cell expressing said trans-spliced RNA; and a carrier suitable for human or veterinary use.
In the afore optional instance said further component may be, e.g., ganciclovir, although other known co-component suicide systems for cell death may be used. Examples are cytosine deaminase-5-fluorocytosine, cytochrome P450-ifosfamide, cytochrome P450-cyclophosphamide, and nitroreductase-5-[aziridin-1-yl]-2,4-dinitrobenzamide.
In an alternative aspect there is provided a cell containing said tsRNA or a vector, ideally a dumbbell-shaped DNA expression vector, comprising or consisting of said tsRNA.
In a further aspect there is provided method of killing a cell comprising transfecting, lipofecting, transducing, electroporating, nucleofecting or transforming said cell with tsRNA, or a vector containing the tsRNA, according to the invention and, optionally where part b (i) of the first aspect of the invention or part c (i) of the second aspect of the invention is deployed, exposing said cell to at least one other component of said suicide system effective to trigger death of a cell expressing said trans-spliced RNA.
In a further aspect there is provided a method of treating a disease comprising transfecting, lipofecting, transducing, electroporating, nucleofecting or transforming a diseased cell with tsRNA, or a vector containing the tsRNA, according to the invention ex vivo or in vivo and, optionally where part b (i) of the first aspect of the invention or part c (i) of the second aspect of the invention is deployed, exposing said cell to at least one other component of said suicide system effective to trigger death of a cell expressing said trans-spliced RNA.
In a further aspect there is provided a method of targeting a diseased cell comprising topical application (including a cream, a gel, a foam, a lotion, ointment or aerosol), intranasal application, alveolar application, systemic application, oral application, intravenous application, intramuscular application, subcutaneous application, cutaneous application, intraperitoneal application, or injection into a tumor with tsRNA, or a vector containing the tsRNA, according to the invention in vivo and, and, optionally where part b (i) of the first aspect of the invention or part c (i) of the second aspect of the invention is deployed, exposing said cell to other components of said suicide system effective to kill said cell.
In a certain methods of the invention said cell is a virally transformed cell. Typically the cell is transformed with a virus selected from the group comprising: Papillomaviruses, human papillomavirus type 16, human papillomavirus type 18, retroviruses, lentiviruses, herpes viruses, adenovirus, adeno-associated virus, Flu virus, Hepatitis virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV), Epstein-Barr virus (EBV), human T-cell lymphotropic virus (HTLV), human immunodeficiency virus (HIV), human immunodeficiency virus type 1 (HIV-1), and human immunodeficiency virus type 2 (HIV-2).
In yet other methods of the invention said cell is a cancer cell such as a hepatocellular carcinoma (HCC) cell, cervical cancer cell, vaginal cancer cell, vulvar cancer cell, penile cancer cell, skin cancer cell, melanoma cell including malignant melanoma cell, squamous-cell carcinoma cell, basal-cell carcinoma cell, Merkel cell carcinoma cell, lung cancer cell, cell bladder cancer cell, breast cancer cell, colon or rectal cancer cell, anal cancer cell, endometrial cancer cell, kidney cancer cell, leukemia cell, acute myelogenous or myeloid leukemia (AML) cell, acute lymphoblastic leukemia (ALL) cell, chronic myeloid leukemia (CML) cell, chronic myelogenous or myeloid leukemia (CML) cell, hairy cell leukemia (HCL) cell, T-cell prolymphocytic leukemia (P-TLL) cell, large granular lymphocytic leukemia cell, adult T-cell leukemia cell, lymphoma cell, myeloma cell, non-Hodgkin lymphoma cell, pancreatic cancer cell, prostate cancer cell, thyroid cancer cell, nasopharyngeal cancer cell, mouth or throat cancer cell, oropharyngeal cancer cell, stomach cancer cell, brain tumour cell, bone cancer cell, and stem cell cancer cell. Ideally said cell is mammalian and most typically human.
According to a third aspect, or a further embodiment, of the invention there is provided a dumbbell-shaped DNA expression vector comprising:
In a further preferred embodiment of the invention said vector comprises at least one internal loop domain. Preferably, said loop domain comprises an abasic site or a nucleotide mismatch.
In a preferred embodiment of the invention said abasic site comprises one or more apurinic/apyrimidinic abasic sites.
In a preferred embodiment of the invention said nucleotide mismatch comprises a tetrahydrofuran-based mimic of an abasic site.
In a preferred embodiment of the invention said nucleic acid molecule to be expressed encodes a therapeutic protein or peptide.
In a preferred embodiment of the invention said therapeutic protein is Cas9, Cas9n, hSpCas9 or hSpCas9n.
In a preferred embodiment of the invention said therapeutic protein or peptide triggers a death signal.
Examples of proteins or peptides that trigger a cellular death signal are known in the art. For example, bacterial toxins such as the cholera toxin or the diphtheria toxin, alpha toxin, anthrax toxin, exotoxin, pertussis toxin, shiga toxin, shiga-like toxin etc are known to induce cell death. Furthermore, apoptotic signals/proteins such as Fas, TNF, caspases (initiator caspases, caspase 2,8,9,10,11,12, and effector caspases, caspase 3,6,7) etc. In addition, enzymes that are able to convert a non-toxic drug into a toxic component: e.g. the herpes simplex virus thymidine kinase (HSVtk) converts the rather non-toxic drug ganciclovir (GCV) into the toxic triphosphate (HSVtk/GCV system). A further example is the Escherichia coli purine nucleoside phosphorylase (PNP)/fludarabine suicide gene system.
In a further preferred embodiment of the invention said therapeutic protein or peptide is the HSVtk.
In an alternative preferred embodiment of the invention said expressed nucleic acid molecule is a therapeutic nucleic acid molecule.
In a preferred embodiment of the invention said therapeutic nucleic acid is a siRNA or shRNA.
In an alternative preferred embodiment of the invention said therapeutic nucleic acid molecule is an antisense RNA oligonucleotide or antisense miRNA.
In a further preferred embodiment of the invention said therapeutic nucleic acid molecule is a miRNA.
In a further preferred embodiment of the invention said therapeutic nucleic acid molecule is a trans-splicing RNA.
In a further preferred embodiment of the invention said therapeutic nucleic acid molecule is a guide RNA, single-guide RNA, crRNA, or tracrRNA.
In an alternative preferred embodiment of the invention said therapeutic nucleic acid molecule is a pre-mRNA or mRNA.
In a further preferred embodiment of the invention said minimal transcription promoter is derived from an RNA polymerase III promoter.
In a preferred embodiment of the invention said RNA 5 polymerase III promoter is a U6 promoter.
In an alternative preferred embodiment of the invention said RNA polymerase III promoter is a H1 promoter.
In an alternative preferred embodiment of the invention said RNA polymerase III promoter is a minimal H1 (mH1) promoter.
In a further alternative preferred embodiment of the invention said RNA polymerase III promoter is a modified mH1 promoter that includes a restriction endonuclease cleavage site and/or an inverted polymerase III transcriptional terminator.
In a further preferred embodiment of the invention said minimal transcription promoter is derived from an RNA polymerase II promoter.
In a preferred embodiment of the invention said RNA polymerase II promoter is a CMV promoter comprising the nucleotide sequence set forth in SEQ ID NO: 240.
In a preferred embodiment of the invention said transcription terminator nucleotide sequence is a RNA polymerase II or RNA polymerase III termination sequence.
In a preferred embodiment of the invention said RNA polymerase III termination sequence comprises one or more motifs comprising the nucleotide sequence TTTTT.
In a preferred embodiment of the invention said DNA nuclear targeting sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1.
In a further preferred embodiment of the invention said enhancer nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1 (full length enhancer: 30 fSV40enh).
In a further preferred embodiment of the invention said vector comprises the intron nucleotide sequence set forth in SEQ ID NO: 241.
In a further preferred embodiment of the invention, said residue or helper function for targeted delivery is a carbohydrate.
In a further preferred embodiment of the invention, said residue or helper function for targeted delivery is a (GalNAc)3 residue.
In an alternative preferred embodiment of the invention, said residue or helper function for targeted delivery is an aptamer.
In a further preferred embodiment of the invention, said aptamer is a CD137 or a PSMA binding aptamer.
In a further preferred embodiment of the invention, said aptamers are non-covalently bound towards a dumbbell loop via complementary base pairing using the sequence set forth in SEQ ID NO: 242.
In a further preferred embodiment of the invention, said enlarged dumbbell loop comprises any of the sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6.
In a further preferred embodiment of the invention, said residue or helper function for targeted delivery is an antibody.
In a further preferred embodiment of the invention, said residue or helper function for targeted delivery is a CD137-binding antibody.
In a further preferred embodiment of the invention, said residue or helper function for targeted delivery is a peptide or carbohydrate such as GalNAc3, ideally more than one GalNAc3 is conjugated to the dumbbell vector.
In a further preferred embodiment of the invention, said residue or helper function for targeted delivery is a cell penetrating peptide.
In a further preferred embodiment of the invention said vector further encodes a detectable marker.
In a preferred embodiment of the invention said detectable marker 5 is a fluorescence marker.
In a preferred embodiment of the invention said fluorescence marker is a fluorescent reporter protein.
The analysis of promoter activity in a tissue can be conveniently monitored by fusing a promoter to a nucleic acid that encodes a “reporter” protein or polypeptide. Examples are well known in the art and include enzymes such as @ glucuronidase. Reporters that are proteinaceous fluorophores are also known in the art. Green fluorescent protein, GFP, is a spontaneously fluorescent protein isolated from coelenterates, such as the Pacific jellyfish, Aequoria victoria. Its role is to transduce, by energy transfer, the blue chemiluminescence of another protein, aequorin, into green fluorescent light. GFP can function as a protein tag, as it tolerates N- and C-terminal fusions to a broad variety of proteins many of which have been shown to retain native function. Most often it is used in the form of enhanced GFP in which codon usage is adapted to the human code. Other proteinaceous fluorophores include yellow, red and blue fluorescent proteins. These are commercially available from, for example, Clontech (www.clontech.com). A yet further example is firefly luciferase.
In a preferred embodiment of the invention said nucleotide sequence with homology, to a part of a mammalian genome, for targeted delivery is implemented into the double-stranded DNA part of the dumbbell vector.
In an alternative preferred embodiment of the invention said nucleotide sequence with homology, to a part of a mammalian genome, for targeted delivery is implemented into the single-stranded loop of the dumbbell vector.
According to a further aspect of the invention there is provided a pharmaceutical composition comprising a dumbbell-shaped vector according to the invention and a suitable carrier.
The dumbbell-shaped vector compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers and supplementary therapeutic agents. The dumbbell shaped vector compositions of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, transdermal, oral, topical, intratracheal, nasal, intravaginal or trans-epithelial. Alternatively, the dumbbell-shaped vector or vector composition of this invention is delivered by physical methods including but not limited to liquid jet-injection, microinjection, microneedles, powder particle injection, gold particle injection, gene gun, electroporation or hydrodynamic injection.
The dumbbell-shaped vector compositions of the invention are administered in effective amounts. An “effective amount” is that amount of the dumbbell-shaped vector that alone, or together with further doses, produces the desired response. In the case of treating a disease, the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
The dumbbell-shaped vector compositions used in the foregoing methods preferably are sterile and contain an effective amount of dumbbell-shaped vector according to the invention for producing the desired response in a unit of weight or volume suitable for administration to a patient. The doses of vector administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Other protocols for the administration of vector compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing. The administration of compositions to mammals other than humans, (e.g. for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.
When administered, the dumbbell-shaped vector compositions of the invention are applied in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. The term “pharmaceutically acceptable” means a non-toxic amount that does not interfere with the effectiveness of the biological activity of the active agent. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents' (e.g. those typically used in the treatment of the specific disease indication). When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
The pharmaceutical compositions containing dumbbell-shaped vectors according to the invention may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
The dumbbell-shaped vector compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the dumbbell-shaped vector into association with one or more accessory ingredients. Compositions containing vectors according to the invention may be administered as aerosols and inhaled. Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of the vectors, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Among the acceptable solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.
For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides.
In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, 5 intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
Preferred features of each aspect of the invention may be as described in connection with any of the other aspects of the invention.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprises”, or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.
Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds, or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.
Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
The present invention will now be described by way of example only with particular reference to the following figures wherein:
The trans-splicing constructs were designed combining various reported and novel molecular features to improve activity and target specificity. The 3′ER ts constructs consisted of a CMV promoter (pEGFP-N1, Clontech acc no. U55762) followed by a binding domain (BD) of 50 bases complementary to the target AFP intron 5. The BD included two mismatches at positions 18 and 19 to inhibit potential antisense (as) effects that can be triggered by longer dsRNA in the nucleus of the cell. Software ‘foldanalyze’ (HUSAR, DKFZ) was used to select short unstructured BDs within the complete antisense RNA structure space that can be directed against the AFP intron 5. Structures of the selected BDs were confirmed by RNA 2° structure (minimum free energy and centroid) predictions using software tools mfold and RNAfold. Such selected BDs were then fused with the rest of the trans-splicing RNA making sure that the BDs remained unstructured upon fusion and were not involved in base-pairing the trans-splicing or coding domains which was achieved by implementing suitable spacers. The selected 3′ splice signal (3′ss) was designed to functionally compete with the cellular cis-splice site and was supported by an intronic splice enhancer (ISE) (McCarthy, et al., 1998; Konczak, et al., 2000; Yeo et al., 2004), a branchpoint (BP) (Eul, 2006) and polypyrimidine tract (Ppt) (Nobel, et al., 1998; Taggart, et al, 2012). The HSVtk cds was preceded with a sequence coding for a proteolytic cleavage site P2A (Kim, et al, 2011) to ensure endogenous release of the native HSV-tk from the AFP-HSVtk fusion protein that initially results from the trans-splicing process. The HSVtk gene is devoid of a start codon and can only be translated after trans-splicing using the translational start of the target message. The HSVtk gene was equipped with an A/G-rich exonic splice enhancer (ESE) generated by using degenerative alternative codons that do not alter the HSV-tk amino acid sequence (Fairbrother, et al, 2002; Jin et al., 2003) (
The 5′ER ts constructs were designed with the same molecular features as p3ER but with different orientation including a translational signal motif along with the CMV promoter. All the structural elements important for translation of eukaryotic mRNA were included: original cap site of AFP (Gibbs, et al. 1987) followed by the consensus Kozak sequence GCCRGCCAUGG (Kozak, 1995, 1999, 2005). Immediately after the translation start signal was the coding domain HSVtk inclusive of the ESE and mini-intron followed by a 5′ss signal (Freund, et al. 2005). The 5′ BD was designed in a similar way with mismatches at positions 24 and 25 to avoid antisense effects a (s-effects). Following the BD, a hammerhead ribozyme (HH Rz) (Saksmerprome, et al. 2004) was incorporated for enhanced cleaving of the BD after delivery into the nucleus. The HH RZ is followed by a long spacer to isolate the polyA from the ribozyme followed by the SV40 polyA.
The strength and nature of the splice sites were predicted using softwares Alternative Splice Site Predictor (ASSP) (Wang, 2006) http://wangcomputing.com/assp/overview.html and Berkeley Drosophila Genome Project Splice Site Prediction (BDGP SSP) (Reese, et al., 1997) http://www.fruitfly.org/seqtools/splice.html using default cut-off values for the splice site predictions. To predict the nature of splice sites in HPV16 genome, ASSP was used to document constitutive or cryptic splice acceptors and donors based on the overall score and confidence generated by the software. The alternative splice sites with confidence >0.89 and score >5.5 and constitutive splice sites with confidence >0.1 and score >7.7 besides the documented splice sites (Johansson, 2013 and Schmitt, et al, 2011) were selected for the trans-splicing analyses.
Plasmid pGFP was cloned by inserting the maxGFP gene (CMV promoter, cds, SV40 ployA site) of the pMAX-GFP (Lonza) vector into the NdeI and BbsI restriction endonuclease cleavage sites of the pVAX1 vector (Addgene). The SV40 enhancer which functions as DNA nuclear localizing signal (dNLS) was inserted into the NdeI and BbsI site upstream of the CMV promoter. The HSVtk positive control plasmid carried a codon optimized HSV1 thymidine kinase coding sequence inserted into the pVAX1 plasmid under the control of the CMV promoter.
Generating dumbbells for trans-splicing from the plasmid vectors was done using the ELAN method of db production. The Enzymatic Ligation Assisted by Nucleases (ELAN) is a three-step process which includes digestion of the transcription cassette from the plasmid, ligation of the closing loops on either side followed by exonuclease treatment to eliminate the unclosed db plasmids.
The stem loops consisting of individual RE site were synthesised by AIT Biotech (Singapore) and was phosphorylated using the following reaction shown in Table 1:
The Stem-loop primers were Stem loop-SpeI and Stem-loop-BamHI.
In the ELAN loop-ligation method, the gene expression cassette was directly cut out from parental plasmid. 50 times more stem-loops were added in the ligation reaction to ensure that most of the gene expressing cassettes could be capped. By-products such as loop dimers were cleaved by the restriction enzymes and were destroyed during the exonuclease treatment. Detailed setups of the reaction are shown in Table 2.
The generated dumbbells were run on a 1% agarose gel to confirm their integrity.
Alternatively to the ELAN method, dumbbells were produced using gap-primer PCR (gpPCR).
At high purity using a process termed 1-2-3 gap-primer PCR. This process represents a 1-tube, 2-enzyme, 3 h procedure that comprises a PCR followed by a ligation. The resulting dumbbells harbor mismatches close to the loop structures, which facilitate nuclear diffusion and result in enhanced gene expression.
Wolfgang Walther (ed.), Gene Therapy of Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2521, https://doi.org/10.1007/978-1-0716-2441-8 18, Springer Science+Business Media, LLC, part of Springer Nature 2022.
Carry out all procedures at room temperature unless otherwise specified.
The gap-PCR primers harbor an invariable universal 5′ domain comprising a 5′ phosphate, a stretch designed to refold and preform the dumbbell loop, the abasic position, and a 3′ domain that is binding toward the DNA template (underlined). In this example, the 3′ ends are complementary to the cloning vector pVAX1
Add 5 times the reaction volume of PB binding buffer, mix well and transfer into the QIAquick spin column. Centrifuge at 12,000×g for 1 min. Discard the flowthrough. Add 700 μL of PE wash buffer to the spin column and centrifuge at 12,000×g for 1 min. Discard the flowthrough before dry spin at 12,000×g for 1 min. Transfer the spin column to a new 1.5 ml microcentrifuge tube and add 50 μL of nuclease-free water for elution. Spin at 12,000×g for 1 min.
Dumbbell-GalNAc3-conjugates
3.5 pmol GalNAc3-DNA or GalNAc3-RNA oligonucleotide was annealed with 3.5 pmol dumbbell DNA in 20 μl 10× hybridisation buffer (1 M NaCl, 0.1 M MgCl2, 200 mM Tris-HCl, pH 7.4) in the presence of 20% v/v of PEG4000. The solution was denatured at 80° C. for 5 min and then incubated at 37° C. for 1 hour. The resulting dumbbell-GalNAc3 conjugates were cleaved with AseI (Thermo Fisher) and GalNAc3 attachment to the conjugation loops was monitored in 1.5% agarose gel shift assays. Dumbbell-conjugates were purified using Sephadex gel permeation chromatography and ethanol precipitation.
GalNAc3-linked oligonucleotides GalNAc3-DNA 5′-GCTATAAGTGTGCATGAGAAC-GalNAc3-3′ and GalNAc-RNA 5′-GCUAUAAGUGUGCAUGAGAAC-GalNAc3-3′ were derived from Microsynth (Switzerland). Underlined positions indicate deoxyribonucletides. Primers for the production of dumbbells were derived from IDT.
Dumbbell-aptCD137-2-conjugates
3.5 pmol of the aptCD137-2 homodimer was annealed with 3.5 pmol dumbbell DNA as described above.
Cells were transfected with plasmids or dumbbell-shaped DNA minimal vectors using Lipofectamine® 3000 following the manufacturer's protocol. In short, 500 ng of DNA and 1 μl of P3000 were diluted in 25 μl of Opti-MEM and then mixed with 1.5 μl Lipofectamine 3000 (diluted in 25 μl of Opti-MEM). The mixture was incubated at room temperature for 5 minutes before adding onto the cells.
RNA was isolated 24 hours post-transfection using RNeasy plus kit (Qiagen) following the manufacturer's protocol. RNA concentrations were measured using NanoDrop 2000.
cDNA Conversion and Real-Time RT-PCR:
500 ng RNA from all samples was converted into cDNA using the First Strand SuperScript RTIII (Invitrogen) kit with 200 ng of random hexamers and 10 μM of dNTPs. The reaction conditions were 25° C. for 5 min, followed by 50° C. for 2 h and enzyme inactivation at 70° C. for 15 min. 20 ng of cDNA was used as template for real time RT-PCR. TaqMan quantification was performed in ABI 7900HT of the cDNAs by designing specific probe and primer sets for each cis- and trans-splicing detection. The number of cycles in over-expression studies and endogenous studies were 40 and 50 respectively. RT-PCR: Reverse transcription PCR was performed on the cDNA samples using Taq DNA polymerase (Fermentas) with 60 cycles of two-step PCR (30+30 cycles or 35+35 cycles) to detect 3′ and 5′ cis and trans-splicing and the bands were visualized on a 1% agarose gel.
For RT-qPCR, Fw_maxGFP (5′-ATCGAGTGCCGCATCACC-3′) SEQ ID No:247 and Rv_maxGFP (5′-ACTCATCGAGCTCGAGATCTGG-3′) SEQ ID No:248 were used. Fw-Beta actin (5′-CTGGCACCCAGCACAATG-3′) SEQ ID No:249 and RP-beta actin (5′-GCCGATCCACACGGAGTACT-3′) SEQ ID No:250 were used as housekeeping gene.
Uptake of Dumbbell-GalNAc3-Conjugates from the Tissue Cell Culture Medium
Uptake of MaxGFP dumbbell-GalNAc3-conjugates
HepG2 cells were trypsinized, washed with 10 ml DMEM, 0.05×106 cells were resuspended in 30 μl DMEM and 3.5 pmol MaxGFP dumbbell-GalNAc3-conjugates dissolved in 20 μl of water were added and incubated with the cells for 4 hour before seeding them again. By adding the dumbbell conjugates to HepG2 cells in suspension, we achieved a higher concentration of dumbbell conjugates in the medium to observe stronger expression of MaxGFP.
0.05×106 HepG2 cells were seeded in 24 wells and 0.35 pmol HSVtk dumbbell-GalNAc3 or -2GalNAc3-conjugates dissolved in 20 μl of water were added after 24 hours to the DMEM culture medium.
qPCR Quantification of Uptaken MaxGFP Dumbbell-GalNAc3 DNA
After 24 hours of exposure, cells were harvested and episomal nucleic acids including uptaken dumbbell-conjugates were isolated using the RNeasy® Plus kit following the manufacturer's protocol. For SYBR Green detection of dumbbell DNA, 1 μl of the episomal nucleic acid sample was mixed with 1×SYBR® Select Master Mix for CFX and each 0.5 μM forward and reverse primers in a 10 μl reaction. All reactions were run in duplicate. Dumbbell vector DNA was quantified using absolute qPCR quantification based on a standard curve created with dumbbell vector DNA.
To check the functional activity of trans-splicing, drug Ganciclovir (GCV) (Sigma) was added to the cells at a concentration of 10 μM, 100 μM and no GCV (internal negative control) 24 hours post-transfection followed by addition of AlamarBlue® cell viability reagent (Thermo Scientific) 24 hours post drug for a duration of 6 days with replacement of fresh media and drug every day after each alamarblue reading. The fluorescence was measured at 230/290 nm after 90 minutes of incubation at 37° C. The positive and negative controls for the assay were designed as mentioned in the manufacturer's protocol.
For FACS analysis, the media was aspirated, and the cells were rinsed once with PBS before trypsinisation with 200 μl of 1× trypsin-EDTA. The trypsinised cells were collected by centrifugation at 4200 rpm for 6 min in 1 ml of media. The pelleted cells were resuspended in 500 μl of 1×PBS. FACS was performed with 10,000 cells for the NTC and >5,000 cells per sample using a LSRFortessa cell analyser, and FACSDiva software v6.1.3 was used for the acquisition of the samples. FlowJo software V7.6.1 was used for data analysis.
To check for apoptosis, cells were harvested 48 hours post 100 μM GCV treatment and stained using Propidium Iodide and Alexa Fluor 647 Annexin V (Life Technologies) in Annexin-binding buffer according to manufacturer's protocol. The samples were gated based on single live cell populations which were positive for GFP. The final % apoptosis values are indicated as (early and late apoptosis+GCV)-(early and late apoptosis−GCV).
GCV was purchased from Sigma as a 100 mg ready-to-mix powder form. To dissolve the powder for master stock for cell culture experiments, 10 mg of GCV was dissolved in 1 ml of 0.1 N/0.1 M HCl (final concentration: 10 mg/ml). For a 10 mM working stock (for conditions ranging from 1-100 μM), 255 μl of 10 mg/ml GCV master stock was diluted in 745 μl of 0.1 N HCl. For 1 mM working stock (for conditions ranging from 0.1 μM and 0.3 μM), 100 μl of 10 mM working stock was further diluted in 900 μl of 0.1 N HCl.
To trigger death of dumbbell-conjugate transfected HepG2 cells, GCV was added to the cells at a concentration of 100 μM, 24 hours post-transfection. The cell death activity was monitored using the AlamarBlue® cell viability reagent every 24 hours for 6 days with addition of fresh media and drug every day. The fluorescence was measured at 530 nm/590 nm after 90 min of incubation.
For tail vein injection, 30 μg of dumbbell-Nanoluc and dumbbell-maxGFP was prepared in 100 μl solution with 0.14 μl/μg DNA of in vivo-JetPEI (Polyplus), SAINT-Vivo (Synvolux) or PBS respectively. 30 μg of dumbbell-Nanoluc/maxGFP-RNA-1-GalNac3 was diluted in 100 μl PBS. For hydrodynamic injection, 30 μg of dumbbell-Nanoluc and dumbbell-maxGFP was diluted in 1.5 mL of PBS. Samples were administered to 4-6 weeks old mice by iv injection. Organs were harvested at day 6 and homogenization of organs was performed prior to RNA isolation with TRIzol. cDNA was converted from 5 μl of RNA and qPCR was carried out.
Error bars represent standard errors of the arithmetic mean (±SEM) of three independent experiments. Unpaired student's t-test was used to determine significance when comparing two groups. GraphPad Prism 9 software was used for the statistical analysis. P values are as indicated on the graphs.
In detail, this invention refers to
This invention concerns novel optimized RNA sequences and structures designed to achieve higher trans-splicing activity and specificity. We designed parental trans-splicing RNA (tsRNA) molecules for 3′ exon labelling comprising some or all of the following molecular features (
Upon cellular delivery, in one embodiment, the trans-splicing based suicide RNAs will bind towards the respective pre-mRNA targets, splice in trans, and enable target cell-specific expression of the HSVtk. Upon co-delivery, the prodrug ganciclovir (GCV) is being phosphorylated by the HSVtk with support from cellular phosphatases triggering the formation of the toxic GCV-triphosphate which leads to cell death (Poddar et al., 2018).
A novel design aspect is the generation of multi-targeting trans-splicing-based suicide RNAs targeting disease-specific plus housekeeping gene derived pre-mRNAs. As opposed to the disease-specific pre-mRNA biomarkers, pre-mRNAs derived from housekeeping genes are constitutively expressed in all cell types. The housekeeping genes include but are not limited to genes involved in gene expression, metabolism, cellular structure, cellular surfaces, signalling, and others. This novel design applies to target cells in which the disease-specific biomarkers are very limited by numbers and/or the level of expression. In such a scenario, trans-splicing towards a housekeeping sequence can trigger basal expression of the death signal but without yet killing the cells. Only additional trans-splicing towards a disease or cell type-specific pre-mRNA biomarker will elevate the expression of the death signal above the threshold that finally kills the cell.
Multi-Targeting Trans-Splicing-Based Suicide RNAs Trigger Superior Levels of Cell Death at 300-Fold Lower GCV Concentration as Compared with Single-Targeting Suicide RNAs
Another embodiment of this invention are trans-splicing-based suicide RNAs targeting hepatoblastoma-derived cells harbouring target binding domains which are complementary to alpha-fetoprotein (AFP), Vascular endothelium growth factor (VEGF), γ-glutamyl transferase (GGT), Hepatocellular carcinoma associated protein 2 (HCCA2), Transforming growth factor beta 1 (TGF-β1), cluster of differentiation 24 (CD24), Cyclin D1 (CCND1), Glypican 3 (GPC3) and Telomerase reverse transcriptase (TERT). Such multi-targeting suicide RNAs triggered superior cell death activity on hepatoblastoma-derived human cells at 300-fold lower GCV concentrations as compared with single-targeting suicide RNAs (
Another embodiment of this invention are novel safety domains, i.e., antisense sequences within the trans-splicing RNAs that are complementary to, at least a part of its splice signal or domain, or its splice donor (SD) and polypyrimidine tract (PPY). These safety domains prevent off-target trans-splicing as any of the target binding domains has to bind to its target first to release the safety domain from the SD and PPY in order to enable on-target trans-splicing. Two designs of safety domains were invented and tested: 1. A continuous safety domain and 2. a segmented safety domain the latter of which was disrupted by the various target binding domains (
Another embodiment of this invention are trans-splicing-based suicide RNAs targeting HBV-positive cells. These suicide RNAs are featured with target binding domains which are complementary to the HBV pre-genomic RNA and AFP or GPC3. Though the expression of the HBV pre-genomic RNA is low in tissue culture cells, the HBV targeting suicide RNAs triggered a comparable cell death activity as the dual HCC targeting suicide RNAs (
Another embodiment of this invention are trans-splicing-based suicide RNAs effectively killing nasopharyngeal cancer cells (
Another embodiment of this invention are trans-splicing-based suicide RNAs targeting EBV-positive cells harbouring target binding domains which are complementary to Epstein-Barr virus pre-mRNAs (i.e., BZLF1, EBNA-3B, LMP1 and LMP2A).
Dumbbell Vectors Expressing Trans-Splicing RNA can Efficiently be Delivered into Various Cell Types of the Epidermis Following Topical Non-Invasive Application Triggering Cell Type Specific GFP Expression or Cell Death
Another embodiment of this invention are trans-splicing RNAs and dumbbell-shaped delivery vectors targeting epidermal cells harbouring target binding domains which are complementary to Keratin 1 (KRT1), Keratin 2 (KRT2), Keratin 10 (KRT10), Keratin 14 (KRT14), Caspase-14 precursor (CASP14), Neuroblast differentiation-associated protein 2 (AHNAK2). Another embodiment of this invention are trans-splicing RNAs targeting basal cells harbouring target binding domains which are complementary to Keratin 15 (KRT15), Collagen 17A1 (COL17A1), Tumour protein 73 (TP73). Another embodiment of this invention are trans-splicing RNAs targeting hair follicle cells harbouring target binding domains which are complementary to Homeobox C13 (HOXC13), Fibroblast growth factor 7 (FGF-7). Another embodiment of this invention are trans-splicing RNAs targeting senescent cells harbouring target binding domains which are complementary to Forkhead Box 04 (FOXO4) and cyclin-dependent kinase inhibitor 2A (p16). All these trans-splicing RNAs were either featured with a GFP gene for imaging or the HSVtk gene to trigger cell death (
Another embodiment of this invention are trans-splicing-based suicide RNAs encoding other death signals than the HSVtk such as CYLD Lysine 63 Deubiquitinase (CYLD), tumor necrosis factor-like weak inducer of apoptosis (TWEAK), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Tumor necrosis factor alpha (TNF-α) pre-mRNA. Transfection of HEK293T cells with plasmids expressing these cell death proteins triggered cell death relative to the controls (
Increasing the Size of One Dumbbell Vector Loop does not Impair Gene Expression
Dumbbell-shaped DNA vectors are unique in the way how they combine double-stranded expression cassettes with single-stranded loops. Another embodiment of this invention are dumbbell vectors with residues for targeted delivery non-covalently attached to the loops via complementary base pairing (
Non-Covalent Conjugation of Dumbbell Vector DNA with GalNAc3 and aptCD137-2 Residues Via Complementary Base Pairing
The residues that were attached were tri-antennary GalNAc residues (GalNAc3) or homo-dimers of CD137-binding aptamers (aptCD137-2). The GalNAc3 residues were attached to the 3′ ends of rather unstructured DNA or RNA oligonucleotides (
GalNAc3-RNA but not-DNA Linkers are Cleavable by RNaseH
RNA in heteroduplexes formed between complementary RNA and DNA can be cleaved by endogenous RNaseH. In the consequence, dumbbell RNA but not DNA linker-conjugates can release the GalNAc3 residue form the dumbbell after delivery into the cytoplasm to facilitate diffusion of the dumbbell through the nuclear pore complex. We investigated the cleavability of our dumbbell-GalNAc3 conjugates by RNaseH using an in vitro assay. Therefore, the conjugates were exposed to RNaseH for 120 min. To better visualise release of the small GalNAc3 residue from the relatively large dumbbell vector DNA using a gel shift assay, the conjugation loops were cleaved off with AseI before loading the samples on a gel. As expected, the gel shift assay indicates cleavability and release of the GalNAc3 residue form the RNA—but not the DNA-linker dumbbell conjugate (
MaxGFP expressing dumbbell vectors were manufactured and proven to be exonuclease resistant (
HSVtk Expressing Dumbbells Featured with Two GalNAc3 Residues at One Conjugation Loop Triggered Death of Hepatoblastoma-Derived Human Tissue Culture Cells Upon Ganciclovir Treatment
To develop dumbbell-GalNAc conjugates for suicide gene therapy of hepatocellular carcinoma (HCC), two GalNAc3 residues were attached via RNA linker oligonucleotides to a HSVtk expressing dumbbell vector (
Dumbbell-shaped DNA vectors increasingly raise attention as a promising versatile naked DNA based delivery vector system for gene therapeutic applications and for genetic vaccination. As opposed to plasmids and DNA minicircles, dumbbells can be covalently or non-covalently conjugated with helper functions for imaging, immune sensing or targeted delivery via the single-stranded loops. The are latter formed by chemically synthesized oligodeoxyribonucleotides which may be chemically modified, and which can either be added by direct ligation or modelled from primers used in a PCR reaction. Loop conjugation of helper functions is not expected to impair the transcriptional activity of dumbbell vectors but may affect cellular and nuclear targeting. We investigated non-covalent linkage of GalNAc3 and aptCD137-2 residues for targeted delivery into hepatocytes and nasopharyngeal cancer cells. Therefore, antisense oligonucleotides (DNA or RNA) were covalently attached to these residues which could then be annealed via complementary base pairing towards extended conjugation loops. Extension of the conjugation loops, though in the absence of residue conjugation, did not impair dumbbell vector-based gene expression (
These data indicate that GalNac3-mediated targeted delivery of gene expression vectors such as dumbbells works, and it works better if more than one GalNac3 residue is conjugated. In principle, dumbbell vectors can be conjugated with more than two GalNac3 residues per conjugation loop and both dumbbell loops can be explored as conjugation loops to further improve delivery into hepatocytes. Though the MaxGFP expression triggered by cellular uptake of dumbbell-GalNAc3 conjugates was readily detectable using flow cytometry, it was scarcely visible under the fluorescence microscope with only single cells showing bright fluorescence. On the other hand, uptake of HSVtk dumbbells effectively killed the targeted cells. This observation that HSVtk-expressing suicide vectors trigger a stronger phenotype than MaxGFP expression vectors may be explained by any or both of the following reasons: 1. A smaller dumbbell DNA cargo load which may be sufficient to kill a cell might not yet efficiently stain it with MaxGFP for detection, or 2. cells which were not primarily targeted by the dumbbell-conjugates might have been killed by the bystander effect that has been reported for the HSVtk/GCV gene-directed enzyme prodrug system. Notably, HepG2 cells express significantly less ASGPR on their surface as compared with primary hepatocytes. In the consequence, one would expect stronger uptake of dumbbell-GalNAc3-conjugates by primary hepatocytes ex vivo or in vivo. In summary, we demonstrated that dumbbell vectors can efficiently be conjugated with helper functions for targeted delivery via cleavable linkers. Our liver cancer-targeting GalNac3-conjugated suicide vectors are currently being tested in patient-derived xenograft (PDX) nude and humanised mouse models of HCC. As opposed to LNPs which can also be conjugated with helper functions including GalNAc3, naked dumbbell-conjugates are significantly smaller and expected to exhibit facilitated diffusion rates in the extracellular matrix. In the consequence, dumbbell-conjugates may identify single cells including cancer cells or metastasis more efficiently providing a minimalistic vector system that can complement or replace existing viral and non-viral carriers for gene therapeutic applications.
Dumbbell-GalNAc3 Conjugates Trigger Strong mRNA Expression in Murine Livers
Mice were injected iv with GFP expressing naked dumbbells (hydrodynamic injection), dumbbell-LNPs or dumbbell-GaNAc3 conjugates. Dumbbell-GalNAc3 conjugates were found to trigger highest levels of GFP mRNA expression in murine livers as quantified by RT-qPCR (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2200546.6 | Jan 2022 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2023/050034 | 1/18/2023 | WO |
