Protein replacement therapy

Abstract

The present disclosure relates to an RNA-based protein replacement therapy for diseases characterised by an abnormality in, or deficiency of one or more proteins, or for which an abnormality in, or deficiency of one or more proteins is implicated In particular, the present disclosure relates to an RNA-based protein replacement therapy for diseases in which a protein deficiency affects vascular cells, and in particular, pulmonary endothelial and / or smooth muscle cells.

Description

[0001] Protein Replacement Therapy

[0002] Field of the Invention

[0003] The present disclosure relates to an RNA-based protein replacement therapy for diseases characterised by an abnormality in, or deficiency of one or more proteins, or for which an abnormality in, or deficiency of one or more proteins is implicated. In particular, the present disclosure relates to an RNA-based protein replacement therapy for diseases in which a protein deficiency affects vascular cells, and in particular, pulmonary endothelial and / or smooth muscle cells.

[0004] Background

[0005] Certain diseases are characterised by a deficiency of one or more proteins. In some such diseases, the protein deficiency affects one or more cell types of the pulmonary and / or cardiovascular system. Examples include pulmonary vascular disease and hereditary hemorrhagic telangiectasia (HHT, also known as Osler-Weber-Rendu syndrome).

[0006] Pulmonary vascular disease (PVD) is a term meaning a condition that affects the blood vessels leading to or from the lungs. PVD is classified into various categories based on the underlying cause, pathology and haemodynamic profile, and includes pulmonary veno-occlusive disease (PVOD), pulmonaiy capillary hemangiomatosis (PCH) (although PVOD and PCH are sometimes difficult to separate clinically) and pulmonary arterial hypertension (PAH).

[0007] HHT is an inherited disorder characterized by malformations of various blood vessels (vascular dysplasia), potentially resulting in haemorrhaging and shunting of blood.

[0008] The indolent nature of these diseases mean that individuals are typically unaware they have them until they present with fully established disease, by which time treatment has limited effects on mortality (as an example, PAH is often fatal in 3-5 years from diagnosis).

[0009] PVOD, PCH, PAH and HHT are characterised by a reduction in one or more proteins. For example, the Bone Morphogenetic Protein Receptor 2 (BMPR2) signalling pathway is central to PAH. BMPR2 is a type II transmembrane serine / threonine kinase belonging to the Tumour Growth Factor (TGF)-p signalling superfamily. BMP signalling through BMPR2 inhibits the proliferation and migration of smooth muscle and endothelial cells, and prevents neointimal formation. Genetic mutations in several components of the BMPR2 pathway have been identified in PAH patients, including in BMPR2, co-receptors such as ALK1, SMAD9 and ENG, as well as the key ligand of BMPR2 in pulmonary arterial endothelial cells, BMP9. In patients with PAH, BMPR2 expression is reduced, which leads to increased activity of TGF-P signalling, resulting in progressive remodelling of the vasculature, with the result that endothelial cells of the pulmonary artery change their differentiation state / identity. Evidence from work on BMP9, the ligand for BMPR2, in endothelial cells showed that targeting BMPR2 signalling increases this cell type and was sufficient to prevent and reverse PAH (Long et al, 2015). However there is currently no clinically selective means of addressing the chronic down-regulation of BMPR2 in PAH and thus no means of restoring the identity of the pulmonary arterial endothelial cells and reversing the vasculature remodelling that occurs.

[0010] PVOD and PCH are characterised by a reduction in the protein general control nonde repressible 2 (GCN2), and HHT is characterised by a reduction in ALK1. It is thought that the reduction or loss of these proteins contributes to lung remodelling.

[0011] Conventional therapies for diseases that arise from defects in a protein-producing gene tend to focus on treatment of symptoms. For example, therapies for PAH target three main signalling pathways which play a key role in regulating vascular cell proliferation and pulmonary vascular tone: prostaglandin analogues (e.g. iloprost and selexipag) target the prostacyclin pathway, stimulating the production of cyclic adenosine monophosphate which induces vasodilation; phosphodiesterase type V inhibitors (e.g. sildenafil and riociguat) target the nitric oxide signalling pathway, promoting vasodilation by increasing the levels of cyclic guanosine monophosphate; and endothelin-1 receptor antagonists (e.g. Bosentan™), which reduce vasoconstriction by targeting endothelin receptors. Although such therapies may contribute to improved life expectancy, they are typically very costly, and importantly, do not address the underlying pathology of the diseases in question.

[0012] Further to this, the routes of administration for current therapies tend to focus on nebulised administration, subcutaneous infusion or daily oral therapy, all of which require administration at least once and typically multiple times a day, or continuous administration.

[0013] There is, therefore, an important unmet clinical need for new treatments for diseases that arise from defects in a protein-producing gene(s), which are more effective, easier and less frequent to administer and less expensive.

[0014] Reynolds et al. (2012) investigated use of an adenovirus vector lung-targeting strategy to deliver BMPR2 to the pulmonary endothelium for the treatment of PAH. The vector was linked to a bispecific antibody conjugate that targeted the virus to angiotensin-converting enzyme (ACE), a membrane-bound protease that is highly expressed on pulmonary endothelial cells. The approach was reported to improve pulmonary endothelial gene delivery and attenuate PAH.

[0015] However, there are concerns over the use of adenosine-associate virus gene therapy, including potential mutagenic genome integrations.

[0016] WO2O23143541 discloses a naked circular RNA comprising a nucleic acid sequence encoding a therapeutic polypeptide for treating a disease or condition. BMPR2 is mentioned as the therapeutic polypeptide, with PAH or POVD as the disease or condition.

[0017] Issues have been identified with the use of circular RNA therapeutically however, including off-target effects and low efficiency.

[0018] In recent years, there has been considerable interest in (linear) mRNA-based therapeutics for protein-replacement therapy, including the use of self-replicating RNA (srRNA). srRNA, also known as self-amplifying replicon RNA, is genetically engineered, linear, single-stranded RNA that is typically synthesized with a 5’ cap, 3’ polyA tail and 5’ and 3’ untranslated regions (UTRs). srRNA encodes the gene of interest together with additional, non-structural genes, derived from either a positive- or negativestrand RNA virus, that encode a replicase. The replicase enables amplification upon delivery of the RNA into a cell. Typically, srRNA constructs are derived from alphaviruses, from which at least one gene encoding structural proteins has been deleted, causing them to be unable to produce infectious progeny. The deleted genes can be replaced with heterologous genes of interest. Such constructs typically comprise four non-structural proteins, a subgenomic promoter, and the gene or genes of interest.

[0019] As such, srRNA can be used as basis for introducing foreign sequences to host cells by replacing viral sequences encoding structural genes or inserting the foreign sequences 5’ or 3’ of the sequences encoding the structural genes. srRNA maintains auto-replicative activity derived from the RNA virus and therefore can replicate in host cells, resulting in an amplification of the RNA encoding the desired gene product. Amplification can lead to very high RNA copy numbers. As a result, therapeutics based on srRNA can typically be dosed at low levels. srRNA can thus be used to enhance efficiency of RNA delivery and / or expression of the encoded gene products for therapeutic and / or prophylactic applications.

[0020] However, despite its therapeutic potential, challenges remain regarding the use of mRNA therapies. These include the need for effective delivery of the therapeutic mRNA specifically to the desired cell(s) or organ(s); successful mRNA translation to the functional protein of interest; and minimization of potential immunogenicity of the therapy.

[0021] In relation to these points, a delivery system is typically required for mRNA-based therapeutics in order to target the mRNA-based therapy to the desired organs or cells, and achieve cytomembrane penetration, lysosome escape, and protein expression, thereby avoiding non-specific delivery. In addition, RNA molecules are inherently fragile as a result of sensitivity to both extracellular and cytosolic ribonucleases (RNases). Further to this, an srRNA construct is typically much larger (typically ~io,ooo nucleotides) than mRNA (typically ~2,ooo nucleotides), and so more difficult to deliver to a cell. A delivery system can aid delivery of such large molecules and help to avoid RNAse degradation. However, difficulties remain with the use of delivery vehicles. For example, targeting of systemically administered lipid nanoparticles (LNPs) co-formulated with mRNA has historically been confined to the liver and spleen, with nanoparticles rapidly recognized by mononuclear phagocytic systems in the liver and spleen by binding to serum proteins.

[0022] Further to this, it is known that administration of RNA based therapeutics can trigger inflammatory responses. The innate immune system contains many microbial recognition molecules, including Toll-like receptors located both at the cell surface and in endosomes, and cytosolic sensing molecules such as RIG-I, PKR. Triggering of these innate mechanisms by exogenous viral and nucleic acid constructs typically leads to induction of type I interferon responses. These can result in modulation of protein expression from the constructs as a result of host protein translational mechanisms being shut down. In addition, activation of the innate immune system may result in generation of anti-drug antibodies against encoded biologies. As different viral species and subspecies may be recognized differently by the innate immune system, the constructs for use in a biotherapeutic may need to be customized, as some viral constructs may be better suited for noninflammatory expression of particular proteins.

[0023] As such, safe and efficacious delivery of mRNA-based therapeutics to specific organs and cells in vivo remains a challenge, and at present there are no RNA-based therapies aimed at addressing pulmonary vascular disease or hereditary hemorrhagic telangiectasia. There is therefore a need for new mRNA-based tools for in vivo expression of gene products, that target specific organs or cells, and in particular vascular cells, in quantities and for a period of time sufficient to produce therapeutic benefit.

[0024] It is amongst the objects of the present disclosure to address these problems and meet the needs discussed here.

[0025] Summary The present inventors have developed an RNA construct containing one or more genes of interest. The construct may be used as the basis for a protein replacement therapy.

[0026] Accordingly, in a first aspect there is provided a self-replicating RNA (srRNA) construct comprising one or more genes of interest, wherein the gene(s) of interest encode for one or more proteins of interest, wherein the proteins of interest are one or more of BMPRII, ALK1, SUR1, Aquaporin-1, ATPase 13A3, BMP-10, Caveolin-1, GCN2, Endoglin, BMP-9, GGCX, TASK-1, VEGFR2, Kallikrein 1, SMAD1, SMAD4, SMAD8 (which is also known as SMAD9), SOX17 and / or TBX4; or an active variant thereof, wherein the construct further comprises a) one or more of a 5' cap, 3' polyA tail and 5' and / or 3' untranslated regions (UTRs), and b) one or more viral retention and replication genes derived from a positive- or negative-strand RNA virus.

[0027] In some embodiments, the gene(s) of interest are one or more of BMPR2, ACVRL1 ABCC8, AQPl, ATP13A3, BMP10, CAV1, EIF2AK4, ENG, GDF2, GGCX, KCNK3, KDR, KLK1, SMAD1, SMAD4, SMAD9, SOX17, TBX4.

[0028] In some embodiments, the virus is an alphavirus, and the viral retention and replication genes are nsPi, nsP2, nsP3 and nsP4.

[0029] In some embodiments, the gene of interest is or includes BMPR2 and / or ACVRL1.

[0030] In some embodiments, the gene(s) of interest is or includes BMPR2. In some embodiments, the gene of interest is or includes ACVRL1. In some embodiments, the gene of interest is BMPR2. In some embodiments, the gene of interest is ACVRL1.

[0031] In some embodiments, the construct further comprises one or more modified nucleosides, wherein the modified nucleosides comprise pseudouridine, Ni-methyl- pseudouridine, 5-methylcytidine, 6-methyladenosine and 2'-0-methylated nucleoside or derivatives thereof.

[0032] In some embodiments, the construct further comprises B18R and / or E3L.

[0033] In some embodiments wherein the construct comprises two or more genes, the genes are separated by a 2A or IRES sequence. In some embodiments, the construct is formulated with, incorporated into, bound to, encased by or adsorbed on a delivery vehicle. In some such embodiments, the delivery vehicle is a liposome, a lipoplex, a lipid nanoparticle or a combination thereof. In some such embodiments, the delivery vehicle is a lipid nanoparticle.

[0034] In a second aspect there is provided a pharmaceutical composition comprising an srRNA construct according to the first aspect, and a pharmaceutically acceptable excipient.

[0035] In a third aspect there is provided an srRNA construct according to the first aspect or a pharmaceutical composition according to the second aspect for use as a medicament.

[0036] In a fourth aspect, there is provided the srRNA construct according to the first aspect, or the pharmaceutical composition according to the second aspect, for use in treating, preventing or ameliorating one or more of pulmonary veno-occlusive disease (PVOD), pulmonary capillary hemangiomatosis (PCH), pulmonary arterial hypertension (PAH) or hereditary hemorrhagic telangiectasia (HHT).

[0037] In a fifth aspect there is provided a method of gene delivery comprising administering an srRNA construct according to the first aspect or a pharmaceutical composition according to the second aspect to an individual in need thereof.

[0038] In a sixth aspect there is provided a method of treating a subject, comprising administering to the subject in need thereof, a therapeutically effective amount of an srRNA construct according to the first aspect, or a pharmaceutical composition according to the second aspect. In some embodiments, the subject has one or more of PVOD, PCH, PAH or HHT.

[0039] In a seventh aspect, there is provided a method of treating, preventing or ameliorating one or more of PVOD, PCH, PAH and HHT in a subject, the method comprising administering, to a subject in need thereof, a therapeutically effective amount of an srRNA construct according to the first aspect, or a pharmaceutical composition according to the second aspect. In an eighth aspect there is provided a kit comprising an srRNA construct according to the first aspect, a pharmaceutical composition according to the second aspect and instructions for use.

[0040] Brief Description of Figures

[0041] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:

[0042] Figure 1: demonstrates expression of BMPR2 in smooth muscle cells (A) and iPSC- derived pulmonary artery like-endothelial cells (B);

[0043] Figures 2A and B: show generalised schematic examples of constructs according to the present disclosure:

[0044] Figure 2A (top figure) shows a monocistronic srRNA construct comprising a 5’ cap, 3’ polyA tail, 5’ and 3’ untranslated regions (UTRs), four viral retention and replication genes, and a single gene of interest; Figure 2A (bottom figure) shows a polycistronic srRNA construct comprising a 5’ cap, 3’ polyA tail, 5’ and 3’ untranslated regions (UTRs), four viral retention and replication genes, and three genes of interest (although it should be noted that more or fewer genes could be included) separated by 2A or IRES;

[0045] Figure 2B (top figure) shows a monocistronic non-self-replicating linear mRNA construct comprising a 5’ cap, 3’ polyA tail and 5’ and 3’ untranslated regions (UTRs), and a single gene of interest; Figure 2b (bottom figure) shows a polycistronic non-self-replicating linear mRNA construct comprising a 5’ cap, 3’ polyA tail, 5’ and 3’ untranslated regions (UTRs), and three genes of interest (although it should be noted that more or fewer genes could be included) separated by 2A or IRES;

[0046] Figure 3: shows a schematic illustration of an example DNA template for an srRNA construct according to the present disclosure comprising IRES, B18R, IRES and E3L (i2592bp);

[0047] Figure 4: shows a schematic illustration of an example DNA template for an srRNA construct according to the present disclosure comprising IRES, B18R, IRES and E3L and the gene of interest, BMPR2 (15709 bp); Figure 5: shows a schematic illustration of an example DNA template for an srRNA construct according to the present disclosure, comprising IRES, B18R, P2A and E3L and the gene of interest, BMPR2 (15159 bp);

[0048] Figure 6: shows a schematic illustration of an example DNA template for an srRNA construct according to the present disclosure, comprising a BMPR2 open reading frame linked to B18R using a self-cleaving T2A peptide sequence. B18R is followed by a triple stop codon signal (14036 bp);

[0049] Figure 7: shows cell count changes in bone endothelial cells (BOECs) from a healthy individual, or an individual with PAH (‘diseased’). Cells transfected with srRNA according to the present disclosure are marked as ‘+sRNA’;

[0050] Figure 8: shows cell count changes in wildtype or isogenic BMPR2+ / - mutant induced pluripotent stem cell-derived arterial endothelial cells (iPSC-ECs). Cells transfected with srRNA according to the present disclosure are marked as ‘+sRNA’; Figure 9: shows cell count changes in wildtype or isogenic BMPR2+ / - mutant induced pluripotent stem cell-derived pulmonary vascular-like smooth muscle cells (iPSC-SMCs). Cells transfected with srRNA according to the present disclosure are marked as ‘+sRNA’;

[0051] Figure 10: shows a schematic illustration of an example DNA template for an srRNA construct according to the present disclosure comprising the gene of interest, human ACVRL1 (i2286bp). The template reflects that of Figure 6, but with the BMPR2 open reading frame replaced by ACVRL1 ORF.

[0052] Detailed Description

[0053] The present disclosure relates to a protein replacement therapy based on the use of an RNA construct to produce expression of a gene product(s). In particular, the therapy aims to provide expression of a gene product(s), the absence or reduction of which detrimentally affects pulmonary cells, and in particular, pulmonary endothelial and / or smooth muscle cells.

[0054] In some embodiments, the therapy is aimed at up regulating or replacing a protein known to be decreased or absent in one or more of pulmonary vascular disease and / or hereditary hemorrhagic telangiectasia (HHT), and in particular, one or more of pulmonary veno-occlusive disease (PVOD), pulmonary capillary hemangiomatosis (PCH), pulmonary arterial hypertension (PAH) and / or HHT. In some embodiments, the therapy is aimed at treating one or more of pulmonary vascular disease and / or hereditary hemorrhagic telangiectasia (HHT), and in particular, one or more of pulmonary veno-occlusive disease (PVOD), pulmonary capillary hemangiomatosis (PCH), pulmonary arterial hypertension (PAH) and / or HHT.

[0055] The RNA construct will comprise one or more genes, the down regulation of which is implicated in or associated with the disease(s) or conditions(s) in question. Example genes, their products and the disease(s) / condition(s) in which the gene products are known to be reduced are shown in Table i.

[0056] Table i.

[0057] In some embodiments, the RNA construct may carry a single gene (mono-cistronic). In alternative embodiments, the RNA construct may carry multiple genes (poly- cistonic). In some embodiments, the RNA construct comprises one or more of the genes in Table i.

[0058] The RNA may be non-self- replicating mRNA. Alternatively, the RNA may be selfreplicating RNA (srRNA). Preferably, the RNA is self-replicating RNA (srRNA).

[0059] Accordingly, in a first aspect there is provided a self-replicating RNA (srRNA) construct comprising one or more genes of interest, wherein the gene(s) of interest encode for one or more proteins of interest, wherein the proteins of interest are one or more of BMPRII, ALK1, SUR1, Aquaporin-1, ATPase 13A3, BMP-10, Caveolin-1, GCN2, Endoglin, BMP-9, GGCX, TASK-1, VEGFR2, Kallikrein 1, SMAD1, SMAD4, SMAD8, SOX17 and / or TBX4; or an active variant thereof; wherein the construct further comprises one or more of a 5' cap, 3' polyAtail and 5' and / or 3' untranslated regions (UTRs), and one or more viral retention and replication genes derived from a positive- or negative-strand RNA virus.

[0060] ‘An active variant’ of a protein of interest is a variant that retains sufficient functionality compared to the protein of interest to have a therapeutic effect. For example, an active variant may retain at least 10%, preferably at least 25%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% of the functionality of the protein of interest. Examples of ‘functionality’ include the role or contribution of the protein in or to one or more of cell proliferation, cell survival, cell differentiation, cell migration, cellular transcriptional profile and / or epigenomic profile or inter- or intra-signalling pathways.

[0061] ‘An active variant thereof includes within its scope a fragment of the protein of interest. In some embodiments, a fragment of the protein of interest has at least 10% of the length of the sequence of the protein of interest, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% and most preferably at least 95% of the length of the sequence of the protein of interest.

[0062] ‘An active variant thereof also includes within its scope a protein sequence that has homology with the sequence of the protein of interest, such as at least 50% identity, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, preferably at least 98%, preferably at least 98.5%, preferably at least 99%, preferably at least 99.5%, or preferably at least 99.8% identity, for example over the full protein of interest sequence or over a region of contiguous amino acid residues representing 10% of the length of the protein of interest sequence, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% and most preferably at least 95% of the length of the protein of interest sequence. Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as “hard homology”).

[0063] The homologous active protein of interest variants may differ from the protein of interest sequence by substitution, insertion or deletion, for example from 1, 2, 3, 4, 5 to 8 or more substitutions, deletions or insertions. The substitutions are preferably 'conservative', that is to say that an amino acid may be substituted with a similar amino acid, whereby similar amino acids share one of the following groups: aromatic residues (F / H / W / Y), non-polar aliphatic residues (G / A / P / I / L / V), polar- uncharged aliphatics (C / S / T / M / N / Q) and polar-charged aliphatics (D / E / K / R). Preferred sub-groups comprise: G / A / P; I / L / V; C / S / T / M; N / Q; D / E; and K / R.

[0064] In some embodiments, a decrease in the expression of the gene(s) of interest and / or the protein(s) of interest is associated with pulmonary veno-occlusive disease (PVOD), pulmonary capillary hemangiomatosis (PCH), pulmonary arterial hypertension (PAH) or hereditary hemorrhagic telangiectasia (HHT).

[0065] In some embodiments, the protein of interest is or includes BMPRII and / or ALK1. In some embodiments, the proteins of interest are BMPRII and ALK1. In some embodiments, the protein of interest is or includes BMPRII. In some embodiments, the protein of interest is BMPRII. In some embodiments, the protein of interest is or includes ALK1. In some embodiments, the protein of interest is ALK1. In some embodiments, the gene(s) of interest are one or more of BMPR2, ACVRL1, ABCC8, AQPl, ATP13A3, BMP1O, CAV1, EIF2AK4, ENG, GDF2, GGCX, KCNK3, KDR, KLK1, SMAD1, SMAD4, SMAD9, SOX17, TBX4.

[0066] Constructs according to the present disclosure comprise one or more of a 5’ cap structure, polyA signals and / or polyA tails, and / or 5’ and / or 3’ untranslated regions (UTRs).

[0067] Capping and structure: the 5’ cap protects the RNA from cellular degradation and is required for translation initiation. The RNA may be co- or post-transcriptionally capped with various capping methods or cap structures and cap structure modifications. Example Cap structures can include ARCA, Cap-o, Cap-1 and CleanCap-i structures, and modifications may include but are not limited to 2'OMe and / or m6A and / or N mG 3'oMe.

[0068] PolyA signals and polyA tails: these are used for poly-adenylating RNA transcripts. Polyadenylation and polyA tail length affects RNA stability and perdurance, they are usually 100-120 adenines in length, although this can be variable. The polyA sequence can be included in the DNA vector used to generate the therapeutic RNA transcript or can be added as a separate reaction after in vitro transcription.

[0069] UTRs: various untranslated regions (UTRs) maybe included in the srRNA and are used to regulate RNA stability and translation. In some embodiments, 5’UTR sequences maybe modified for inclusion / absence of start / stop codons and sequence that minimises secondary structure. In some embodiments, 3’UTR sequences maybe modified for optimal sequences for translation and for secondary structure regulate RNA translation, stability or degradation.

[0070] In some embodiments UTRs may include a gene of interest’s endogenous UTRs, or UTRs from viruses such as alphavirus or other organism, which may be added in various arrangements or as fusions. Examples include a- and p-globin gene UTRs and the fusion UTR of the mitochondrially encoded 12S rRNA (mtRNRi) 3’ UTR and the Aminoterminal Enhancer of Split 3’ UTR.

[0071] In some embodiments, constructs comprise a 5’ cap structure. In some embodiments, constructs comprise polyA signals and / or polyA tails. In some embodiments, constructs comprise polyA signals. In some embodiments, constructs comprise polyA tails. In some embodiments, constructs comprise 5’ and / or 3’ UTRs. In some embodiments, constructs comprise 5’ UTRs. In some embodiments, constructs comprise 3’ UTRs. In some embodiments, constructs comprise a 5’ cap, preferably a 5’ cap and a polyA tail, preferably further including a polyA signal, preferably further including a 3’ UTR, preferably further including a 5’ UTR.

[0072] In some preferred embodiments, constructs according to the present disclosure comprise a post-transcriptionally added modified 5’ cap, alphavirus or SV40 polyA 3’ polyA signal and tail and alphavirus, globin or SV405’ and 3’ untranslated regions (UTRs).

[0073] In some embodiments, the RNA construct comprises genetic material from a virus. In preferred embodiments, the virus belongs to the Alphavirus genus of the Togaviridae family. Alphaviruses are small, enveloped RNA viruses with a singlestranded, positive-sense RNA genome. The alphavirus genome is approximately 11- 12 kb long, comprising a 5’ prime cap, a 3’ poly(A) tail, and two open reading frames (ORFs): a 4 kb frame encoding the structural polyprotein and a 7 kb frame encoding the nonstructural proteins (nsPs). The 4 kb frame encodes the viral structural proteins such as the capsid protein CP, El glycoprotein, E2 glycoprotein, E3 protein, and 6K protein. The non- structural polyprotein (nsP) is cleaved into four different proteins (nsPi, nsP2, nsP3, and nsP4) which are necessary for the transcription and translation of viral mRNA inside the cytoplasm of host cells.

[0074] In some preferred embodiments, the virus belongs to an alphavirus species belonging to VEEV / EEEV group, or the SFV group, or the SINV group. Examples of suitable alphaviruses include Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), Everglades virus (EVEV), Mucambo virus (MUCV), Pixuna virus (PIXV), Middleburg virus (MIDV), Chikungunya virus (CHIKV), O’Nyong-Nyong virus (ONNV), Ross River virus (RRV), Barmah Forest virus (BF), Getah virus (GET), Sagiyama virus (SAGV), Bebaru virus (BEBV), Mayaro virus (MAYV), Una virus (UNAV), Sindbis virus (SINV), Aura virus (AURAV), Whataroa virus (WHAV), Babanki virus (BABV), Kyzylagach virus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu virus (NDUV), Madariaga virus (MADV), and Buggy Creek virus. In some embodiments, the virus is Eastern equine encephalitis virus (EEEV), Chikungunya virus (CHIKV), Sindbis virus (SINV), Venezuelan equine encephalitis virus (VEE), Madariaga virus (MADV), Western equine encephalitis virus (WEEV), or Semliki Forest virus (SFV).

[0075] In preferred embodiments, the virus is the Venezuelan Equine Encephalitis virus.

[0076] In embodiments wherein the RNA is srRNA, the construct further comprises one or more viral retention and replication genes. In some such embodiments, the construct comprises four viral retention and replication genes. For example, in VEEV, these genes are known as nsPi, nsP2, nsP3 and nsPq (where nsP means non- structural protein). nsPi is required for RNA capping. nsP2 is involved in RNA replication, capping and procession of the non-structural polyprotein. nsP3 is required for RNA synthesis. nsPq is necessary for functioning of the viral replicase complex.

[0077] In some embodiments, the gene of interest is or includes BMPR2 and / or ACVRL1. In some embodiments, the genes of interest are or include BMPR2 and ACVRL1. In some embodiments, the genes of interest are BMPR2 and ACVRL1. In some embodiments, the gene of interest is or includes BMPR2. In some embodiments, the gene of interest is or includes ACVRL1. In some embodiments, the gene of interest is BMPR2. In some embodiments, the gene of interest is ACVRL1.

[0078] In some embodiments, the construct comprises one or more 2A or IRES sequences. In some embodiments wherein the construct contains more than one gene, or more than one gene of interest, the genes and / or genes of interest may be separated by one or more of 2A or IRES sequence(s). 2A peptides are typically short (-18-25 amino acid) peptides derived from viruses. They are referred to as ‘self-cleaving’ peptides, and will produce multiple proteins from the same transcript. For example, 2A peptides can be used to express two separate proteins from a single open-reading frame. Suitable 2A peptides include be P2A, E2A, F2A or T2A. IRES (‘internal (or ‘intra’) ribosome entry site’) are translational enhancers which allow translational machinery to start protein synthesis by internal initiation; they allow the design of polycistronic constructs expressing several genes from a single mRNA, by allowing the simultaneous expression of two proteins separately from the same RNA transcript. Use of 2A peptides can confer an advantage over the use of IRES sequences due to their smaller size, which can help in construct creation. Accordingly, in some embodiments wherein the construct contains more than one gene, or more than one gene of interest, the genes or genes of interest are separated by a 2A sequence; preferably a T2A sequence.

[0079] In some embodiments, constructs according to the present disclosure comprise modified nucleosides, such as those aimed at increasing expression and / or half-life of the RNA and / or reduce immunogenicity. Examples include but are not limited to pseudouridine, Ni-methyl-pseudouridine, 5-methylcytidine, Mi-methyladenosine and 2,-0-methylated nucleoside or modifications or derivatives thereof.

[0080] In some embodiments, constructs according to the present disclosure comprise one or more features aimed at reducing or avoiding inflammation responses. In some embodiments, constructs according to the present disclosure may comprise factors known to reduce or prevent an inflammatory response and / or reduce immunogenicity, such as factors known to reduce or prevent an inflammatory response and / or reduce immunogenicity upon administration of an RNA-based therapy. In some embodiments, constructs according to the present disclosure may comprise B18R and / or E3L. B18R is a protein of the Vaccinia virus which binds to type I interferons and inhibits activation of interferon-mediated signal transduction. E3L protects a virus from the immune system complement attack by inhibiting the classical and alternative complement activation pathway response process. In some embodiments, constructs may comprise B18R. In some embodiments, constructs may comprise E3L.

[0081] Alternatively, or in addition, constructs according to the present disclosure may be transcribed using modified ribonucleoside tri-phosphate (rNTPs), with the aim of reducing the recipient cell’s interferon response to the exogenous RNA . Examples of modified nucleosides include but are not limited to pseudouridine, Ni-methyl- pseudouridine, 5-methylcytidine, M>-methyladenosine and 2'-0-methylated nucleoside or modifications or derivatives thereof. Figures 2A and B show schematic generalised representations of exemplary RNA constructs according to the present disclosure. Conserved sequence elements at the 5’ and 3’ end act as promoters for transcription.

[0082] In some embodiments, expression of the gene(s) of interest occurs in cells found in or associated with the pulmonary and / or cardiovascular system. In some embodiments, expression of the gene(s) of interest occurs in one or more of pulmonary endothelium cells, smooth muscle cells, fibroblasts, myofibroblasts, adipose cells, mesenchymal cells, pericytes or immune cells and / or blood cells found in or associated with the pulmonary or cardiovascular system.

[0083] In some embodiments, expression of the gene(s) of interest is limited to cells found in or associated with the pulmonary and / or cardiovascular system. In some such embodiments expression of the gene(s) of interest is limited to one or more of pulmonary endothelium cells, smooth muscle cells, fibroblasts, myofibroblasts, adipose cells, mesenchymal cells, pericytes or immune cells and / or blood cells found in the pulmonary or cardiovascular system. In some embodiments, expression of the gene(s) of interest is limited to vascular cells. In some embodiments, expression of the gene(s) of interest is limited to pulmonary endothelium cells and / or smooth muscle cells.

[0084] In some preferred embodiments, the RNA is srRNA and the gene of interest is BMPR2 and / or ACVRLi. In some such embodiments expression of the gene(s) of interest is limited to vascular cells. In some embodiments, the srRNA produces sustained expression of the gene of interest in vascular cells. In some embodiments, the srRNA produces sustained expression in pulmonary endothelial cells and / or smooth muscle cells. In some such embodiments wherein the gene of interest is BMPR2, BMPR2 expression occurs or is limited to vascular cells. In some embodiments, the srRNA produces sustained expression of BMPR2 in vascular cells. In some embodiments, the srRNA produces sustained expression of BMPR2 in pulmonary endothelial cells and / or smooth muscle cells.

[0085] Figure 3 shows a schematic illustration of a DNA template for an srRNA construct according to the present disclosure comprising IRES, B18R, IRES and E3L. The sequence for the template of Figure 3 can be found in SEQ ID NO:i. One or more genes of interest may be inserted in between the Ndel or Asci restriction sites at 5’ end and / or Fsel or Notl restriction sites at the 3’ end.

[0086] Figure 4 shows a schematic illustration of a DNA template for an srRNA construct according to the present disclosure comprising IRES, B18R, IRES and E3L and the gene of interest, BMPR2. The sequence for the construct of Figure 4 can be found in SEQ ID N0:2.

[0087] Figure 5 shows a schematic illustration of a DNA template for an srRNA construct according to the present disclosure comprising IRES, B18R, P2A, and E3L and the gene of interest, BMPR2. The sequence for the construct of Figure 5 can be found in SEQ ID NO:3.

[0088] SEQ ID NO: 4 provides the sequence for a DNA template for an srRNA construct according to the present disclosure comprising IRES, B18R, P2A and E3L.

[0089] Figure 6 shows a schematic illustration for a DNA template for an srRNA construct according to the present disclosure comprising a BMPR2 open reading frame linked to B18R using a self-cleaving T2A peptide sequence. The B18R is followed by a triple stop codon signal. The sequence for the construct of Figure 6 can be found in SEQ ID NO: 5.

[0090] The BMPR2 open reading frame, as disclosed in Figure 6, may be replaced by the open reading frame of any of the other genes of interest disclosed herein, to produce a DNA template for an alternative srRNA construct according to the present disclosure.

[0091] Figure 10 shows a schematic illustration for a DNA template for an srRNA construct according to the present disclosure comprising human ACVRL1. The template reflects that of Figure 6, but with the BMPR2 open reading frame replaced by ACVRLi ORF. The sequence for the construct of Figure 10 can be found in SEQ ID NO: 7. SEQ ID NO: 6 provides the sequence for an alternative DNA template for an srRNA construct according to the present disclosure comprising human ACVRL1 and B18R and E3L separated by IRES sequences.

[0092] In some embodiments, RNA constructs according to the present disclosure may be formulated with, incorporated into, bound to, encased by or adsorbed on a delivery vehicle. In some embodiments the delivery vehicle is a liposome, a lipoplex, a lipid nanoparticle, or combinations thereof. Alternative delivery vehicles may be include a polymer nanoparticle, a polyplex, a conjugate of a bioactive ligand, or a combination of any thereof. Such delivery vehicles are known, as the skilled person would appreciate. In preferred embodiments, the RNA construct or pharmaceutical composition is encapsulated in a lipid nanoparticle (LNP). The LNP may be a modifiable ionizable liponanoparticle (LNP). The use of LNPs as delivery vehicles is known, as is their use to deliver RNA constructs to specific cells in vivo. LNPs can be optimised for the selected route(s) of administration. In some embodiments, the RNA construct of the present disclosure maybe incorporated into an LNP which provides effective delivery to vascular cells, and in particular, pulmonary endothelial and / or pulmonary smooth muscle cells. An additional advantageous feature of the selected LNP would be low toxicity.

[0093] In a second aspect, there is provided a pharmaceutical composition comprising the RNA construct according to the first aspect and a pharmaceutically acceptable excipient.

[0094] In a third aspect, there is provided the RNA construct according to the first aspect, or the pharmaceutical composition according to the second aspect, for use as a medicament.

[0095] In a fourth aspect, there is provided the RNA construct according to the first aspect, or the pharmaceutical composition according to the second aspect, for use in treating, preventing or ameliorating one or more of pulmonaiy vascular disease or hereditaiy hemorrhagic telangiectasia (HHT), and in particular, one or more of pulmonary veno-occlusive disease (PVOD), pulmonaiy capillaiy hemangiomatosis (PCH), pulmonary arterial hypertension (PAH) or HHT. In a fifth aspect there is provided a method of gene delivery comprising administering an RNA construct according to the first aspect or a pharmaceutical composition according to the second aspect to an individual in need thereof.

[0096] In a sixth aspect, there is provided a method of treating a subject, the method comprising administering to a subject in need thereof, a therapeutically effective amount of the RNA construct according to the first aspect, or the pharmaceutical composition according to the second aspect. In some embodiments, the subject has one or more of PVOD, PCH, PAH or HHT.

[0097] In a seventh aspect, there is provided a method of treating, preventing or ameliorating one or more of PVOD, PCH, PAH and HHT in a subject, the method comprising administering, to a subject in need thereof, a therapeutically effective amount of the RNA construct according to the first aspect, or the pharmaceutical composition according to the second aspect.

[0098] Constructs, compositions or medicaments according to the present disclosure may take a number of different forms depending, in particular, on the manner in which they are to be administered. For example, the construct, composition or medicament may be provided in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, transdermal patch, suspension, polyplex or emulsion, or any other suitable form that may be administered to a person or animal.

[0099] Constructs, compositions or medicaments according to the present disclosure may be formulated for use in a number of ways. For example, oral administration maybe required, in which case the construct, composition or medicament maybe formulated to be ingested orally, for example in the form of a tablet, capsule or liquid. Alternatively, the construct, composition or medicament may be formulated for inhalation. The construct, composition or medicament may alternatively be formulated for topical use, for example as creams or ointments for application to the skin. Alternatively, the construct, composition or medicament may be formulated for systemic delivery. The construct, composition or medicament may be provided in a lyophilised format for reconstitution. In some embodiments, the construct, composition or medicament is formulated for one or more of intranasal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, intrathecal administration, intraocular, rectal, systemic and oral administration. In some embodiments, the construct, composition or medicament is formulated for systemic administration, or administration by inhalation.

[0100] Constructs, compositions or medicaments according to the present disclosure may be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin for release of the construct, composition or medicament over days, weeks or months.

[0101] In some preferred embodiments, compositions according to the invention are administered to a subject systemically, or by inhalation. Systemic injection may be into the blood stream, muscle or skin and may be intravenous, subcutaneous, intradermal, intramuscular, intrathecal, epidural or intraperitoneal.

[0102] A ‘subject’ may be a vertebrate, mammal or domestic animal. Hence constructs, compositions and medicaments according to the present disclosure may be used to treat any mammal, for example livestock (e.g. a horse), pets or may be used in other veterinary application. Preferably, however, the subject is a human.

[0103] A ‘therapeutically effective amount’ of constructs, compositions or medicaments according to the present disclosure is any amount which, when administered to a subject, is the amount needed to produce a therapeutic effect.

[0104] The term ‘pharmaceutically acceptable excipient’ as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of a compound(s) of interest to a subject. As an example, a pharmaceutically acceptable excipient includes but is not limited to diluents, additives and carriers; a pharmaceutically acceptable carrier includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents.

[0105] In an eighth aspect there is provided a kit comprising one or more constructs, compositions or medicaments as disclosed here, together with instructions for use. In some instances, the instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet) may instead be provided, for example a web address where the instructions can be viewed and / or from which the instructions can be downloaded. In some embodiments, the kits further include one or more means useful for the administration of the disclosed construct, compositions or medicaments to a subject. For example, in some embodiments, one or more nebulisers, syringes and / or catheters.

[0106] Advantages

[0107] As exemplified in the Examples, the inventors have surprisingly demonstrated that the disclosed RNA constructs can be delivered to and expressed in pulmonary cells; and can reduce proliferation rates, in comparison to controls, of adult blood outgrowth endothelial cells (BOECs) from an individual with PAH; isogenic BMPR2+ / - mutant induced pluripotent stem cell-derived arterial endothelial cells (iPSC-ECs); and isogenic BMPR2+ / - mutant induced pluripotent stem cell-derived pulmonary vascular-like smooth muscle cells (iPSC-SMCs).

[0108] These data indicate that RNA constructs according to the present disclosure can be used to increase the level of specific proteins in pulmonary cells and reduce cellular proliferation rates, thereby offering the potential to prevent and / or reverse the vasculature remodelling that occurs in pulmonary vascular diseases such as PVOD, PCH, PAH and HHT. These findings are especially advantageous given the understanding that the products of the genes of interest are diminished in the specified pulmonary diseases, and when considering the current paucity of effective treatments for these diseases, and in particular the absence of a means of addressing the underlying pathology of these diseases.

[0109] The disclosed constructs, compositions and medicaments thus offer numerous advantages, particularly over current treatments of pulmonary vascular disease and HHT. Firstly, RNA-based therapeutics are known for use in humans. Administration of compositions according to the present disclosure thus constitutes an accepted means of therapy. The use of an RNA-based therapeutic offers safety advantages over DNA-based therapies, as protein expression is cytosolic, thus avoiding nuclear entry and excluding potential insertional mutagenesis risks.

[0110] In addition, RNA therapeutics based on an alphavirus, as disclosed here, can confer advantages, as they can generate large amounts of subgenomic mRNA, from which large amounts of the protein(s) of interest in can be expressed. In addition, in contrast to, for example, retroviruses, alphavirus RNA is not subjected to reverse transcription and integration into the host genome.

[0111] Additionally, the inclusions in the construct aimed at reducing the subject’s innate inflammation response have an addition benefits: blocking the interferon (IFN) pathway has been shown to have a positive effect on reducing pathogenesis of PAH. As such it is considered that the incorporation of features such as B18R and / or E3L into the construct may provide a double benefit, by both reducing the IFN inflammatory response associated with administration of the RNA construct, and having a direct effect on disease symptoms.

[0112] The use of a delivery system, such as a nanoparticular delivery system allows targeting of the RNA-based therapy to vascular cells, and in particular to pulmonary endothelial cells and / or smooth muscle cells, thereby limiting off-target effects.

[0113] Further to this, the disclosed RNA-based protein replacement therapy has the potential to specifically upregulate the protein reduced in the disclosed conditions. For example, the disclosed srRNA-based protein replacement strategy for PAH has the potential to specifically upregulate BMPR2 signalling, the central gene that is down-regulated in PAH. As a result, off-target effects are limited or eliminated, as is the possibility of genomic integration, as discussed above. This constitutes a significant advantage, particularly over adenosine-associate virus (AAV) gene therapy, as safety concerns surrounding the use of AAV vectors include lack of target specificity, potential mutagenic genome integrations, small cloning capacity, and potential necessity for helper Ad or herpesviruses for transduction and humoral responses. The constructs of the present disclosure thus have the potential to provide a clinically selective means of achieving sustained and specific upregulation of one or more genes of interest in vascular cells; an example being the sustained and specific upregulation of BMPR2 in pulmonary endothelial and / or smooth muscle cells. The present disclosure thus provides the potential for preventing and / or reversing pulmonary vascular diseases associated with reduced expression of specific protein(s). Currently, no other therapy achieves this.

[0114] Further to this, the possibility of retainment of the srRNA construct for a prolonged period of time (for example up to 4 weeks) provides the potential for a treatment based on a single intravenous injection at ~4-week intervals, or at the time of an acute clinical event, rather than daily or continuous infusion as is the case with other PVD therapies, and thus constitutes a far less invasive, and far more cost effective method of treatment.

[0115] As such, a single administration of a construct, composition or medicament according to the present disclosure may be enough to dramatically slow chronic deterioration in the specified diseases or conditions, or to halt or reverse the pulmonary remodelling that occurs in the specified diseases or conditions.

[0116] As the points outlined above demonstrate, the present disclosure constitutes a significant and highly advantageous development for treating pulmonary vascular diseases such as PVOD, PCH, PAH and HHT, which are characterised by a reduction in one or more proteins.

[0117] The following Examples are provided to illustrate embodiments of the present invention and should not be construed as limiting thereof.

[0118] Example 1 - Background work (published in Eminli et al, 2021).

[0119] In this previous work, an RNA-based reprogramming method was used to generate induced pluripotent stem cells (iPSCs) from human late-outgrowth endothelial progenitor cell (EPCs). The work involved delivery of mRNA and self-replicating mRNA to EPCs. EPCs (also known a blood outgrowth endothelial cells or BOECs) are endothelial cells that have are well established for use as surrogates for pulmonary arterial endothelial cells (PAECs), having extremely similar identity and biology including BMPR2 signalling and proliferation and apoptosis responses. They have also been isolated from people with BMPR2 and other PAH associated mutations, and when carrying these mutations, they exhibit PAH associated disease phenotypes akin to PAECs from patients.

[0120] Example 2 - Cloning of RNA expression constructs: initial work

[0121] An mCherry (a red fluorescent protein derived from Discosoma sp.)-tagged BMPR2 srRNA construct was created as follows:

[0122] A full-length human BMPR2 open reading frame fused to mCherry was cloned into a plasmid carrying a VEE virus construct. The construct was transcribed and the transcript 5’capped and poly-adenylated to produce the srRNA construct. o.75ug of the construct was transfected into 25K iPSC-derived pulmonary artery like endothelial cells (ECs) (generated according to the teaching of Eminli et al) and smooth muscle cells (SMCs) generated using MessengerMax and supplemented with B18R protein to test its expression.

[0123] The tagged BMPR2 was expressed and localised to the cell membrane in iPSC- derived pulmonary artery like smooth muscle cells (Figure 1A) and iPSC-derived pulmonary artery like endothelial cells (Figure 1B).

[0124] Example 3 - Refinement of RNA Constructs

[0125] A generalised schematic illustration of constructs according to the present disclosure is provided in Figure 2.

[0126] It is anticipated that B18R and / or E3L could be cloned into the construct or supplemented in a second construct. As an alternative to, or in addition, transcribing the RNA using one or more modified ribonucleoside tri-phosphate (rNTPs) as discussed herein will be tested, as this has also been shown to reduce the IFN response in cells. Modified nucleosides may include but are not limited to: pseudouridine, Ni-methyl-pseudouridine, 5-methyIcytidine, N6-methyladenosme and 2'-0-methylated nucleoside or derivates or other suitable modifications thereof.

[0127] Figures 3-5 provide schematic illustrations of envisaged constructs according to the present disclosure: Figure 3 (SEQ ID NO:1) illustrates a DNA template for an srRNA construct comprising IRES, B18R, IRES and E3L; Figure 4 (SEQ ID N0:2) illustrates a DNA template for an srRNA construct comprising IRES, B18R, IRES, E3L and a gene of interest, BMPR2; Figure 5 (SEQ ID NO:3) illustrates a DNA template for an srRNA construct comprising IRES, B18R, P2A, E3L and a gene of interest, BMPR2.

[0128] Example 4 - Evaluation of delivery and expression of RNA constructs Work will be undertaken to evaluate the expression and delivery of RNA:LNPs according to the present disclosure to establish the most effective combinations.

[0129] As a control, full-length BMP9 and BMP9:T2A:GFP will be cloned into separate constructs. BMPR2, GFP and B18R or E3L will also be closed into a standard nonself-replicating mRNA generating expression construct for comparison with the srRNAs generated.

[0130] Example 4a: Reversal of PAH disease-associated cellular phenotypes in BMPR2+ / - vascular cells in vitro using BMPR2 srRNA

[0131] To test the potential therapeutic efficacy of the developed constructs, the constructs will be administered to PAH patient-derived BMPR2+ / - EPCs and pulmonary artery smooth muscle cells (PASMCs). An assessment will be undertaken to establish if diseased-associated cellular phenotypes of hyper-proliferation and reduced apoptosis and reduced BMPR2 signalling is normalised.

[0132] To complement these studies, induced pluripotent stem cell (iPSCs)-derived pulmonary-like vascular cell models of PAH9 will also be used: previous work developed induced pluripotent stem cell (iPSC)-derived pulmonary-like vascular cell models of PAH (Kiskin et al. 2018). These models allow the transition of pulmonary artery-like endothelial cells (iPSC-ECs) and smooth muscle cells (iPSC-SMCs) from a disease-free to a diseased-state by exposing the cells to a secondary factor, such as tumour-necrosis factor alpha. These models offer the opportunity to monitor the effect of expression of mRNA according to the present disclosure upon PAH- associated cellular disease phenotypes, and in particular, to track any reversal of PAH- associated cellular disease phenotypes resulting from treatment with mRNA therapies according to the present disclosure. It is anticipated that the following studies will be undertaken:

[0133] Isogenic wild-type and BMPR2+ / - iPSCs-derived pulmonary artery-like ECs and SMCs will be used to assess when the disclosed RNA constructs achieve normalisation of diseased-associated cellular phenotypes of hyper-proliferation and reduced apoptosis and reduced BMPR2 signalling. BMP9 protein and RNA will be used as controls in these studies for comparing normalisation of disease phenotypes and the enhancement of BMPR2 expression with BMPR2 srRNA constructs according to the present disclosure.

[0134] The above detailed work has been carried out and is detailed in Example 4b, below.

[0135] The effect of the BMPR2 RNA:LNP on the transcriptomes of vascular cells will also be assessed using 10X Chromium single cell RNAseq.

[0136] Example 4b: Reversal of PAH disease-associated cellular phenotypes in BMPR2+ / - vascular cells in vitro using BMPR2 srRNA sRNA was generated using the vector shown in Figure 6 (SEQ ID NO: 5). In the vector, BMPR2 open reading frame was linked to B18R using a self-cleaving T2A peptide sequence. The B18R was followed by a triple stop codon signal.

[0137] The cells used in the experiment were either: (1) adult blood outgrowth endothelial cells (BOECs) from a healthy individual or an individual with PAH; (2) wildtype or isogenic BMPR2+ / - mutant induced pluripotent stem cell-derived arterial endothelial cells (iPSC-ECs); or (3) wildtype or isogenic BMPR2+ / - mutant induced pluripotent stem cell-derived pulmonary vascular-like smooth muscle cells (iPSC- SMCs) using previously published iPSC-EC and -SMC differentiation protocols from Kiskin et al. 2018.

[0138] In each experiment, either 20,000 endothelial cells (ECs) or 30,000 smooth muscle cells (SMCs) were transfected one time on day o with o.5Ug / 100K cells of BMPR2 expressing srRNA, as detailed above. Transfected cells were maintained for 7, 14, 21 or 28 days, then detached from the culture plates into a single cell suspension and cell numbers were counted using a haemocytometer. Data is shown in Tables 2-4, below; and corresponding Figures 7-9.

[0139] Table 2: cell count data for adult blood outgrowth endothelial cells (BOECs) from a healthy individual (‘healthy’) or an individual with PAH (‘diseased’). Cells transfected with srRNA are marked as ‘+sRNA’. Data in Table 2 is shown in Figure 7. Table 2

[0140] Table 3: Cell count data for wildtype or isogenic BMPR2+ / - mutant induced pluripotent stem cell-derived arterial endothelial cells (iPSC-ECs). Cells which were transfected with srRNA are marked as ‘+sRNA’. Data in Table 3 is shown in Figure 8.

[0141]

[0142] Table 3

[0143] Table 4: cell count data for wildtype or isogenic BMPR2+ / - mutant induced pluripotent stem cell-derived pulmonary vascular-like smooth muscle cells (iPSC- SMCs). Cells which were transfected with srRNA are marked as ‘+sRNA’. Data in Table 4 is shown in Figure 9.

[0144] Table 4 The data provided in Tables 2-4 (exemplified in Figures 7-9) shows that an RNA construct according to the present disclosure (a BMPR2-srRNA construct) reduced the proliferation rates of diseased and BMPR2+ / - mutant ECs and SMCs over a 28 day period. In all the cell models tested, reduction in proliferation occurred by day 7 but was more pronounced on day 14. Reduction in proliferation rate was also observed on day 21 and day 28 and BMPR2-srRNA transfected diseased and mutant cell proliferation rates were approaching normal levels by day 28 with the dose of sRNA used.

[0145] In contrast, the effect of BMPR2-srRNA on the proliferation rate of healthy or isogenic wildtype ECs and SMCs was minimal.

[0146] Example 4c: Reversal of PAH diseased-associated cellular phenotypes in BMPR2+ / - vascular cells in vitro using ALKisrRNA

[0147] Experiments will be undertaken to test the potential therapeutic efficacy of constructs comprising human ACVRL1. An assessment will be undertaken to establish if such constructs can normalise increased cell proliferation in the cell types detailed in Example 4b. sRNA will be generated using the template shown in Figure 10. The template reflects that of Figure 6, but with the BMPR2 open reading frame replaced by ACVRLi ORF. The sequence for the construct of Figure 10 can be found in SEQ ID NO: 7. In the disclosed sequence, the insert contains hACVRLi and B18R separated by a T2A self-cleaving peptide. Alternative 2A peptides could equally be used. Alternatively, or in addition, E3L could be included. In alternative embodiments, the vector could link hACVRLi to B18R and / or E3L using one or more IRES sequences (as per SEQ ID NO:6).

[0148] Example 4d: Animal models

[0149] PAH is complex multifactorial disease involving the whole cardiopulmonary system and also other interacting systems such the immune system. As such, it is anticipated that in vivo animal model work will be undertaken to replicate the in vivo interactions and the nutrient and mechanical environments that occur in disease. In this regard, it is anticipated that the following two models animal will be used.

[0150] The CDHs-tdTom reporter mouse model uses a vascular endothelial cell cadherin promoter to drive fluorescence in endothelial cells.

[0151] A rat model mimics human PAH pathologies more closely than mouse, including vessel muscularisation. The rat Sugen54i6 / hypoxia PAH models the accepted gold standards for developing new therapeutic treatments: the combination of Sugen 5416 (SU5416; a vascular endothelial growth factor receptor antagonist) and chronic hypoxia causes pronounced pulmonary hypertension (PH) with angioobliterative lesions in rats. This combination also leads to exaggerated PH in mice.

[0152] The various embodiments described herein are presented only to assist in understanding and teaching the claimed features. These embodiments are provided as a representative sample of embodiments only, and are not exhaustive and / or exclusive. It is to be understood that advantages, embodiments, examples, functions, features, structures, and / or other aspects described herein are not to be considered limitations on the scope of the invention as defined by the claims or limitations on equivalents to the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope of the claimed invention. Various embodiments of the invention may suitably comprise, consist of, or consist essentially of, appropriate combinations of the disclosed elements, components, features, parts, steps, means, etc, other than those specifically described herein. In addition, this disclosure may include other inventions not presently claimed, but which may be claimed in future.

[0153] References

[0154] Eminli et al. N Biotechnol. 2021 Jul 25;63:i-9. doi:io.ioi6 / j.nbt.2O2i.O2.ooi. Epub 2021 Feb 12. PMID: 33588094;

[0155] Kiskin et al. Am J Respir Crit Care Med. 2018 Jul i5;i98(2):27i-275. doi: i0.n64 / rccm.20i80i-0049LE. PMID: 29547009; PMCID: PMC6058985; Long et al. Nat Med. 2Oi5;2i(7):777-85. doi: 10.1038^111.3877. Epub 2015 Jun 15. PMID: 26076038; PMCID: PMC4496295;

[0156] Reynolds et al. 2012. Eur Respiratory J. 201239: 329-343.

Claims

Claims1. A self-replicating RNA (srRNA) construct comprising one or more genes of interest, wherein the gene(s) of interest encode for one or more proteins of interest, wherein the proteins of interest are one or more of BMPRII, ALK1, SUR1, Aquaporin-1, ATPase 13A3, BMP-10, Caveolin-i, GCN2, Endoglin, BMP-9, GGCX, TASK-1, VEGFR2, Kallikrein 1, SMADi, SMAD4, SMAD8, SOX17 and / or TBX4, or an active variant thereof, wherein the construct further comprises: a) one or more of a 5' cap, 3' polyA tail and 5' and / or 3' untranslated regions (UTRs), and b) one or more viral retention and replication genes derived from a positive- or negative-strand RNA virus.

2. An srRNA construct according to claim 1, wherein the gene(s) of interest are one or more of BMPR2, ACVRL1, ABCC8, AQP1, ATP13A3, BMP10, CAV1, EIF2AK4, ENG, GDF2, GGCX, KCNK3, KDR, KLKl, SMADi, SMAD4, SMAD9, SOX17, TBX4.

3. An srRNA construct according to claim 1 or claim 2, wherein the gene(s) of interest is or includes BMPR2 and / or ACVRL1.

4. An srRNA construct according to any one of claims 1 to 3, wherein the gene of interest is BMPR2.

5. An srRNA construct according to any of claims 1 to 4, wherein the virus is an alphavirus, and wherein the viral retention and replication genes are nsPi, nsP2, nsP3 and nsP4.

6. An srRNA construct according to any one of claims 1 to 5, further comprising one or more modified nucleosides, wherein the modified nucleosides comprise pseudouridine, Ni- methyl-pseudouridine, 5-methylcytidine, 6-methyladenosine and 2'-0-methylated nucleoside or derivatives thereof.

7. An srRNA construct according to any one of claims 1 to 6, further comprising B18R and / or E3L.

8. An srRNA construct according to any one of claims 1 to 7, wherein the construct comprises two or more genes, and wherein the genes are separated by a 2A or IRESsequence.

9. An srRNA construct according to any one of claims 1 to 8, wherein the construct is formulated with, incorporated into, bound to, encased by or adsorbed on a delivery vehicle. to. An srRNA construct according to claim 9, wherein the delivery vehicle is a liposome, a lipoplex, a lipid nanoparticle or a combination thereof, optionally wherein the delivery vehicle is a lipid nanoparticle.

11. A pharmaceutical composition comprising an srRNA construct according to any one of claims 1 to 10, and a pharmaceutically acceptable excipient.

12. An srRNA construct according to any one of claims 1 to 10, or a pharmaceutical composition according to claim 11, for use as a medicament.

13. An srRNA construct or pharmaceutical composition according to claim 12, for use in treating, preventing or ameliorating one or more of pulmonary veno-occlusive disease (PVOD), pulmonary capillary hemangiomatosis (PCH), pulmonary arterial hypertension (PAH) or hereditary hemorrhagic telangiectasia (HHT).

14. A method of gene delivery comprising administering an srRNA construct according to any one of claims 1 to 10, or a pharmaceutical composition according to claim 11 to an individual in need thereof.

15. A method of treating a subject, comprising administering to the subject in need thereof, a therapeutically effective amount of an srRNA construct according to any one of claims 1 to 10, or a pharmaceutical composition according to claim 11, optionally wherein the subject has one or more of PVOD, PCH, PAH or HHT.

16. A method of treating, preventing or ameliorating one or more of PVOD, PCH, PAH and HHT in a subject, the method comprising administering, to a subject in need thereof, a therapeutically effective amount of an srRNA construct according to any one of claims 1 to10, or a pharmaceutical composition according to claim 11.

17. A kit comprising an srRNA construct according to any one of claims 1 to 10, a pharmaceutical composition according to claim 11 and instructions for use.

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