Polyomavirus gene delivery vector particles containing a nucleic acid sequence encoding a polypeptide having phosphatase activity - Patent Application 20070229933

Abstract

The present invention relates to polyomavirus gene delivery vector particles comprising a nucleic acid sequence encoding a polypeptide having phosphatase activity. In particular, the present invention relates to polyomavirus gene delivery vector particles comprising a nucleic acid sequence encoding a polypeptide having alkaline phosphatase activity and / or further comprising a nucleic acid sequence encoding a growth factor protein such as insulin and / or insulin-like growth factor or a precursor thereof. Furthermore, the present invention relates to polyomavirus gene delivery vector particles according to the present invention and compositions comprising the polyomavirus gene delivery vector particles of the present invention for use in methods of treatment by removal or inactivation of damage associated molecular patterns at a site of inflammation in a subject.

Description

[Technical field]

[0001] The present invention relates to polyomavirus gene delivery vector particles.The present invention further relates to a polyomavirus gene delivery vector particle according to the present invention for use in a method of treatment or prophylactic treatment of inflammation in a subject, for example by removal or inactivation of damage associated molecular patterns at the site of inflammation in the subject.Furthermore, the present invention relates to a composition comprising the polyomavirus gene delivery vector particle of the present invention. [Background technology]

[0002] Autoimmune diseases and allergies are associated with chronic or relapsing inflammation and immunity against self- or non-self-proteins, termed autoantigens or allergens, respectively. To date, autoimmune diseases and allergies cannot be cured. Symptoms can be masked or alleviated by general suppression of the patient's immune system. Long-term use of common immunosuppressants is often accompanied by severe side effects, such as increased susceptibility to pathogenic infections and the development of other immune-related diseases, such as cancer, inflammatory diseases, degenerative diseases, dystrophies, autoimmune diseases, and allergies. Therefore, novel therapies for autoimmune diseases and allergies that leave the patient's immune system intact are urgently needed.

[0003] Suppressing chronic or relapsing inflammation is an attractive approach to prevent inflammatory, degenerative, dystrophic, autoimmune tissue destruction, and allergic reactions. In patients with inflammatory, degenerative, dystrophic, autoimmune, and allergic diseases, inflammation can be suppressed by restoring immune tolerance to primary autoantigens (pSAg) or allergens, respectively. However, tolerization approaches to autoantigens or allergens have been less effective so far. Moreover, in most autoimmune diseases, pSAg has not been identified so far.

[0004] Another approach acting on the innate immune level to treat autoimmune diseases where pSAg has not been identified is to stop the progression of the disease by removing alarm molecules named damage-associated molecular patterns (DAMPs) released from damaged cells. Extracellular adenosine triphosphate (ATP) released from pyroptotic cells is one of the most potent DAMPs. Extracellular ATP promotes inflammation, tissue damage and mortality (Cauwels, Anje, et al. “Extracellular ATP drives systemic inflammation, tissue damage and mortality.”. Cell death & disease 5.3 (2014): e1102-e1102).

[0005] An attractive approach to remove extracellular ATP is its conversion to adenosine diphosphate (ADP), monophosphate (AMP), or adenosine (ADO) by phosphatases present at the site of inflammation. Removal of extracellular ATP molecules prevents further activation of innate and adaptive immune responses that attack and destroy cells in the affected tissue, slowing or halting the progression of autoimmune diseases.

[0006] Extracellular ATP is sensed by membrane-bound, G-protein-coupled P2 receptors (see also Figure 1). After binding, mitochondria start to produce excess ROS, leading to assembly of cytoplasmic Nod-like receptor protein 3 (NLRP3) proteins into inflammasomes and the initiation of a local inflammatory response. Extracellular ATP is degraded to adenosine (ADO) by two membrane-bound ectonucleotidases, CD39 (ecto-apyrase) and CD73 (ecto-5'-nucleotidase). Both nucleotidases are present on the surface of T regulatory cells (Tregs) and prevent immune cell activation. Although extracellular ATP is highly inflammatory, its dephosphorylated derivatives, ADP, AMP, and ADO, are sensed by membrane-bound P1 receptors and counteract inflammation and promote the restoration of immune tolerance.

[0007] US Patent Application Publication No. 2009 / 0130092 discloses a method for treating or preventing immune inflammatory, thrombotic or ischemic disorders in a subject by inhibiting leukocyte infiltration to the site of inflammation, the method comprising administering to the subject an effective amount of ecto-apyrase and / or ecto-5'-nucleotidase protein to the site of inflammation. One drawback of such methods is that the proteins have a relatively short half-life in vivo. This means that the proteins must be administered repeatedly to the patient throughout their life to maintain low levels of inflammation.

[0008] The problem of the short half-life of ectoapyrase protein and / or ecto-5'-nucleotidase protein in vivo can be circumvented by using viral gene delivery vectors to express ectoapyrase protein and / or ecto-5'-nucleotidase protein (soluble form or fusion protein thereof) at the site of inflammation.

[0009] Among the viral gene delivery vectors currently used for such purposes, lentiviral (LV) and adeno-associated viral (AAV) vectors are the most widely used, and for both vectors, such replication-defective viral vectors have been shown to be non-immunogenic or tolerogenic in hosts naive to the cognate virus.

[0010] LV vectors randomly integrate into the host genome, increasing the risk of insertional mutagenesis. Furthermore, LV vector particles are rapidly degraded when administered in vivo. For these reasons, LV vectors are primarily used for ex vivo transduction of leukocytes and / or their precursor cells to treat blood-related genetic disorders, lysosomal storage diseases, or cancer.

[0011] AAV vectors are mainly used for in vivo gene therapy. For example, WO 2014 / 003553 discloses the use of AAV vectors to express ectoapyrase and / or ecto-5'-nucleotidase proteins or their fusion proteins at sites of inflammation. US Patent Application Publication No. 2015 / 0190481 and Antonioni (Antonioli, Luca, et al. "CD39 and CD73 in immunity and inflammation." Trends in molecular medicine 19.6 (2013): 355-367) deal with ectoapyrase and / or ecto-5'-nucleotidase coding sequences in viral gene delivery vectors for treating inflammatory diseases. Since ectoapyrase and / or ecto-5'-nucleotidase proteins remain bound to the cell surface of transduced cells, the phosphatase activity in inflamed tissues is very low and the therapeutic effect is limited. Matsumoto et al. aimed to restore TNALP activity in a model animal of human pediatric hypophosphatasia using an AAV vector encoding secreted tissue non-specific alkaline phosphatase (TNALP), thereby alleviating characteristic disease symptoms such as epileptic seizures in treated animals (Matsumoto, Tae, et al. “Rescue of severe infantile hypophosphatasia mice by AAV-mediated sustained expression of soluble alkaline phosphatase.” Human gene therapy 22.11 (2011):1355-1364).

[0012] However, before receiving treatment with AAV vectors, the majority of humans have already encountered wild-type AAV and its helper virus (the adenovirus that causes the common cold) and have already acquired strong humoral and cellular immune memory against AAV capsid proteins. Clinical studies with recombinant AAV vectors have revealed that in the majority of patients administered vector particles, innate and adaptive immune responses against the virus and transgene-encoded proteins are elicited, leading to a decline in the expression levels of the therapeutic transgene over time and clearance of the transduced cells from the body, preventing re-administration of the vector. The few treated patients who showed long-term transgene expression were likely never infected with AAV and therefore immunologically naive to the vectors used in the trials. For these reasons, the efficacy of in vivo gene therapy with AAV vectors is limited. Summary of the Invention

[0013] Detailed explanation In view of the above, there is a need for novel treatments for autoimmune diseases and / or allergies that are highly effective and / or reduce patient discomfort while leaving the patient's immune system intact. In a first aspect, the present invention provides a polyomavirus gene delivery vector particle comprising a nucleic acid sequence encoding a polypeptide having phosphatase activity. It has been found that the polyomavirus gene delivery vector particle of the present invention effectively protects cells exposed to ATP from undergoing pyroptosis. Cells transduced with the polyomavirus gene delivery vector particle of the present invention are effectively protected from ATP-induced pyroptosis.

[0014] As used herein, the term "viral gene delivery vector particle" refers to a transduction-competent viral particle that comprises exposed capsid proteins on the particle surface surrounding a viral vector genome that incorporates a transgene construct.

[0015] As used herein, the term "transgene construct" refers to a nucleic acid sequence that encodes a polypeptide or protein and that has been incorporated into a viral gene delivery vector particle.

[0016] The term "polyomavirus gene delivery vector particle" as used herein refers to a virus vector particle derived from polyomavirus. Polyomavirus (family: Polyomaviridae) naturally infects mainly mammals and birds, causing chronic asymptomatic infection. Each polyomavirus species strictly replicates in its natural host. Of the approximately 120 polyomaviruses known to date, only 14 are known to infect humans. These polyomaviruses are called human polyomaviruses. Other polyomaviruses do not infect humans and are called non-human polyomaviruses. This means that humans have no immune memory against non-human polyomaviruses. As a result, the polyomavirus gene delivery vector particle of the present invention is non-immunogenic in humans. Administration of the polyomavirus gene delivery vector particle of the present invention to humans induces an immune tolerance response directed to the viral proteins and proteins encoded by the transgene construct. Typically, polyoma virus gene delivery vectors are safe, highly efficient for gene delivery, and non-immunogenic / tolerogenic in humans.

[0017] In particular, the present invention relates to polyomavirus gene delivery vector particles comprising a nucleic acid sequence encoding a polypeptide having phosphatase activity, including replication-deficient polyomavirus gene delivery vector particles, thus providing gene delivery vector particles that are defective in one or more functions essential for the replication of the viral genome or the synthesis and assembly of viral particles.

[0018] In a preferred embodiment of the present invention, the polyomavirus gene delivery vector particle may be derived from a primate polyomavirus, preferably a simian polyomavirus, such as the simian polyomavirus Simian Virus 40 (SV40). SV40 vectors, preferably replication-defective SV40 vectors, have been found to be an attractive alternative to AAV vectors for clinical gene therapy. SV40 is a polyomavirus with a 45-nanometer icosahedral capsid containing a 5.25-kb-long circular double-stranded DNA genome. The virus replicates strictly in its natural host, monkeys, causing a chronic, asymptomatic infection. SV40 particles enter infected cells via the caveolae-endosomal pathway, but in contrast to other viruses, are able to evade the lysosomal compartment, avoiding exposure to the host immune system.

[0019] Replication-deficient SV40 vectors were generated by deleting the coding regions of two early nonstructural proteins called large T antigen (LTag) and small T antigen (STag), giving 2.7 kb of space for cloning transgenes encoding therapeutic proteins or RNAs. SV40 vectors have been introduced into a wide range of cell types in vivo, and their therapeutic potential has been demonstrated in animal models of human disease. Because humans are immunologically naive to SV40, replication-deficient SV40 vectors are nonimmunogenic and tolerogenic for clinical applications. The non-immunogenicity in humans and ability to induce immune tolerance to the transgene protein make SV40 vectors highly attractive for use in gene therapy (Toscano, MG, et al. "Generation of a vero-based packaging cell line to produce SV40 gene delivery vectors for use in clinical gene therapy studies." Molecular Therapy-Methods & Clinical Development 6 (2017): Toscano, MG, and De Haan, P. "How simian virus 40 hijacks the intracellular protein trafficking pathway to its own benefit... and ours. "Frontiers in Immunology 9 (2018): 1160; Vera, M. and Fortes, P. "Simian virus-40 as a gene therapy vector." DNA and cell biology 23.5 (2004): 271-282).

[0020] The term "polypeptide having phosphatase activity" as used herein refers to a phosphatase having phosphatase activity or an active derivative thereof, i.e. a polypeptide (e.g. a protein) capable of dephosphorylating a compound. The polypeptide of the present invention may be a secreted form of a polypeptide having phosphatase activity. Particularly good results are observed by selecting a polypeptide having alkaline phosphatase activity. The polypeptide of the present invention may be selected from the group of alkaline phosphatases, which are found across a large number of organisms, both prokaryotes and eukaryotes, and have the same general function but different structural forms suited to the environment in which they function. Preferably, the polypeptide having alkaline phosphatase activity is selected from the group of alkaline phosphatases of mammals, such as humans, or active derivatives thereof.

[0021] In a first aspect of the present invention, the present invention provides the insight that a drawback of the use of ecto-apyrase and / or ecto-5'-nucleotidase to suppress inflammation is that the phosphatase activity of ecto-apyrase and ecto-5'-nucleotidase is low, especially compared to that of alkaline phosphatase. By providing polyomavirus gene delivery vector particles capable of expressing four types of membrane-bound alkaline phosphatase, namely germ cell alkaline phosphatase (GCALP; SEQ ID NO:1), placental alkaline phosphatase (PALP; SEQ ID NO:2), intestinal alkaline phosphatase (IALP; SEQ ID NO:3), and tissue non-specific alkaline phosphatase (TNALP; SEQ ID NO:4), a highly effective autoimmune therapy and / or allergy therapy can be provided. In particular, it has been found that the phosphatase activity of alkaline phosphatase is higher than that of ecto-apyrase and ecto-5'-nucleotidase. That is, as used herein, the term "polypeptide having alkaline phosphatase activity" refers to an alkaline phosphatase or a derivative thereof that has higher phosphatase activity than ecto-apyrase and ecto-5'-nucleotidase.

[0022] In particular, it has been found that polyomavirus gene delivery vector particles comprising a nucleic acid sequence encoding a secreted alkaline phosphatase or its active derivative provide the most effective method of protecting cells from undergoing pyroptosis after exposure to ATP. In a preferred embodiment, the secreted alkaline phosphatase or its active derivative is selected from a secreted variant of human PALP (SPALP), a secreted variant of human IALP (SIALP), a secreted variant of human GCALP (SGCALP), a secreted variant of human TNALP (STNALP) or their active derivatives. More preferably, the alkaline phosphatase activity-bearing polypeptide comprises a nucleic acid sequence encoding a SPALP.

[0023] It should be noted that since the present invention relates to a polyomavirus gene delivery vector particle comprising a nucleic acid sequence encoding a polypeptide having alkaline phosphatase activity, alkaline phosphatase derivatives may also be used to possess phosphatase activity. As a result, the nucleic acid sequence preferably comprises a nucleic acid sequence identical (at least in part) to SEQ ID NO:5 (SPALP), although the nucleic acid sequence may have substantial nucleic acid sequence homology with the target nucleic acid sequence (encoding a polypeptide having phosphatase activity). Preferably, the nucleic acid sequence encoding a polypeptide having phosphatase activity comprises a nucleic acid sequence (at least in part) having at least 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to the amino acid sequence SEQ ID NO:5 (SPALP).

[0024] The term "nucleic acid sequence homology" as used herein indicates the presence of homology between two polynucleotides. Polynucleotides have "homologous" sequences when the sequences of nucleotides in the two polynucleotides are the same or the sense sequence of one polynucleotide and the antisense sequence of the other polynucleotide are the same when aligned for maximum correspondence. Sequence comparison between two or more polynucleotides is generally performed by comparing at least a portion of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The length of the comparison window is generally about 20 to 200 contiguous nucleotides.

[0025] The "percentage of sequence identity" for the polynucleotide sequence of the present invention, such as at least 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity, can be determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window can include additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences (which does not include additions or deletions). The percentage is calculated as follows: (a) determine the number of positions where the same nucleobase occurs in both sequences to obtain the number of matched positions; (b) divide the number of matched positions by the total number of positions in the comparison window; and (c) multiply the result by 100 to obtain the percentage of sequence identity (also called sequence homology). The optimal alignment of sequences for comparison can be performed by computerized implementation of known algorithms or by inspection. Ready-to-use sequence comparison and multiple sequence alignment algorithms include the Basic Local Alignment Search Tool (BLAST) (Altschul SF et al., Basic local alignment search tool. J. Mol. Biol: Altschul SF et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25: 3389-3402, 1997) and the ClustalW program, respectively, available on the Internet. Other suitable programs include GAP, BESTFIT, and FASTA from the Wisconsin Genetics Software Package (Genetics Computer Group (GCG), Madison, Wl, USA).

[0026] Homology between nucleic acid sequences can be determined by reference to the ability of the nucleic acid sequences to hybridize to each other upon denaturation (e.g., hybridization at 50°C to 65°C for 12 to 16 hours under conditions of 400 mM NaCI, 40 mM PIPES pH 6.4, 1 mM EDTA, followed by washing) (Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989 or Current Protocols in Molecular Biology, Second Edition, Ausubel, F. et al. eds., John Wiley & Sons, 1992).

[0027] In general, those skilled in the art are fully capable of designing the construction and protocol of polyomavirus gene delivery vectors suitable for use in the present invention, i.e., for expressing polypeptides having phosphatase activity.Suitable vectors can be selected or constructed to contain suitable regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.For further details, see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989.

[0028] In one embodiment of the present invention, the polyomavirus gene delivery vector particle may essentially consist of a nucleic acid sequence encoding a polypeptide having alkaline phosphatase activity. In other words, the polyomavirus gene delivery vector particle of the present invention may be substantially free or free of a nucleic acid sequence encoding an ectonucleotidase, such as a secreted ectonucleotidase. The phosphatase activity of ectonucleotidase has been found to be too low compared to alkaline phosphatase, particularly to suppress inflammation. In particular, the polyomavirus gene delivery vector particle of the present invention may be substantially free or free of a nucleic acid sequence encoding an ectoapyrase and an ecto-5'-nucleotidase.

[0029] In a second aspect, the present invention relates to a polyomavirus gene delivery vector particle comprising a nucleic acid sequence encoding a polypeptide having phosphatase activity, the particle further comprising a nucleic acid sequence encoding a growth factor protein, which is a naturally occurring protein capable of stimulating cell proliferation, wound healing and cell differentiation. Examples of growth factor proteins include adrenomedullin, angiopoietin, autocrine motility factor, bone morphogenetic protein, ciliary neurotrophic factor family, colony stimulating factor, epidermal growth factor, ephrins, erythropoietin, fibroblast growth factor, GDNF family of ligands, growth differentiation factor-9, hepatocyte growth factor, hepatoma-derived growth factor, insulin, insulin-like growth factor (IGF), interleukins, keratinocyte growth factor, migration stimulating factor, macrophage stimulating protein, myostatin, neuroglin, neurotrophins, placenta growth factor, platelet-derived growth factor, renalase, T cell growth factor, thrombopoietin, transforming growth factor, tumor necrosis factor-alpha, and vascular endothelial growth factor.

[0030] The phosphatases expressed by the polyomavirus gene delivery vector particles of the present invention have been found to be particularly active in combination with nucleic acid sequences encoding insulin and / or IGF, including insulin-like growth factor 1 (IGF1; SEQ ID NO:6) and insulin-like growth factor 2 (IGF2; SEQ ID NO:7).

[0031] Preferably, the nucleic acid sequence encoding insulin and / or IGF or its precursors comprises a nucleic acid sequence encoding a secreted form of insulin and / or IGF or its precursors, and similar to the nucleic acid sequence encoding a polypeptide having phosphatase activity, the nucleic acid sequence encoding insulin and / or IGF or its precursors comprises (at least in part) a nucleic acid sequence having at least 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to the amino acid sequence SEQ ID NO:6 (IGF1) or SEQ ID NO:7 (IGF2).

[0032] In one embodiment of the present invention, a polyomavirus gene delivery vector particle may comprise a first nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having phosphatase activity and a second nucleic acid molecule comprising a nucleic acid sequence encoding insulin and / or IGF or precursors thereof.

[0033] In an alternative embodiment of the invention, a polyomavirus gene delivery vector particle may comprise a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide possessing phosphatase activity and a nucleic acid sequence encoding insulin and IGF or precursors thereof.

[0034] In further embodiments of the invention, the polyomavirus gene delivery vector particle may comprise a nucleic acid sequence encoding a growth factor binding protein, such as insulin growth factor binding protein (IGFBP, which includes IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6 and IGFBP7), or a precursor thereof.

[0035] As used herein, the term "nucleic acid molecule" includes both DNA molecules, such as cDNA or genomic DNA, and RNA molecules, such as mRNA, as well as analogs of DNA or RNA produced using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0036] In a third aspect of the invention, the invention relates to a polyomavirus gene delivery vector particle of the invention for use in a method of therapeutic or prophylactic treatment (e.g. prevention) of inflammatory, degenerative, dystrophic, autoimmune disease and / or allergy. In one embodiment, the invention relates to a polyomavirus gene delivery vector particle of the invention for use in a method of therapeutic or prophylactic treatment by removal or inactivation of damage associated molecular patterns at a site of inflammation in a subject. In particular, the invention may relate to a polyomavirus gene delivery vector particle according to the invention for use in a method of therapeutic or prophylactic treatment by removal of extracellular ATP in a subject. For example, the polyomavirus gene delivery vector particle of the invention may be used in a method of treating a subject with inflammatory, degenerative, dystrophic, autoimmune disease and / or allergy.

[0037] Examples of diseases suitable for treatment with the polyomavirus gene delivery vector particles of the present invention include sporadic retinal degenerative diseases such as age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy (DR). For example, AMD is generally associated with chronic inflammation of macular retinal pigment epithelial (RPE) cells and subsequent choroidal vascular hyperplasia under the inflamed RPE cells, resulting in irreversible T cell-mediated macular damage and loss of central vision and blindness. It has been reported that ATP released from the RPE, neurons, and endothelial cells of the retina plays an important role in the development of AMD, glaucoma, and DR, respectively (Ventura, Ana Lucia Marques, et al. "Purinergic signaling in the retina: From development to disease." Brain research bulletin 151 (2019): 92-108).

[0038] Other examples of diseases suitable for treatment with the polyomavirus gene delivery vector particles of the invention include chronic obstructive pulmonary disease (COPD), asthma, arthritis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, and rheumatoid arthritis and osteoarthritis.

[0039] To treat a subject, the polyomavirus gene delivery vector particles are administered to the subject.Preferably, the polyomavirus gene delivery vector particles are administered to the site of inflammation, for example, by intravenous injection, intrapulmonary injection, intravitreal injection, or subretinal injection.The polyomavirus gene delivery vector particles can be administered to a mammalian subject that is naive to polyomavirus.However, preferably, the polyomavirus gene delivery vector particles are administered to a subject, where the subject is a human subject.

[0040] In a fourth aspect of the invention, the invention relates to a composition comprising an effective amount of a polyomavirus gene delivery vector particle of the invention.The invention further relates to a composition comprising an effective amount of a polyomavirus gene delivery vector particle for use as a medicament.

[0041] In an embodiment of the invention, the composition may further comprise insulin, an IGF, such as IGF1 and IGF2, and / or an IGFBP or precursors thereof. The phosphatase expressed by the polyomavirus gene delivery vector particles of the invention has been found to be particularly active in combination with insulin, an IGF, such as IGF1 and IGF2, and / or an IGFBP or precursors thereof.

[0042] As used herein, the term "effective amount" refers to a dosage sufficient to effectively treat a subject compared to no treatment. The term "effective amount" refers to an amount that has a desired therapeutic effect, i.e., a "therapeutically effective amount."

[0043] The term "therapeutically effective amount" as used herein refers to an amount sufficient to cure, alleviate, or partially arrest the clinical symptoms of a given disease and its complications. An amount sufficient to accomplish this is defined as a "therapeutically effective amount" or "effective amount." The effective amount for each purpose will vary depending on the weight and general condition of the subject as well as the severity of the disease or injury. It will be understood that the determination of the appropriate dosage is accomplished using routine experimentation, constructing a matrix of values ​​and testing various points within the matrix, all of which is within the ordinary skill of a trained physician or veterinarian.

[0044] In the context of the present invention, a (therapeutically) effective amount can be expressed in terms of the number of polyomavirus gene delivery vector particles. Since the effective amount is highly dependent on the actual number of vector particles, the actual number of vector particles administered to the subject can also be determined. [Brief description of the drawings]

[0045] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] experiment We investigated the possibility of using a simian virus 40 (SV40)-derived gene delivery vector (hereinafter referred to as "SVec vector") encoding a secreted mutant of human placental alkaline phosphatase (SPALP) for the treatment of AMD by intravitreal administration.

[0047] The gene delivery vectors used were prepared using the methods disclosed in WO 2010 / 122094 and Toscano (Toscano, MG, et al. "Generation of a vero-based packaging cell line to produce SV40 gene delivery vectors for use in clinical gene therapy studies." Molecular Therapy-Methods & Clinical Development 6 (2017): 124-134).

[0048] In vitro AMD disease model AMD is an idiopathic retinopathy, and no established animal disease model is available. An in vitro cell-based model developed by Phenocell SAS was used to obtain proof-of-principle for SVec-based dry AMD treatment. An overview of the in vitro dry AMD model developed by Phenocell SAS is shown in Figure 2.

[0049] This model relies on human induced pluripotent stem cells that differentiate (mature) into RPE-like cells after treatment with the myosin inhibitor blebbistatin (Maruotti, J., et al. "A simple and scalable process for the differentiation of retinal pigment epithelium from human pluripotent stem cells. "Stem cells translational medicine 2.5 (2013): 341-354; Frank, D. and Vince JE "Pyroptosis versus necroptosis: similarities, differences, and crosstalk. "Cell Death & Differentiation 26.1 (2019): 99-114).

[0050] After this 15-day maturation phase, RPE-like cells are incubated for another 15 days with the retinal photosensitizer A2E (A2E loading), which is a retinoid component of lipofuscin and acts as a DAMP to induce oxidative stress and generate reactive oxygen species (ROS) in cells.

[0051] Irradiation with blue light for 4 days further promotes the generation of reactive oxygen species and induces pyroptosis in RPE-like cells. To gain insight into the inflammatory process underlying blue light-induced pyroptosis in RPE-like cells, we measured total ROS production and pyroptotic cell death on day 34. In addition, we measured the expression of inflammatory genes such as IL-1b, IL-6, IL-8, IL-18, TNF-α, IFN-γ, caspase 1, C3, IGF-1, and GFAP by PCR or ELISA on day 34.

[0052] Efficient and long-term transfection of mouse retinal cells with GFP-expressing SV40 vectors To demonstrate that SV40-derived vectors can be introduced into retinal cells of C57BL / 6 mice, we used an SV40 vector expressing jellyfish green fluorescent protein (GFP) (SVGFP). Expression of GFP cDNA is driven by the SV40 early promoter. The dose of vector particles (vector genome, VG) was 10 7 From 10 8 Two types of VG were tested, administered via two routes: subretinal and intravitreal. Vectors were administered in one eye, and the control eye was injected with the same volume of PBS. One month later, GFP expression was measured by fluorescent funduscopy. GFP fluorescent signals were detected in the retinas of mice that received SVGFP vectors in the subretinal space. The signals were widespread over the mouse eyeball. GFP fluorescent signals were also detected in the retinas of mice that received a high dose of vector intravitreally, but not in mice that received a low dose of vector.

[0053] GFP fluorescence in the retinas of mice that received a high dose of vector in the subretinal space was monitored by funduscopy for up to 12 months. GFP fluorescent signals were detectable at all measurements up to 12 months. Qualitatively, the spread and intensity of the fluorescence remained comparable up to 10 months. At the end of the experiment, the intensity of the fluorescent signal was slightly reduced but still detectable.

[0054] To investigate which cell types were preferentially targeted after subretinal or intravitreal administration of SVGFP particles, mouse eyes were processed to obtain frozen sections and evaluated by confocal or structured illumination (apotome) microscopy. In both cases, DAPI staining was used to reveal the tissue structure. First, an anti-GFP antibody was used to confirm that the signal obtained in the 488 nm channel detecting GFP fluorescence belonged to that of the GFP protein. The presence of GFP was confirmed in the mouse retina. The location of the GFP signal indicated that retinal pigment epithelial (RPE) cells were predominantly transduced. This was confirmed using an antibody against the RPE65 protein, which marks the RPE cell layer. It was observed that GFP was not found in the photoreceptor layer. However, at the doses tested, both photoreceptors and Müller cells were easily transduced with SVGFP, although the efficiency was tentative.

[0055] Second, slides from mice that received intravitreal injections of SVGFP particles were also single-labeled with antibodies to identify the RPE and photoreceptor layers. GFP signal was absent in either the RPE or photoreceptor layers, but the majority of transduced cells appeared to be microglia. Microglia were widely distributed within the outer and inner nuclear layers and were also close to the ganglion cell layer. GFP expression was also seen in cells of the ganglion cell layer. Electroretinograms of the eyes of injected mice revealed that transduction of retinal cells did not impair their visual function.

[0056] Biodistribution analysis of SVGFP vector in mice To evaluate whether SVGFP particles could be introduced into other cell types outside the retina after subretinal or intravitreal administration, genomic DNA was isolated from different tissues and qPCR was performed with primers specific for sequences in the late region of the SV40 genome. The same setup was used as for the measurement of vector particle numbers. Thus, genomic DNA isolated from brain, kidney, lung, spleen, liver and eye was used as template. SVGFP-specific DNA sequences could only be detected in DNA samples isolated from vector-injected eyes (Figure 3A). SVGFP particles were not detected in any of the DNA samples isolated from the remaining tissues tested. In brain tissues of mice from the subretinal administration group, a non-significant increase in the presence of SVGFP vector particles was detected (Figure 3B). However, further confocal analysis of immunostained slides prepared from brain tissues of these mice did not confirm the presence of the vector (data not shown).

[0057] Analysis of anti-SV40 neutralizing antibodies As observed with other gene therapy vectors, the presence or induction of neutralizing antibodies against the vector itself may reduce the expression level of the transgene and result in the elimination of transduced cells from the body. Therefore, we examined whether antibodies against SV40 capsid components could be detected in both subretinal and intravitreal injections and whether these antibodies had neutralizing activity. VP1 is the major capsid protein of SV40 particles. First, we performed Western blots using lysates from SuperVero cells (Amarna Therapeutics) transfected with SVGFP as a source of VP1 capsid protein. Nontransfected SuperVero cells were used as a negative control. A commercial antiserum containing an antibody against the VP1 capsid protein was used as a positive control for the detection of VP1 (Figure 4A, middle lane). No presence of VP1 was observed when membranes containing SuperVero cell lysates were incubated with serum samples from both subretinal and intraocular SVGFP-injected mice (Figure 4A). The same membranes were incubated with VP1 commercial serum or anti-GAPDH antibody to confirm that the membranes contained VP1 protein from the SuperVero cell lysate (Fig. 4B ).

[0058] Second, neutralization assays were performed with COS-1 cells. COS-1 cells were transduced at an MOI of 100 with SVLuc particles preincubated with different dilutions of serum from subretinal and intravitreal SVGFP-treated animals (Figures 4C and 4D). Vector particles were also preincubated with dilutions of serum from untreated control mice. A positive control for neutralizing antibody activity using a commercial rabbit anti-VP1 serum is shown in Figure 4E. No reduction in the transduction ability of SV40 vector particles was observed when the SV40 vector particles were preincubated with serum from animals transduced with the SV40 vector, indicating that SV40 vector particles do not induce the generation of neutralizing antibodies after intraocular administration.

[0059] Analysis of SV40 main capsid protein VP1 in target cells The SV40 early genes, which encode viral structural proteins, are expressed only in macaque cells expressing the viral Large T antigen during vector particle production in SuperVero packaging cells (Toscano, MG, et al. "Generation of a vero-based packaging cell line to produce SV40 gene delivery vectors for use in clinical gene therapy studies." Molecular Therapy-Methods & Clinical Development 6 (2017): 124-134). To obtain confirmation that viral structural genes were not expressed in vivo, we determined the presence or absence of VP1 capsid protein in the eyes of SVGFP-treated mice. Protein samples from SVGFP-transfected and non-transfected mice eyes and from SVGFP-transfected SuperVero cells were used as controls to confirm the absence of VP1 capsid protein in the eyes of mice administered vector particles. Only the transgene protein delivered by the SV40 vector (in this case GFP) was detected in lysates from the eyes of treated mice (Figure 5).

[0060] In vitro study of the ability of SVec vectors encoding anti-inflammatory phosphatases to suppress blue light-induced pyroptosis in RPE cells We constructed SVec vectors encoding SCD39L4 and SPALP, resulting in pSVSCD39L4 (pAM396) and pSVSPALP (pAM397), respectively. Human pluripotent stem cells were nucleofected with pSVGFP DNA, and GFP expression was measured after 30 days by fluorescence microscopy. The induced RPE-like cells maintained stable GFP expression, indicating that the experimental setup was valid.

[0061] Human pluripotent stem cells were nucleofected with pSVGFP DNA. Cells were then matured, loaded with A2E, and irradiated with blue light 30 days later. Cell death by pyroptosis was measured 34 days after nucleofection. Quercetin protected cells from pyroptosis and was used as a positive control in the experiment. After A2E loading and blue light irradiation, a strong decrease in cell viability was observed (from 32% in vehicle conditions to 0.01% in A2E conditions). The loss of viable cells was significantly counteracted by quercetin treatment (15.5% viable cells).

[0062] Without blue light exposure, RPE-like cells maintained a healthy phenotype (91.7% viable cells), whereas exposure to quercetin restored cell viability to 32.5%. Human pluripotent stem cells were then nucleofected with pSVGFP, pSVSCD39L4 (pAM396) or pSVSPALP (pAM397) DNA. Cells were matured, loaded with A2E, exposed to blue light, and ROS production and cell death were measured 34 days after nucleofection. In addition, production of inflammatory markers was measured by PCR or ELISA.

[0063] Nucleofection of RPE-like cells with pSVSPALP (pAM397) DNA significantly improved cell viability after blue light exposure, resulting in 35.2% viable cells (slightly above vehicle levels), even better than cells treated with quercetin (15%) (Figure 6). Measurements of reactive oxygen species and production of inflammatory markers 34 days after nucleofection showed no differences between untreated and nucleofected cells (results not shown). Overall, RPE-like cells containing the vector pSVSPALP, but not pSVCD39L4, are effectively protected from pyroptosis after blue light exposure.

[0064] Combination of pSVSPALP with pSVIGF1 enhances therapeutic efficacy Nucleofection of RPE-like cells with the DNA combination of pSVSPALP and pSVIGF1 resulted in a significant synergistic rescue of cell viability after blue light exposure 41 days after nucleofection, yielding 46% viable cells (slightly below vehicle (untreated) control levels), even better than cells treated with the positive control, quercetin (Figure 7). These results were also confirmed by a second assay (Cell Titer Glo assay, Promega) measuring intracellular ATP as a marker of cell viability. Here, the combination of pSVSPALP and pSVIGF1 outperformed nucleofection of pSVSPALP or pSVIGF1 alone (Figure 8). Overall, RPE-like cells containing the vectors pSVSPALP, pSVIGF1 were protected from pyroptosis after blue light exposure. However, cell cultures of RPE-like cells treated with both pSVSPALP and pSVIGF1 were significantly better protected from pyroptosis after blue light exposure than those treated with either pSVSPALP or pSVIGF1 alone.

[0065] conclusion In conclusion, we have shown that an SVec vector encoding jellyfish green fluorescent protein (SVGFP) was injected into the eye of mice, which expressed GFP in the retina and was monitored for up to one year after administration, and was efficiently introduced into ganglion cells and microglia without side effects or vision loss. We have shown that an SVec vector encoding secreted placental alkaline phosphatase (SVSPALP) effectively protected RPE-like cells from pyroptosis after blue light exposure. This protection is thought to be based on the conversion of ATP released from inflamed cells to the inactive metabolites ADP and AMP by SPALP released from RPE-like cells.

[0066] Taken together, these experimental results suggest that the SVec vector is a promising gene delivery vector for the treatment of ophthalmic diseases. In addition, SVSPALP can effectively stop retinal inflammation, making it a promising vector for treating not only AMD, but also glaucoma, DR, and other autoimmune diseases associated with local tissue inflammation.

[0067] In addition to the above, a replication-deficient SV40 gene delivery vector encoding a soluble form of human PALP, called SPALP (SVSPALP), was found to effectively protect Vero cells from pyroptosis after ATP exposure. Furthermore, retinal pigment epithelial cells expressing SPALP encoded by SVSPALP are protected from blue light-induced pyroptosis. This vector was further used in mouse models of age-related macular degeneration, nonalcoholic steatohepatitis, arthritis, and asthma to stop inflammation and prevent disease progression.

Claims

1. A polyomavirus gene delivery vector particle comprising a nucleic acid sequence encoding a polypeptide possessing phosphatase activity.

2. The polyomavirus gene delivery vector particle of claim 1 , wherein the polyomavirus gene delivery vector particle comprises a replication-deficient polyomavirus gene delivery vector particle.

3. 3. The polyomavirus gene delivery vector particle of claim 1 or 2, wherein the polyomavirus gene delivery vector particle is derived from a primate polyomavirus, preferably a simian polyomavirus, such as the simian polyomavirus Simian Virus 40 (SV40).

4. The polyomavirus gene delivery vector particle of any one of claims 1 to 3, wherein the nucleic acid sequence encoding a polypeptide having phosphatase activity comprises a nucleic acid sequence encoding a secreted form of a polypeptide having phosphatase activity.

5. A polyomavirus gene delivery vector particle according to any one of claims 1 to 4, wherein the nucleic acid sequence encoding a polypeptide having phosphatase activity comprises a nucleic acid sequence encoding a polypeptide having alkaline phosphatase activity, such as a mammalian, particularly human, alkaline phosphatase or an active derivative thereof.

6. The polyomavirus gene delivery vector particle of claim 5, wherein the nucleic acid sequence encoding a polypeptide having alkaline phosphatase activity comprises a nucleic acid sequence encoding a secreted variant of human placental alkaline phosphatase (PALP), human intestinal alkaline phosphatase (IALP), human germ cell alkaline phosphatase (GCALP), human tissue non-specific alkaline phosphatase (TNALP) or an active derivative thereof, preferably a secreted variant of human placental alkaline phosphatase (SPALP).

7. 7. The polyomavirus gene delivery vector particle of claim 5 or 6, wherein the nucleic acid sequence encoding a polypeptide having alkaline phosphatase activity comprises a nucleic acid sequence having at least 70% sequence identity to the amino acid sequence SEQ ID NO:5 (SPALP).

8. 8. The polyomavirus gene delivery vector particle of any one of claims 5 to 7, with the proviso that the polyomavirus gene delivery vector particle does not comprise a nucleic acid sequence encoding an ectonucleotidase, such as a secreted ectonucleotidase.

9. The polyomavirus gene delivery vector particle of any one of claims 1 to 8, wherein the particle further comprises a nucleic acid sequence encoding a growth factor protein or a precursor thereof.

10. The polyomavirus gene delivery vector particle of claim 9, wherein the particle comprises a first nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having phosphatase activity and a second nucleic acid molecule comprising a nucleic acid sequence encoding a growth factor protein or a precursor thereof.

11. 10. The polyomavirus gene delivery vector particle of claim 9, wherein the particle comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having phosphatase activity and a nucleic acid sequence encoding a growth factor protein or a precursor thereof.

12. The polyomavirus gene delivery vector particle of any one of claims 9 to 11, wherein the nucleic acid sequence encoding a growth factor protein or a precursor thereof comprises a nucleic acid sequence encoding a secreted form of the growth factor protein or a precursor thereof.

13. The polyomavirus gene delivery vector particle of any one of claims 1 to 12, wherein the particle further comprises a nucleic acid sequence encoding a growth factor binding protein or a precursor thereof.

14. A polyomavirus gene delivery vector particle according to any one of claims 1 to 13 for use in a method for the (prophylactic) treatment of a disease, an inflammatory disease, a degenerative disease, a dystrophic disease, an autoimmune disease and / or an allergy.

15. A polyomavirus gene delivery vector particle according to any one of claims 1 to 13 for use in a (prophylactic) method of treatment by removal or inactivation of damage associated molecular patterns at a site of inflammation in a subject.

16. A polyomavirus gene delivery vector particle according to any one of claims 1 to 13 for use in a (prophylactic) method of treatment by removal of extracellular adenosine triphosphate (ATP) in a subject.

17. A polyomavirus gene delivery vector particle according to any one of claims 1 to 13 for use in a method of treating a subject with an inflammatory, degenerative, dystrophic, autoimmune disease and / or allergy.

18. 18. A polyomavirus gene delivery vector particle for use according to any one of claims 14 to 17, said use comprising administering to a subject the polyomavirus gene delivery vector particle according to any one of claims 1 to 13.

19. 20. The polyomavirus gene delivery vector particle for use according to claim 18, wherein the polyomavirus gene delivery vector particle is administered by intravenous, intrasynovial, intrapulmonary, intravitreal or subretinal injection.

20. The polyomavirus gene delivery vector particle for use according to any one of claims 14 to 19, wherein the subject is a human.

21. A composition comprising an effective amount of the polyomavirus gene delivery vector particle of any one of claims 1 to 13.

22. 22. The composition of claim 21 for use as a medicament.

Images