A pharmaceutical formulation that combines hydroxychloroquine or its analogs with nucleic acid-based therapeutic ingredients to suppress excessive inflammation triggered by the introduction of nucleic acids

Abstract

The invention provides the use of formulation of pharmaceutical ingredients that combine various types of nucleic acids with hydroxychloroquine or its analogs. The use of these pharmacological combinations can reduce excessive inflammation in vitro and in vivo. This approach of reducing excessive inflammation can be exploited in various fields, where nucleic acid-based drugs are used as therapeutics or as a preventive medicine approach.

Description

[0001] A pharmaceutical formulation that combines hydroxychloroquine or its analogs with nucleic acid-based therapeutic ingredients to suppress excessive inflammation triggered by the introduction of nucleic acids

[0002] FIELD OF THE INVENTION

[0003] A pharmaceutical formulation, consisting of hydroxychloroquine or its analogs and nucleic acid-based therapeutics, diminishes excessive inflammation triggered by nucleic acid delivery or therapy in various fields of medicine.

[0004] BACKGROUND OF THE INVENTION

[0005] Nucleic acid-based therapeutics are various chemically-modified or nonmodified poly-RNA or poly-DNA molecules that act mainly on the gene or RNA level. They possess highly accurate targeting moiety making them useful in preventive and curative applications for treating diverse diseases such as, for example, cancer, genetic diseases, or infectious diseases. Several nucleic acid therapeutics have been EMA or FDA-approved. The widest emergence of nucleic acid-based therapeutics arose due to the COVID-19 pandemic as several COVID-19 vaccines were based on mRNA delivered by lipid nanoparticles (LNPs).

[0006] Nucleic acids used for human therapy can be in the form of antisense oligonucleotides (ASO), aptamers, DNA, where DNA can be ssDNA or dsDNA, messenger RNA (mRNA), miRNA, short interfering RNA (siRNA) or packed into viruses. Nucleic acid-based therapeutics can be delivered by different delivery strategies, for example, viral vectors (in particular AAV viral vectors), electroporation of nucleic acids, using polyplexes or by using lipid nanoparticles (LNPs). Nucleic acidbased medical products have been registered and approved or are in clinical trials for treating and preventing various diseases.

[0007] The most widely used nucleic acid therapeutics have been C0VID19 vaccines, for example, LNP-delivered mRNA vaccines BNT162b2, mRNA-1273 and AV vaccine AZD1222. Nucleic acid therapeutics are registered to be administered by subcutaneous, intramuscular, intrathecal, subretinal, intravenous, or intravitreous delivery routes. The final outcome of nucleic acid administration is often a translation of a therapeutic protein with long-lasting curative or preventive effects, gene inhibition, addition, replacement, or genome editing.

[0008] The side effect of delivery of nucleic acid therapeutics is the activation of cellular nucleic acid-sensing receptors, such as membrane Toll-like receptors (TLR3, TLR7, and TLR8), and cytosolic receptors dsRNA sensor MDA5, RIG-1 receptors, STING / cGAS that detect single or double-stranded RNA or DNA. Stimulation of nucleic acid sensors activates the inflammatory cytokine and chemokine production that results in mediating cellular and humoral immunity. Some degree of simulation is often desired to trigger the activation of the innate immune system to provide a better response to vaccination. However, excessive inflammation in the nucleic acid-treated organism can occur, leading to moderate or severe side effects that are characterized by elevated levels of proinflammatory cytokines and chemokines, for example, IL-6, CCL2, TNFa, IFNy, and many others. This can result in transient fever, muscle aches and general poor well-being, seen also in C0VID19 preventive immunization, where many recipients exhibited strong side effects that in rare cases led to life-threatening situations. Strong response may also lead to a decreased vaccine uptake and acceptance, which may lead to general vaccine hesitancy. Excessive activation of innate immune receptors by introduced mRNA can be to some degree reduced by the selection of the sequence of mRNA and the introduction of modified nucleotides that decrease activation of nucleic acid receptors, such as pseudomethyluridine replacing the uridine.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides the formulation of pharmaceutical ingredients that combine nucleic acids that act as therapeutics or as preventive medicine delivered into cells or organisms in a naked form or by using different delivery vehicles, such as LNPs with hydroxychloroquine or its analogs to reduce the excessive inflammation and decrease the reactogenicity of nucleic acid therapeutics and unwanted side effects of the therapy of nucleic acids. In this invention, we introduce vaccines and other nucleic acid therapeutics that combine chloroquine or its analog hydroxychloroquine to diminish the inflammation triggered by nucleic acid therapeutics or preventive medicine. Chloroquine (CQ) and hydroxychloroquine (HCQ) are chemical compounds belonging to 4-Aminoquinolline that are widely used as antimalarial agents, also for treating rheumatoid arthritis, systemic lupus erythematosus, etc. CQ and HCQ act through binding to nucleic acids and decrease their recognition by cellular receptors of nucleic acids. It has been proposed that HCQ binds at lower concentrations to the minor groove of dsDNA whereas at higher concentrations it exhibits dsDNA intercalating properties. One of the additional possible mechanisms of CQ and HCQ action may be interference of Toll-like receptor signaling by modification of the lysosomal pH, leading to a change in the processing of TLR9 and TLR7. Upon binding of CQ or HCQ to nucleic acid, steric hindrance of binding to TLR9 can occur. Another proposed mode of action of CQ and HCQ is interference with ligand binding to cGAMP synthase, resulting in inhibition of activation of the innate immune response.

[0011] The formulation of chloroquine (CQ) or hydroxychloroquine (HCQ) in combination with therapeutic nucleic acids can be used to diminish excessive inflammation. A pharmaceutic formulation using nucleic acids can be either arranged as two separate consecutive deliveries, with HCQ or CQ followed by nucleic acids, or as a single formulation of HCQ or CQ and nucleic acids. The separate formulation (herein also referred to as "kit”) comprises hydroxychloroquine or its analogs that are to be delivered first in the oral or other forms of delivery, 3 to 48 hours before the delivery of the formulation comprising nucleic acids, where the HCQ or CQ bind to the therapeutic nucleic acids in the organism and diminish the inflammatory response. In a single formulation hydroxychloroquine or its analogs is combined with nucleic acidcontaining compounds at the same time in a single mixture, resulting in the immediate binding CQ or HCQ to nucleic acids. All proposed pharmaceutical compositions result in lowering inflammation triggered by the delivery of nucleic acids without CQ or HCQ.

[0012] This disclosure provides the use of CQ or HCQ as a part of a pharmaceutical composition where binding of CQ or HCQ to any form of nucleic acid (e.g. mRNA, dsDNA, ASO, miRNA, siRNA etc.) delivered in a naked form or by other formulations (e.g. in viruses, LNPs etc.) in the use for infection treatment or prevention, cancer treatment, genetic diseases therapy or any other use where nucleic acids are the drug of use. Compositional single-formulation use or a two-dose regime of the proposed pharmaceutical composition results in anti-inflammatory properties, while surprisingly and importantly it does not decrease the efficiency of nucleic acid therapeutics.

[0013] The present invention can be generalized not only on COVID-19 vaccines based on nucleic acids but on other nucleic acid treatments and prophylaxis. This invention of reducing excessive inflammation through pharmaceutical formulation that combines HCQ could also be used in ASO treatment (e.g. CMV infections, Duchenne muscular dystrophy, TTR amyloidosis, spinal muscular atrophy, hypercholesterolemia etc.), RNA-LNPs (e.g. C0VID19, TTR amyloidosis etc.), AAV vectors (LPL deficiency, spinal muscular atrophy, MS, Leber congenital amaurosis etc.), adenoviral vectors (C0VID19, etc.).

[0014] Figure legends

[0015] Figure 1: Hydroxychloroquine (HCQ) does not interfere with the translation of the protein of interest based on the mRNA.

[0016] Expression of firefly luciferase from cap-0 or cap-1 modified (wherein additionally pseudo-UTP was used) or non-modified mRNA was not altered when HCQ was added (A). Pro-inflammatory cytokine IL-6 production was suppressed by the addition of HCQ regardless if the mRNA used for translation of the protein of interest comprised pseudomethyluridine or cap type(B).

[0017] Figure 2: Formulation of vaccine with HCQ suppresses inflammatory cytokine production in vivo.

[0018] Mice that were injected with a prime dose of a single formulation of HCQ and LNPs containing SARS-CoV-2 Spike mRNA, wherein non-modified cap-1 mRNA was packed within LNP, showed decreased IL-6, CCI2, and IFNy secretion in mice sera upon receiving a single formulation of HCQ and LNPs, containing SARS-CoV-2 Spike mRNA.

[0019] Figure 3: Formulation of mRNA / LNP vaccine with added HCQ suppresses the inflammatory cytokine production in vivo at all stages of the immunization regime. Mice that were injected with a booster dose of a single formulation of HCQ and LNPs containing SARS-CoV-2 Spike mRNA, wherein non-modified cap-1 mRNA was packed within LNP, showed decreased IL-6, CCI2, and IFNy secretion upon receiving a single formulation of HCQ and LNPs, containing SARS-CoV-2 Spike mRNA also in the next stage of the immunization cycle.

[0020] Figure 4: A pharmaceutical formulation that combines HCQ and SARS-CoV- 2 Spike mRNA-packed LNP has no detrimental effect on antibody production.

[0021] The secretion of total IgG against SARS-CoV-2 was equivalent in mice that were immunized with single pharmaceutical formulation, wherein HCQ and SARS-CoV-2 Spike cap-1 non-modified mRNA, packed within LNP were used or immunized solely with LNP containing SARS-CoV-2 Spike cap-1 non-modified mRNA.

[0022] Figure 5: Comparable cytotoxic T cell killing from mice immunized with nucleic acids in the presence or absence of formulation with HCQ. Mice were immunized with a single pharmaceutical formulation that combines HCQ and SARS-CoV-2 Spike mRNA-packed LNP or with SARS-CoV-2 Spike mRNA-packed LNP alone. The cytotoxic effect of isolated CD8+ T cells was comparable when a single pharmaceutical formulation that combines HCQ and SARS-CoV-2 Spike mRNA-packed LNP was used for immunization or only SARS-CoV-2 Spike mRNA- packed LNP was used.

[0023] Figure 6: Hydroxychloroquine (HCQ) does not interfere with the translation of the protein of interest based on the AAV delivery. Pro inflammatory cytokine IL6 production, produced after AAV9 (adeno- associated virus) treatment, was strongly suppressed by the addition of HCQ (A). HCQ does not interfere with the translation of the protein of interest from the AAV9 vector. Expression green fluorescent protein was not altered when HCQ was added into the cells treated with AAV9-eGFP vector (B). Figure 7: A HCQ prophylaxis attenuates AAV9-induced inflammatory cytokine production in vivo and blood-brain barrier (BBB) damage. Mice that were stereotactically injected with an AAV9 viral vector into the right cortex of the brain showed decreased IL-6 cytokine after the mice were treated with HCQ prophylactically (A). CD31 length change, a BBB damage marker, was reduced in mice that were treated with HCQ (B). Loss of AQP4 positive area was reduced in the mice that were treated with HCQ (C). The occurrence of GFAP-positive cells, a BBB damage marker, was reduced in the mice that were treated with HCQ (D). The occurrence of IBA1 -positive cells was reduced in mice that were treated with HCQ (E).

[0024] Definitions

[0025] The term »chloroquine« as used herein relates to a drug, belonging to 4- aminoquinolines, with a flat aromatic core structure and basic side chain, making chloroquine a weak base. The basic side chain contributes to the accumulation of the drug in intracellular compartments. Chloroquine occurs as an R- and S- isomer. Chloroquine is a drug that is usually administered into organisms as a phosphate salt with 0,7-0, 8 bioavailability with 39% liver metabolism to desethylchloroquine, with 51 % of renal clearance and 58% of unmetabolized excretion. Chloroquine has approximately 41 ±11 days of lifetime.

[0026] The term »hydroxychloroquine« as used herein relates to a drug, belonging to 4-aminoquinolines, with a flat aromatic core structure and basic side chain with an adjacent OH group, making hydroxychloroquine a weak base. The basic side chain contributes to the accumulation of the drug in intracellular apartments. Hydroxychloroquine occurs as an R- and S- isomer. Hydroxychloroquine is a drug that is usually administered in the form of a sulfate or phosphate salt with 0,7-0, 8 bioavailability with 18% liver metabolism to desethylchloroquine and 16% of Desethylhydroxychloroquine, with 21 % of renal clearance and 62% of unmetabolized excretion. Chloroquine has approximately 45±15 days of lifetime.

[0027] The term »pharmaceutical composition« as used herein relates to any form of composition or formulation using any form of nucleic acid and hydroxychloroquine or its analogs to suppress excessive inflammation in a single composition or as a two- dose regimen, where hydroxychloroquine or its analogs is administered first and the nucleic acid are given latter to allow their combination in the organism as soon as the nucleic acid is administered.

[0028] The term »inflammation« as used herein relates to a biological process within the living organism, tissue, or a group of organized cells responding through the immune response to a stimulus that is perceived as dangerous or foreign by the organism. Inflammation that can occur in an acute or chronic form is characterized by the onset of heat, pain, redness, swelling, and loss of function. Inflammation characterization is cell-type dependent and cytokine secretion dependent as used herein.

[0029] The term »cytokine« as used herein relates to a small secreted protein with a molecular weight of up to 40 kDa. Cytokines are secreted generally by B- cells, T- cells, macrophages, mast cells, endothelial cells, fibroblasts, or any other stromal cell to modulate the immune system by interacting with cell cytokine receptors. Cytokines may be produced and secreted by more than one type of cell. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Based on the structure we divide them into IL-1 family, IL-17 family, cysteine knot cytokines, and a-helix bundle family. Based on their function, type 1 cytokines (e.g. TNFa, IFN- y, etc) promote cellular immune response, whereas type 2 cytokines (IL-4, IL-10, IL- 13, etc) promote antibody response.

[0030] The term »IL-6« as used herein relates to a cytokine named interleukin-6 with a human, mouse, or any other origin, composed of four a-helices, secreted mainly by T-cells, monocytes, endothelial cells, and fibroblasts. IL-6 exhibits pro- and antiinflammatory properties. IL-6 is bound to the membrane or soluble IL-6 receptor with resulting downstream signaling. IL-6 is used as a biomarker for inflammation.

[0031] The term »CCL2« as used herein relates to a cytokine named chemokine (C-C motif) ligand 2 with human, mouse, or any other origin, secreted mainly by monocytes, macrophages, dendritic cells, or other cells. CCL2 is bound to CCR2 or CCR4 receptor. CCL2 promotes the recruitment of immune cells to the site of inflammation.

[0032] The term »IFNy« as used herein relates to a cytokine named interferon-y with a human, mouse, or any other origin, composed of six a-helices. IFNy is mainly secreted by T-cells, NK cells, B-cells, and antigen-presenting cells. IFNy is bound to IFNGR1 and IFNGR2 receptors. IFNy exhibits inflammation promotion, antiviral, and anti-cancer properties. The term »vaccine« as used herein relates to the preparation introduced into the body of a human or animal that stimulates adaptive immunity to a particular infectious disease or other antigen through cellular immunity or the production of specific antibodies developed after vaccination. The term vaccine as used herein contains protein components that are based on the sequence of selected viral proteins but can also be other molecules or molecular patterns.

[0033] The term »cancer« as used herein relates to a disease where abnormal cell growth with versatile known or unknown etiology invades or spreads to other parts of the living organism. The disease is characterized by different clinical symptoms based on different diagnostic measurements. Treatment of cancer as used herein can be based on the use of nucleic acids, delivered into the organism in the naked form or by other delivery vehicles.

[0034] The term »genetic disease« as used herein relates to a disease or a group of diseases with genetic etiology, characterized as a pathological change in the genome of the organism resulting in specific clinical symptoms or phenotype of the organism. Treatment of genetic disease as used herein can be based on the use of nucleic acids, delivered into the organism in the naked form or by other delivery vehicles.

[0035] The term »nucleic acids« used herein refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length and is not limited to single (- ss), double (-ds) or higher order chains of DNA or RNA, genomic DNA, cDNA, DNA- RNA hybrids, or polymers with a phosphorothioate polymer backbone made from purine and pyrimidine bases or other natural, chemical or biochemically modified, synthetic or derived nucleotide bases.

[0036] The term »ASO« as used herein relates to antisense modified or non-modified single-stranded oligonucleotides, normally ranging from 13-25 nucleotides targeted to specific RNA transcripts that are degraded by RNAse H-mediated cleavage to sterically hinder the cleavage to increase translation of protein of interest or by inhibiting the interaction between the RNA transcript and enzymes resulting in the loss of effective translation of the protein of interest.

[0037] The term »siRNA« as used herein relates to generally 19-25 nucleotides long double-stranded RNA with 5' phosphorylation and 3’ nucleotide 3' hydroxylated overhangs catalyzed by the Dicer enzyme. Activation and modification of siRNAcomplexes result in specific degradation of mRNAs of interest to prevent translation of protein of interest and expression of the gene of interest. The term »miRNA« as used herein relates to generally 22 nucleotide long noncoding RNA to inhibit translation of the protein of interest through binding of miRNA on UTR of mRNA or by promoting targeted mRNA of interest of cleavage by recruiting enzyme complexes responsible for mRNA degradation.

[0038] The term »mRNA« as used herein relates to a single-stranded processed or non-processed RNA strand, made by biological processes or synthetically from nonmodified or modified nucleotides, whereas pseudomethyluridine can be one form of modified nucleotides. mRNA can have a 5' cap and 3' polyadenylation signal to effectively translate the protein of interest.

[0039] The term »cap« as used herein relates to enzymatic modification of 5' nucleotide of mRNA transcript, whereas cap-0 or m7G cap denotes N7-methyl guanosine connected to 5' nucleotide of mRNA transcript through 5’ to 5’ triphosphate linkage, whereas cap-1 or m7GpppNm-, where Nm stand for any nucleotide with a 2’0 methylation. Cap-1 mRNA evades innate immune response, resulting in lesser innate immune system signaling.

[0040] The term »organism«, as used herein relates to any living organism.

[0041] Concerning a pathological target such as a virus or an infected cell, the term »antigen« refers to a compound such as a polypeptide, polypeptide complex, glycoprotein, nucleic acid, or the like, which elicits an immune response.

[0042] The term »delivery vehicle« as used herein relates to any form of active or passive assistance in introducing nucleic acids as used herein into cells, tissue, or organism.

[0043] The term »LNP« for lipid nanoparticles as used herein relates to non-modified or modified lipid nanoparticles that act as a delivery vehicle for a desired cargo, where cargo can be nucleic acids. LNPs are composed of various formulations and ratios of lipids, synthesized by using different preparation steps, whereas the aqueous phase that normally contains nucleic acids is effectively encapsulated within the lipid compartments.

[0044] The term »antibody« as used herein relates to a protective protein produced by B lymphocytes in response to the presence of a foreign substance, called an antigen, where the antibody is complementary to and binds to the antigen and can neutralize its activity. The term »immune system« as used herein, relates to an organ system in higher-developed organisms composed of specific cell subtypes, which act to neutralize and eliminate foreign molecules from the organism.

[0045] The term »immune response« as used herein relates to the ability of an immune system of any living organism to respond to the administered antigen to formulate appropriate antibodies by antibody-producing cells, e.g. plasma cells to achieve specific protection against any foreign pathogens.

[0046] The term »immunization« as used herein relates to the administration of prepared vaccine into a living organism or its body via oral or different parenteral inoculation routes, e.g. intranasal, intramuscular, subcutaneous, intradermal. Vaccines can be delivered via a needle injection, inhalation, or by the use of different traumatic or non-traumatic devices after vaccine administration. The first dosage of the administered vaccine into the organism is referred to as prime and any following administrations of vaccines are known as boost.

[0047] The term »mice« as used herein relates to C57BI6 / J OlaHsd mice, an inbred laboratory-bred strain or any mice laboratory strain that is commonly used in life science in vivo studies, appropriate blood draw, and final termination of the study.

[0048] The term »Spike« as used herein relates to the Spike glycoprotein of SARS- Cov-2 (UniProt: PODTC2) which attaches the virion to the cell membrane through interaction with the host receptor and mediates fusion of the virion with cellular membranes.

[0049] The term Endpoint titer (EPT) as used herein relates to ELISA-based or any other experimental determination and calculation of specific antibodies towards different antigens. EPT is depicted as the final serum dilution or titer that presents the absorbance value in this test above the calculated cutoff value.

[0050] The term »T cell«, as used herein, relates to lymphocytes, the T-subset (e.g. CD4+ and CD8+ T cells) of white blood cells, a specific mononuclear immune cell population that interacts in the adaptive immune system by recognizing antigen peptides bound to major histocompatibility complex (MHC) molecules with a T cell receptor. Recognition of antigen peptides via a TCR activates signaling pathways, which result in cytokine signaling and a cytotoxic effect.

[0051] The term »cytotoxic« as used herein relates to cell destruction or cell killing by specific cytotoxic T cells, e.g. CD8 positive T cell subset via MHCI, bound to a specific antigen. Antigen-specific CD8+ T cell recognition of specific antigens bound to MHCI results in cell death activation and subsequent antigen-infected cell destruction.

[0052] The term »specific lysis« as used herein relates to the calculated percentage of antigen-infected cell death via antigen-specific CD8 cytotoxic T cells. The percentage of specific lysis is calculated based on the bioluminescence values of measured cells. Bioluminescence values are presented as Average Radiance (p / s / cm2 / sr).

[0053] The term »cell« as used herein refers to a eukaryotic cell, a cellular or multicellular organism (cell line) cultured as a single cell entity that has been used as a recipient of nucleic acids and includes the daughter cells of the original cell that has been genetically modified by the inclusion of nucleic acids. The term refers primarily to cells of higher-developed eukaryotic organisms, preferably vertebrates, preferably mammals.

[0054] The term »pseudotyped virus SARS-CoV-2« as used herein relates to VSV-G virus, where surface G protein was replaced with SARS-CoV-2 Spike protein.

[0055] The term »PBMC« as used herein refers to peripheral blood mononuclear cells, meaning cells with round nuclei, normally T cells, B cells, NK cells, and monocytes, derived from the peripheral blood of the human or any other mammal.

[0056] The term »cells« also refers to human cell lines and plant cells. Naturally, the descendants of one cell are not necessarily completely identical to the parents in morphological form and its DNA complement, due to the consequences of natural, random, or planned mutations. "Genetically modified host cell" (also "recombinant host cell") is a host cell into which the nucleic acid has been introduced. The eukaryotic genetically modified host cell is formed in such a way that a suitable nucleic acid or recombinant nucleic acid is introduced into the appropriate eukaryotic host cell. The invention hereafter includes host cells and organisms that contain nucleic acid according to the invention (transient or stable) bearing the operon record according to the invention. Suitable host cells are known in the field and include eukaryotic cells. It is known that proteins can be expressed in cells of the following organisms: human, rodents, cattle, pork, poultry, rabbit, and the like. Host cells may include cultured cell lines of primary or immortalized cell lines.

[0057] The term » protein« «, used herein refers to the polymeric forms of amino acids of any length, which expresses some structure and function, for instance, localizing to a specific location, binding to a specific DNA sequence, facilitating and triggering chemical reactions, and transcription regulation.

[0058] The term "recombinant" used herein means that a particular nucleic acid (DNA or RNA) is a product of various combinations of cloning, restriction, and / or ligation leading to a construct having structurally coding or non-coding sequences different from endogenous nucleic acids in a natural host system.

[0059] The insertion of the vectors into the host cells is carried out by conventional methods known from the field of science, and the methods relate to transformation or transfection and include: chemically induced insertion, electroporation, micro-injection, DNA lipofection, cellular sonication, gene bombardment, viral DNA input, as well as other methods. The entry of DNA may be transient or stable. Transient refers to the insertion of a DNA with a vector that does not incorporate the DNA of the invention into the cell genome. A stable insertion is achieved by incorporating the DNA of the invention into the host genome. The insertion of the DNA of the invention, in particular for the preparation of a host organism having stably incorporated DNA of the invention, can be screened by the presence of markers. The DNA sequence for markers refers to resistance to antibiotics or chemicals and may be included on a DNA vector of the invention or a separate vector.

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061] The present invention provides the formulation of pharmaceutical ingredients that combines nucleic acids that act as a therapeutic or as a preventive medicine and hydroxychloroquine or its analogs to reduce excessive inflammation and unwanted side effects of the therapy of nucleic acids delivered into cells or organisms.

[0062] Nucleic acids that act as a drug can be administered in naked form or by using different delivery vehicles, where different viral or non-viral modes of delivery can be used. The pharmaceutical composition can be administered into the organism by different administration routes, whereas mostly oral and injectable route of administration is used. The pharmaceutical composition can be used as a single mixture, where nucleic acids and hydroxychloroquine are combined into a single administration compound, or can be used as a separate administrative regime. Later, meaning that nucleic acid is administered separately as hydroxychloroquine or its analogs but in all cases the effect of this pharmaceutical composition is used to diminish excessive inflammation, caused by the nucleic acid as a drug of choice for treating or preventing different un-physiological states of the organism.

[0063] The effect of chloroquine and hydroxychloroquine treatment on reducing inflammation and side effects due to the nucleic acid treatment was determined in human cell cultures and in vivo in experimental animals and this is used for pharmaceutical formulations of therapeutics containing nucleic acids for vaccines, gene therapies or other drugs.

[0064] A pharmaceutical formulation for therapy of humans or animals using nucleic acids that combines hydroxychloroquine or its analogs with nucleic acids in the form of RNA, DNA in the form of naked nucleic acids, nucleic acids in nanoparticle formulations such as lipid nanoparticles, viral vectors, in particular AAV vectors, whereas the formulation can be separated into the hydroxychloroquine or its analogs that are to be introduced before nucleic acids or a single pharmaceutical formulation comprising hydroxychloroquine or its derivatives and nucleic acids, to decrease inflammation.

[0065] A pharmaceutical formulation of drugs for human or animal use separated into two or more distinct components delivered at different times (herein also referred to as "kit”), the first one comprising hydroxychloroquine or its analogs and the second formulation that comprises nucleic acids, where the first formulation is applied 1 -48 hours before the formulation that contains nucleic acids.

[0066] A pharmaceutical formulation of drugs for human or animal use that combines hydroxychloroquine or its analogs with nucleic acids which are either RNA, DNA, or their derivatives, produced either by chemical, enzymatic synthesis or in cells and delivered as naked nucleic acids, combined with nanoparticles such as lipid nanoparticles or viral vectors, in particular AAV vectors.

[0067] A pharmaceutical formulation of drugs for human or animal use that combines oral delivery of hydroxychloroquine or its analogs that is applied 1 -48 hours before any nucleic-acid-based formulation.

[0068] A pharmaceutical formulation that combines hydroxychloroquine or its analogs with polymeric nucleic acid-based drugs, produced synthetically or naturally, for simultaneous delivery to suppress inflammation. A pharmaceutical formulation that combines hydroxychloroquine or its analogs for oral delivery or any other route of administration that is applied 1 - 48 hours before the vaccination based on mRNA, DNA, or viral vectors (in particular AAV vectors) against COVID-19 or other infectious diseases.

[0069] A pharmaceutical formulation that combines hydroxychloroquine or its analogs into the vaccine formulations based on mRNA, DNA or viral vectors (in particular AAV vectors) against COVID-19, influenza, RSV, malaria, mumps, rubella, polio, or other infectious diseases.

[0070] A pharmaceutical formulation that combines hydroxychloroquine or its analogs into the pharmaceutical formulations based on any form of nucleic acids, administered in the naked form or by using a delivery vehicle to suppress inflammation, occurring after nucleic acid treatment in treating cancer disease or cancer-related disease.

[0071] A pharmaceutical formulation that combines hydroxychloroquine or its analogs into the pharmaceutical formulations based on any form of nucleic acids, administered in the naked form or by using a delivery vehicle to suppress inflammation, occurring after nucleic acid treatment in treating genetic diseases.

[0072] A pharmaceutical composition or kit for a vaccine for use in the prevention or treatment Covid 19 comprising (i) hydroxychloroquine or its derivative, for a decreased inflammation and other side effects and (ii) mRNA-lipid nanoparticles or adenovirus or other nucleic acids coding for the viral components.

[0073] A pharmaceutical composition or kit for a vaccine for use in the prevention or treatment Covid 19 comprising the following components: (i) hydroxychloroquine or its derivative and (ii) mRNA-lipid nanoparticles or adenovirus or other nucleic acids coding for viral components and where the component (i) is applied from 1 hour to 2 days before, preferably 1 day before the component (ii), to suppress excessive inflammation or other nondesired side effects of vaccination.

[0074] The implementation examples that will be described in more detail are designed to best describe the invention. These descriptions have no intention of limiting the scope of the invention and its applicability but are merely intended to provide a better understanding of the invention and its use. EXAMPLES

[0075] Example 1 : mRNA nucleic acid treatment-induced cytokine secretion is abrogated by the addition of hydroxychloroquine

[0076] Modification of mRNA as a therapeutics (e.g use of vaccine against known antigen) is important for its use, wherein an introduction of cap-1 on 5' nucleotide of the mRNA and introduction of pseudomethyluridine into the mRNA sequence can diminish to some extent the recognition of the mRNA by the innate immune system, evading the signaling of innate receptors and diminishing the inflammatory cytokine production. However, therapeutics based on modified nucleic acids or modified nucleotide sequences may still produce strong undesired effects, which, as we proposed, could be further decreased in a formulation that adds HCQ or CQ to nucleic acid therapeutics.

[0077] To determine the anti-inflammatory effect of hydroxychloroquine on differently modified or non-modified nucleic acids, mRNA, coding for firefly luciferase (fLUC) was synthetically prepared by in vitro transcription reaction. The mRNAs were prepared by in vitro transcription (IVT) with T7 RNA polymerase according to the manufacturer’s instructions (Ribomax LargeScale RNA production System, Promega P1300). Purified PCR fragments for DNA templates, with already encoded 102bp long polyA tail were used. Additionally, cap-0 or cap-1 was synthetically added by the products of a commercial vendor (NEB) to the 5' nucleotide of the mRNA, wherein cap-1 makes the nucleic acid less recognizable by the immune system leading to decreased cytokine production upon treatment. For integration of modified uracil nucleotides, Pseudo-UTP (Jena Bioscience NU-1139S) and N-methylpseudo-UTP (Jena Bioscience NU890-S) we added 20 pL of each, replacing non-modified UTP in the reaction mixture entirely. After DNasel treatment of the IVT reaction mix, we performed capping of mRNA, using the Vaccinia capping system (NEB M2080S) for the formation of Cap-0 and additionally, Cap 2-O-methyltransferase for the formation of Cap-1 (NEB, NE- M0366S). mRNA was purified according to the manufacturer’s instructions (Phenol- Chlorophorm extraction) or with NEB’s Monarch RNA Cleanup Kit NEB T2040L. mRNA’s integrity and correct size were assessed using Agilent’s 2100 Bioanalyzer with Agilent RNA 6000 Pico kit (5067-1513, 5067-1514), and concentration and A260 / A280 ratios were measured on Nanodrop. Firstly, we tested if modification of the mRNA influences translation of the protein of interest upon delivering the mRNA into the tested cells or subject and whether the hydroxychloroquine diminishes inflammation in all forms of nucleic acid treatment of cells.

[0078] We electroporated human PBMC cells, Ficoll-isolated from the peripheral blood of the healthy donor, approved by the national ethics committee. Modified or non-modified mRNA for fLUC translation was electroporated into human PBMC in the amount of 1 ,5 pg of mRNA / 106of cells using 100 pl tips and a Neon electroporation device. Cells were afterward grown at 37 ° C and 5% CO2 in RPMI1640 cell medium, supplemented with 10% of FBS. At the same time, 50 pM of hydroxychloroquine sulfate (MedChemExpress) was administered to the cells. Twenty-four hours later the expression of firefly luciferase was determined by bioluminescence measurement upon providing the cells with 500nM of luciferin (PerkinElmer). The human IL-6 cytokine production, depicting the inflammatory status within the cells, was measured by ELISA test from the commercial vendor according to their instructions (Invitrogen).

[0079] Results: Figure 1 shows the effect of the mRNA modification on the translation of the protein of interest and the effect of hydroxychloroquine on lowering the cytokine secretion from treated cells. In Figure 1A the level of bioluminescence is presented resulting from the translation of firefly luciferase from fLUC mRNA electroporated cells. Bioluminescence values are presented as Average Radiance (p / s / cm2 / sr), which were determined using Living Image® software. In all cases, where PBMC were electroporated from N-pseudomethyluridine modified mRNA, containing cap-0 or cap- 1 the level of fLUC expression was the same with no statistical difference, even in those biological replicates where hydroxychloroquine sulfate was added, demonstrating that mRNA in vitro transcription yielded efficient translation of protein of interest within treated cells. The addition of hydroxychloroquine sulfate did not interfere with the efficient translation of the protein of interest, in this case, the translation of fLUC. In Figure 1 B the levels of human IL-6 cytokine, secreted from the mRNA electroporated cells measured by ELISA, are shown. The results are presented as values in pg / ml of tested cell supernatant. The PBMC, electroporated with nonmodified fLUC mRNA, containing cap-0 that lacks the properties of evading the innate immune system, secreted the highest values (app. 200 pg / ml) of human IL-6 cytokine, compared to cells treated with modified mRNA that exhibit lower values (app. 100 pg / ml) of human IL-6 cytokine. The addition of hydroxychloroquine sulfate strongly diminished (values app to 5 pg / ml) the secretion of human IL-6 cytokine in all cases, where hydroxychloroquine sulfate was added. Hydroxychloroquine sulfate addition therefore significantly diminished the inflammatory response, observed by IL-6 measurement in PBMC, regardless of the form or the modification of the nucleic acid.

[0080] Example 2: Pharmaceutical composition composed of hydroxychloroquine and nucleic acids diminishes the inflammation triggered by the nucleic acid therapeutics in a living organism

[0081] The anti-inflammatory effect of hydroxychloroquine sulfate was tested using experimental animals. C57BI / 6J OlaHsd mice were intramuscularly immunized with LNP mRNA (4 pg / animal), expressing SARS-CoV2 Spike protein of interest, combined with hydroxychloroquine sulfate (60mg / kg of body weight of the animal), combined into a single pharmaceutical composition. This type of LNP mRNA has been used as a Covid19 vaccine. All animal studies were approved by the appropriate national government institutions taking into account all the ethical considerations regarding animal studies. Laboratory animals were housed in IVC cages (Techniplast), and fed standard chow (Mucedola), and tap water was provided ad libitum. Mice were maintained in a 12-12 hours dark-light cycle. All animals, used in the study, were healthy; accompanied by a health certificate from the animal vendor. Immunization was carried out under general inhalation of 1 ,8% MAK isoflurane anesthesia (Harvard Apparatus). Vaccine (prime; 1stdose) was administered using a 30G needle into m. tibialis anterior after the appropriate area preparation. The boost (2nddose) was administered 3 weeks later. The blood was drawn 6 and 24 hours after prime. The blood was taken again before the 1stboost and again 6 and 24 hours after the 1stboost. Blood was drawn from the lateral tail vein using Microvette 300 (Sarstedt). Three weeks after the boost, the experiment was terminated. The final blood was taken and spleens were harvested from animals. Mouse sera were prepared by centrifugation of blood samples 3000 RPMZ 20 min at 4°C. In mouse sera IL-6, CCL2, and IFNy values were determined by ELISA upon vendors instructions (Invitrogen) to monitor the inflammatory state of the living organism. Graphs present values of determined cytokines as a mean value with corresponding SEM value; each dot represents a single animal.

[0082] The pharmaceutical composition was composed of the hydroxychloroquine sulfate and non-modified Spike mRNA, containing cap-0 (prepared as described in Examplel ), encapsulated into LNPs; all combined into a single mixture. For lipid nanoparticle preparation lonisable cationic aminolipid heptatriacont-6,9,28,31 - tetraene-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA; MC3, HY-112251 ; MedChem Express), 1 ,2-diastearoyl-sn-glycero-3-phosphocholine (DSPC; Sl- 850365P; Sigma), cholesterol (Choi; C8667; Sigma), 1 ,2-dimyristoyl-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000; 880151 P; Avanti lipids) were used. Lipid nanoparticles, encapsulating mRNA, were prepared using Precision Nanosystems Ignite microfluidic mixing platform. Lipid formulation in ethanol was prepared at molar ratios of 50.5: 10:38: 1 .5 (MC3 / DSPC / Cholesterol / DMG-PEG2000). Thus prepared lipid mix with a final lipid concentration of 12.5 mM and mRNA concentration of 0.174-0.200 mg / mL in 25 mM citrate buffer (pH 4.0) were injected into the NxGen cartridge at flow rate ratios 1 :3 with a final flow rate of 12 mL / min. The N / P ratio was 4 (cationic ionizable lipid to anionic mRNA). The final product was diluted 10 times in phosphate buffer saline (pH 7.4) and concentrated the diluted product was at 2000 g for 30 min. LNPs were filtered through 0.22 pm membranes and stored at 4°C or at -80°C (diluted 2x with 10% sucrose solution). Size and polydispersity index were measured using a dynamic light scattering device (Malvern). LNPs, containing Spike mRNA, had an 88, 5 nm Z-average size with 0,161 polydispersity index and 96% encapsulation efficiency and this size did not change substantially in the presence of HCQ.

[0083] Results: The levels of mouse inflammatory cytokines, CCL2 (figure 2A), IFNy (figure 2B), and IL-6 (figure 2C) from the first dose of the vaccine, were significantly lowered in animals that were immunized with a single pharmaceutical formulation, containing LNP with Spike non-modified mRNA and hydroxychloroquine sulfate, compared to animals, treated just with non-modified Spike mRNA LNP vaccine. The decrease of cytokine values is seen in blood taken 6 hours after the first dose of the vaccine as well as 24 hours after the first dose of the vaccine, but in lower values of depicted cytokines in all cases where hydroxychloroquine sulfate was added to LNP based vaccine as a part of a pharmaceutical composition to diminish excessive inflammation.

[0084] Figure 3 clearly shows that the addition of hydroxychloroquine sulfate into a single pharmaceutical composition for antiCOVID19 vaccination based on Spike mRNA diminishes the inflammation, characterized by the increase of pro-inflammatory cytokines CCL2 (Figure 3A), IFNy (Figure 3B) and IL-6 (Figure 3C) also in the booster dose. The decrease in cytokine secretion is seen 6 hours and 24 hours after the booster dose of the mRNA LNP-based vaccine. The value at 0 hours, which means just before the booster dose was administered, demonstrates the absence of inflammation in the animals that may result because of the prime dose of the administered vaccine.

[0085] Example 3: Pharmaceutical composition composed of hydroxychloroquine and nucleic acid maintains the vaccine efficiency

[0086] To investigate the effect of hydroxychloroquine on the antigen presentation and efficiency of a vaccine, based on the mRNA from the pharmaceutical composition due to the potential hydroxychloroquine accumulation within lysosomes, autophagosomes, and changing the local pH that might be expected to lead to the inhibition of the MHC class II expression, antigen presentation, and immune activation, specific anti- Spike total IgGs were determined in experimental animals. Total anti-Spike IgG was produced in animals as a result of an adaptive immune system activation and antibody production upon immunization of the animals with Spike mRNA LNP-based vaccine alone or with a pharmaceutical formulation, composed of non-modified Spike mRNA LNP and hydroxychloroquine sulfate. The immunization protocol is described in Example 1 above. In mouse sera, anti-Spike SARS CoV-2 -specific and total IgGs were determined by ELISA test to test the immunogenicity of vaccines and determine the effect of hydroxychloroquine on antibody production. ELISA tests were performed to determine Endpoint titer. High-binding half-well plates (Greiner) were used. To determine specific anti-Spike SARS CoV-2 total IgG recombinant Spike SARS CoV-2 protein was used for coating the ELISA plates. Recombinant proteins were coated in PBS buffer at the amount of 50 ng of designed protein per well. The total volume of the coated solution was 50 pl / well. Coated plates were incubated overnight at 4°C. The next day plates were washed with PBS+0,05 % Tween20 using an ELISA plate washer (Tecan). Next, plates were blocked for 1 h at RT with ELISA diluent (PBS+3%FBS) solution. Afterward, the plates were again washed. The serial dilution (factor of 10) of mice sera was added to plates, where each dilution presented a certain titer value. For mouse sera, ELISA diluent was used. Mouse sera were incubated at 4°C overnight. The next day, plates were washed and afterward, goat anti-mouse IgG (H+L)-HRP antibodies (Jackson ImmunoResearch), were diluted 1 :3000 in ELISA diluent solution. Plates were incubated for 1 h at RT. Next plates were washed. After the final wash, TMB substrate was added and the reaction was stopped with the addition of acid solution (3M H3PO4). Absorbance (A450 nm, A620 nm) was measured by Synergy Mx microtiter plate reader (Biotek). Next Endpoint titers (EPT) were calculated. The endpoint titer was determined as the dilution above the value of the cutoff.

[0087] From the absorbance data of control animals (non-treated animals) the cutoff value was determined (Frey et al. 1998) by the formula:

[0088] Cutoff = x + SD * ; where x is the mean of absorbance samples, SD is the standard deviation and f is the standard deviation multiplier. For a confidence level of 95% value 2,335 was used as the standard deviation multiplier.

[0089] Graphs present values of calculated EPT; each dot represents a single animal.

[0090] Results: Figure 4 shows the values of endpoint titer against Spike SARS-CoV- 2 antibodies, whereas Figure 4A represents the values of EPT three weeks after a prime dose, whereas Figure 4B shows the values of EPT three weeks after the boost immunization. In both cases, where only a non-modified Spike mRNA LNP-based vaccine alone or a pharmaceutical composition, composed of non-modified Spike mRNA LNP and hydroxychloroquine sulfate was administered to the animals, the EPT is similar with no hydroxychloroquine sulfate effect on antibody production, demonstrating the comparable efficiency with a decreased inflammatory response. Example 4: Cellular response is maintained upon treatment of the living organism with a pharmaceutical composition composed of therapeutic nucleic acid and hydroxychloroquine

[0091] To determine the effect of hydroxychloroquine on the antigen-specific presence of cytotoxic T-cells and its action, regarding cytotoxicity that can kill virus-infected cells, mice spleens from immunized animals that were administered either with non-modified Spike mRNA LNP-based vaccine alone or with a pharmaceutical composition, composed of non-modified Spike mRNA LNP and hydroxychloroquine sulfate were harvested at the end of the immunization protocol, described in Example 1 .

[0092] Single-cell suspension from spleens was obtained using tissue dissociator gentleMACS™ Dissociator, gentleMACS C tubes, and MACS buffer according to the manufacturer’s instruction (Miltenyi Biotec). CD8+ T cells from mouse spleen cell suspension were isolated using CD8a+ T Cell Isolation Kit according to the manufacturer’s instruction (Miltenyi Biotec). CD8 T Cells were isolated based on the negative selection using LS columns, obtaining up to 108of the maximal number of labeled cells. To determine Spike-specific cytotoxicity mouse NIH-3T3 cells cells were seeded into 24-well plates (1 *105 / well); the next day cells were transfected with pCG1 - hACE2 and pCMV-TMPRSS2 plasmids. The next day, cells were infected with Spike pseudovirus. The following day isolated CD8a+T cells (1 *105 / well) were added to the RPMI1640 cell medium. After 24 hours, bioluminescence triggered by a pseudovirus was determined using IVISIII (Perkin Elmer) after the addition of D-luciferin (500 pg / ml), demonstrating the Spike-specific cytotoxicity of CD8+T cells, isolated from Spike mRNA LNP-based vaccine alone or with a pharmaceutical composition, composed of non-modified Spike mRNA LNP and hydroxychloroquine sulfate vaccinated animals. Bioluminescence values are presented as Average Radiance (p / s / cm2 / sr), which were determined using Living Image® software. From Average Radiance values (ARV) the percentage of infected NIH-3T3 specific lysis was calculated using the formula: spontaneous death ARV — test ARV

[0093] % specific lysis = 100% spontaneous death ARV — maximal killing ARV Results: Figure 5 demonstrates that higher cytotoxicity was observed in NIH- 3T3 infected cells, which were treated with CD8+ T cells from mice that were immunized with non-modified Spike mRNA LNP-based vaccine alone or immunized with the pharmaceutical composition comprising mRNA and hydroxychloroquine compared to control cells, derived from non-immunized animals. There was no significant decrease of specific lysis seen in cells, originating from spleens of nonmodified Spike mRNA / LNP-based vaccine immunized animals compared to cells, originating from spleens of pharmaceutical composition immunized animals, implicating no significant effect of hydroxychloroquine as a part of pharmaceutical composition on cell-mediated cytotoxicity. Mean % of all specific lysis off non-modified Spike mRNA LNP based vaccine vaccinated animals isolated CD8 cells exhibited more than 90% Spike-specific cytotoxic activity, compared to mRNA-HCQ pharmaceutical composition-immunized animals, where approximately 80% of specific lysis was observed. Each dot in the graphs presents spleen cells from a designated animal that was immunized with an appropriate vaccine or pharmaceutical composition.

[0094] Example 5: AAV treatment induces cytokine secretion which is abrogated by the addition of hydroxychloroquine.

[0095] Adeno-associated virus (AAV) delivered nucleic acids can induce activation of cellular nucleic acid-sensing receptors, leading to the production of inflammatory cytokines that can trigger adverse effects. AAVs are the clinically used delivery tool for gene therapies for the treatment of a range of diseases, including genetic diseases, and for the delivery of genome editing tools. To determine the anti-inflammatory effect of hydroxychloroquine on nucleic acids, delivered by AAV, a mouse neuroblast cell line, Neuro-2A, was transduced with 106pfu of AAV9-eGFP (Origene). Twenty-four hours later the expression of enhanced green fluorescent protein (eGFP) as a reporter from the AAV9 was determined using flow cytometry. The mouse IL-6 cytokine production, demonstrating the inflammatory status within the cells, was measured by ELISA test from the commercial vendor according to their instructions (Invitrogen).

[0096] Results: Figure 6 shows the effect of hydroxychloroquine on the expression of the protein of interest translation from nucleic acid, delivered by the AAV9 viral vector, and the effect of hydroxychloroquine on lowering the inflammatory cytokine secretion from treated cells. In figure 6A the levels of mouse IL-6 cytokine, secreted from the AAV9-eGFP transduced cells, measured by ELISA, are shown. The results are presented as values in pg / ml of tested cell supernatant. The Neuro-2A cells, transduced with 106pfu of AAV9-eGFP viral vector, secreted the highest values (app- 250 pg / ml) of IL-6 cytokine, compared to other control cells treated with only HCQ or PBS buffer (app.75 pg / ml). The addition of hydroxychloroquine sulfate diminished (values app. 80 pg / ml) the secretion of IL-6 cytokine in all cases, where hydroxychloroquine sulfate was added. Hydroxychloroquine sulfate addition significantly diminished the inflammation, assessed by mouse IL-6 measurement in Neuro-2A cells, treated with AAV9-eGFP viral vector. In Figure 6B the percentage of eGFP-positive Neuro-2A cells is presented resulting from the translation of eGFP from AAV9-eGFP transduced cells. The enhanced green fluorescence in Neuro-2A cells was determined using flow cytometry (Aurora) and FlowJo software. In all cases, where Neuro-2A cells were transduced with AAV9-eGFP, the level of eGFP expression was very similar with no statistical difference, including biological replicates where hydroxychloroquine sulfate was added, demonstrating that AAV9-eGFP transduction yielded efficient translation of protein of interest within treated cells. The addition of hydroxychloroquine sulfate did not interfere with the efficient translation of the protein of interest, in our case, the translation of eGFP, suggesting that it does not decrease efficacy.

[0097] Example 6: A pharmaceutical composition composed of AAV-nucleic acids and hydroxychloroquine as a prophylactic diminishes the inflammation triggered by the AAV-delivered nucleic acid therapeutics and attenuates blood-brain damage in a living organism triggered by AAV treatment.

[0098] AAV9 viral vectors, due to the high tropism to astrocytes and neurons are used as a delivery vector for treating versatile genetic diseases. Some therapeutic interventions, such as for the treatment of neurodevelopmental conditions but also others, require direct administration of the AAV9 viral vector into the brain. A high dose of an AAV9 vector administration directly into the brain can result in blood-brain barrier damage, characterized by infiltration of a higher number of microglia cells (IBA1 positive cells), astrocytes (GFAP positive cells), loss of water channel aquaporin-4 (AQP4) staining and shortening, leakiness of endothelial cells, depicted as CD31 positive cells with a shortage in length. To determine the anti-inflammatory effect of hydroxychloroquine on nucleic acids, delivered by AAV9 and to identify the role of HCQ in attenuating the BBB damage, AAV9-eGFP (Origine) was administered into the right cortex by stereotactic injection of 1 ,5*1 O10AAV9-eGFP pfu / animal using a stereotactic device (RWD). C57BI / 6J OlaHsd mice were administered daily for three consecutive days before the AAV9-eGFP injection and two days after the injection of the AAV9-eGFP with HCQ (30 pg / animal) intraperitoneally. All animal studies were approved by the appropriate national government institutions taking into account the ethical considerations regarding animal studies. Laboratory animals were housed in IVC cages (Techniplast), and fed standard chow (Mucedola), and tap water was provided ad libitum. Mice were maintained in a 12-12 hours dark-light cycle. All animals, used in the study, were healthy; accompanied by a health certificate from the animal vendor. AAV9-eGFP injection was carried out under the general inhalation of 1 ,8% MAK isoflurane anesthesia (Harvard Apparatus). The blood was drawn 24 hours after the AAV9-eGFP injection and 30 days later when the experiment was terminated. Mouse sera were prepared by centrifugation of blood samples at 3000 RPM / 20 min at 4°C. In mouse sera, IL-6 values were determined by ELISA upon vendor instructions (Invitrogen) to monitor the inflammatory state of the living organism. The graphs present values of determined cytokines as a mean value with the corresponding SEM value; each dot represents a single animal. OCT frozen brain samples were sectioned using cryostat at 10 pm. Next, immunohistochemistry was performed by staining tissue brain sections with antibodies recognizing IBA1 (Cell Signaling Technology), GFAP (Cell Signaling Technology), AQP4 (Cell Signaling Technology), and CD31 (Santa Cruz Biotechnology). Tissue sections were visualized by confocal microscopy. Pictures were quantified by the software Imaged.

[0099] Results: The levels of mouse inflammatory cytokine, IL6 (figure 7A) were significantly lowered in animals that were given HCQ compared to animals, treated just with AAV9-eGFP. The decrease of cytokine values is seen in blood taken 24 hours after the AAV9-eGFP and 30 days after the AAV9-eGFP administration, demonstrating the effect of HCQ on suppressing the inflammation due to the AAV9 injection. Figure 7B clearly shows that the prophylactic administration of hydroxychloroquine sulfate diminishes the BBB damage as the length of CD31- positive cells was higher in HCQ-treated animals. The AQP4 staining (figure 7C) revealed that the addition of HCQ into the animals attenuated the level of BBB damage. The lower degree of BBB damage was demonstrated by fewer infiltrating GFAP-positive cells (figure 7D) and IBA1 -positive cells (figure 7E).

Claims

CLAIMS1 . Hydroxychloroquine or an analog or derivative thereof, for use in a method of prevention or treatment of a medical condition or disease comprising administration of at least one nucleic acid to a subject, wherein the hydroxychloroquine or the analog or derivative thereof is administered simultaneously or before administration of the at least one nucleic acid to reduce or avoid inflammation, in particular inflammation caused by administration of the at least one nucleic acid.

2. Hydroxychloroquine or an analog or derivative thereof for the use of claim 1 , wherein the hydroxychloroquine or the analog or derivative thereof is administered 1 -48 h, preferably 5-24 h, before administration of at least one nucleic acid.

3. Hydroxychloroquine or an analog or derivative thereof for the use of claim 1 or 2, wherein at least one nucleic acid is selected from the group consisting of single- or double-stranded DNA, RNA, in particular mRNA, miRNA, or siRNA, antisense oligonucleotides (ASO) and aptamers, and wherein the nucleic acid can be in a naked form or packed e.g. in lipid nanoparticles (LNPs), or viral vectors.

4. Hydroxychloroquine or an analog or derivative thereof for the use of any one of claims 1 -3, wherein at least one nucleic acid is administered by subcutaneous, intramuscular, intrathecal, subretinal, intravenous, oral or intravitreous delivery routes.

5. Hydroxychloroquine or an analog or derivative thereof for the use of any one of claims 1 -4, wherein the hydroxychloroquine or the analog or derivative thereof is provided for oral administration, preferably wherein both the hydroxychloroquine or the analog or derivative thereof and the at least one nucleic acid are orally administered.

6. Hydroxychloroquine or the analog or derivative thereof for the use of any one of claims 1 -5, wherein the medical condition or disease is selected from the group consisting of infectious diseases, in particular viral infections, such as Covid 19, influenza, RSV, malaria, mumps, rubella, and polio, cancer or cancer-related diseases and genetic diseases.

7. A pharmaceutical composition or kit comprising:(i) hydroxychloroquine or an analog or derivative thereof, and(ii) at least one nucleic acid, in a single formulation or in two separate formulations.

8. The pharmaceutical composition or kit according to claim 7, wherein at least one nucleic acid is selected from the group consisting of single- or doublestranded DNA, RNA, in particular mRNA, miRNA, or siRNA, antisense oligonucleotides (ASO) and aptamers, and wherein the nucleic acid can be in naked form or packed e.g. in lipid nanoparticles (LNPs), or viral vectors, in particular AAV viral vectors.

9. The pharmaceutical composition or kit according to claim 7 or 8, which is adapted for oral administration.

10. The pharmaceutical composition or kit according to any one of claims 7-9, for use in medical therapy or as a vaccine.

11. The pharmaceutical composition or kit for use according to claim 10, wherein the component (i) is administered orally or as an injection at least 1 hour but not more than 2 days before the component (ii), or wherein components (i) and (ii) are administered simultaneously.

12. The pharmaceutical composition or kit for use according to claim 10 or 11 , for the prevention or treatment of an infectious disease, in particular a viral infection such as Covid 19, influenza, RSV, malaria, mumps, rubella, and polio, cancer, or cancer-related diseases, or a genetic disease.

13. A pharmaceutical composition or kit for use as a vaccine in the prevention or treatment of Covid 19, the pharmaceutical composition or kit comprising (i) hydroxychloroquine or a derivative thereof, for a decreased inflammation and other side effects and (ii) mRNA-lipid nanoparticles or adenovirus or other nucleic acids coding for the viral components.

14. A pharmaceutical composition or kit for use as a vaccine in the prevention or treatment of Covid 19, the pharmaceutical composition or kit comprising the following components: (i) hydroxychloroquine or a derivative thereof, and (ii) mRNA-lipid nanoparticles or adenovirus or other nucleic acids coding for viral components and wherein the component (i) is applied from 1 -48 h, preferably 5-24 h before the component (ii), to suppress excessive inflammation or other nondesired side effects of vaccination.

15. A method of reducing or inhibiting inflammation caused by the administration of a nucleic acid to a subject, comprising a step of administering hydroxychloroquine or an analog or derivative thereof to the subject, simultaneously with or before administration of the nucleic acid, in particular 1 -48 h before administration of the nucleic acid, preferably 5-24 h before.

Images