Modified Flavivirus Nucleotide Sequence, Therapeutic Agent, Viral Vector, Obtaining Process and Treatment Method

Description

1 / 62 Descriptive Report of Invention Patent Modified Flavivirus Nucleotide Sequence, Therapeutic Agent, Viral Vector, Use, Obtaining Process and Treatment Method Field of Invention

[0001] The present invention is situated in the fields of molecular biology, biotechnology, nanotechnology, synthetic biology, pharmacology, vaccinology, medicine, virology, genetics, and oncology. More specifically, the invention provides a modified flavivirus nucleotide sequence, a therapeutic agent, a viral vector and its production process, its use, and a treatment or prophylaxis method that utilizes said sequence, agent, or vector. The invention is associated with the so-called iOZ Platform, which leverages the potential of the Zika virus (ZIKV) viral genome to provide advanced therapeutic approaches, also offering targeted and effective treatments for complex diseases, including the treatment of malignant and other types of tumors. In one embodiment, the therapeutic agent of the invention is a viral vector for gene therapy. In another embodiment, the therapeutic agent of the invention is a non-natural RNA sequence, useful for vaccines and / or gene therapy.One embodiment of the therapeutic method invention is an oncolytic therapy. Another embodiment of the therapeutic method invention is a gene therapy based on the use of the modified virus, viral vector, or modified RNA of the invention. Yet another embodiment of the invention is a viral vector platform for gene editing. Background of the Invention

[0002] Neurological disorders are diseases of the central and peripheral nervous systems, which include the brain, spinal cord, and associated nerves. Most neurological and / or genetic diseases lack effective therapies for cure or treatment. This is due to the complexity of the CNS, the limited regenerative capacity of this tissue, and the low capacity of Petition 870250015956, dated 26 / 02 / 2025, p. 7 / 88 2 / 62 conventional medications cross the blood-brain barrier (BBB).

[0003] The World Health Organization (WHO) defines Brain Health as a dynamic state of brain function encompassing cognitive, sensory, socio-emotional, behavioral, and motor areas. This state allows each individual to reach their maximum potential throughout life, regardless of the presence or absence of diseases or disorders. However, various conditions can affect the brain and nervous system, impacting brain development, damaging brain structures, or impairing brain function. These conditions can arise at any point in life and include, for example, congenital conditions and chronic diseases of the nervous system, which require medication and therapies.

[0004] Brain diseases, including malignant tumors and neurodegenerative conditions, represent one of the greatest global health challenges. Within the scope of oncolytic therapy technology, the study of the problems of aggressive brain tumors, such as glioblastoma, is included, which have very low survival rates, with less than 5% of patients surviving beyond five years after diagnosis. These tumors are resistant and unresponsive to many conventional treatments, as the blood-brain barrier limits the effective reach of most traditional and modern drugs and therapies available to patients.

[0005] Glioblastoma (GBM) is the most common and malignant form of primary brain tumor. Despite available treatments such as surgery, radiation, and chemotherapy, GBM remains lethal, with a patient survival period of less than 2 years. Therefore, there is a need for new, more effective and less aggressive therapies. The present invention provides an alternative to these problems of current therapies, meeting the aforementioned needs.

[0006] Glioma stem cells (GSCs) have self-renewal, tumorigenic and differentiation potential, and are therefore similar to cells Petition 870250015956, dated 26 / 02 / 2025, page 8 / 88 3 / 62 neural progenitor cells (NPCs). GSCs play critical roles in the progression, recurrence, and therapeutic resistance of GBM, and are important targets for therapeutic processes for GBM.

[0007] SHANK3 haploinsufficiency, a genetic disease that causes neurodevelopmental disorders, is responsible for Phelan-McDermid Syndrome (PMS). The clinical picture of PMS is quite heterogeneous and is characterized by the presence of global developmental delay, absence or delay of speech, facial dysmorphisms, hypotonia, and Autism Spectrum Disorder (ASD). Furthermore, clinical studies in PMS patients over 10 years of age have revealed the occurrence of severe psychiatric conditions that appear to trigger severe regression of acquired cognitive abilities, which would be consistent with a progressive neurological disorder. Studies in mouse models for PMS syndrome, with the reversible knockout of the SHANK3 gene, allow the restoration of the expression of this gene in vivo, considerably improving the symptoms of PMS in affected animals.

[0008] Oncolytic virotherapy has been identified as a promising therapeutic approach for cancers, with several clinical studies completed and underway, including products already approved for human use by the FDA (Food and Drug Administration) for the treatment of melanoma, head and neck cancer, and malignant glioma. The fundamental concept of oncolytic virotherapy is to treat tumors with Oncolytic Viruses (OVs) that replicate specifically in tumor cells and cause cell apoptosis, leading to tumor destruction. OVs can be classified into two main groups: naturally occurring viruses and those that are genetically modified using molecular biology techniques.

[0009] OVs offer an excellent platform for tumor therapy. The viral genome can be modified to increase viral tropism and target them to neoplastic cells, improve selective viral replication in these cell types and lytic potential, as well as increase the host's antitumor immunity. These characteristics form the basis for the applications of OVs. Petition 870250015956, dated 26 / 02 / 2025, page 9 / 88 4 / 62 in cancer treatment. The ability to genetically modify the virus to promote antitumor immunity, increase tumor sensitivity to conventional drugs or radiation therapies, as well as ensure patient safety, are all important advantages of OVs.

[0010] Most OVs replicate and selectively destroy tumor cells through cell lysis and stimulation of the host's antitumor immune response. In the case of the Herpes virus, although it does not have the ability to replicate and directly destroy tumor cells, it can enter the tumor cell and activate the immune system that attacks the tumor, which is why it is used as an oncolytic therapy and has been the basis for the only two products currently approved for use in humans.

[0011] Within this spectrum, ZIKV is known for its ability to produce an oncolytic effect on Central Nervous System (CNS) cells with a dual mechanism of action: (i) direct lysis of tumor cells due to its ability to replicate in the tumor, leading to cell death by necrosis and / or apoptosis (an ability not present in OVs originating from Herpes virus), and (ii) the ability to activate the immune system to directly combat the tumor, being considered an effective therapy with a rapid response and minimal side effects, factors that lead to fewer clinical interventions in the patient.

[0012] Within the scope of viral vector technology, its use is also being studied in genetic conditions and neurodegenerative diseases, such as Alzheimer's and Parkinson's, which affect millions of people and still lack effective treatments. These disorders alter the activity of brain cells and can cause their progressive degradation, resulting in profound disabilities, loss of cognitive function, and a significant economic impact on the health system.

[0013] Viral vectors have applications in the development of treatments for genetic diseases, including rare diseases and diseases related to the central nervous system, such as neurodegenerative diseases. In addition, viral vectors can also be used to aid in the treatment of tumors. No Petition 870250015956, dated 26 / 02 / 2025, page 10 / 88 5 / 62 In the context of rare diseases, viral vectors offer new hope for patients who often lack effective treatment options. These diseases are usually caused by specific genetic mutations that result in the production of defective proteins (non-functional or with deleterious effects), or in the absence or low levels of essential proteins.

[0014] The use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated enzyme (CAS) in genome editing has also become one of the most promising tools for treating human genetic diseases, including neurodisorders, cancer, cardiovascular and blood diseases. In the latter case, there is already an FDA-approved product for use in humans diagnosed with transfusion-dependent beta-thalassemia or sickle cell disease.

[0015] The choice and implementation of safe and effective delivery systems in the therapeutic application of CRISPR / Cas is a significant obstacle. In this context, viral vectors are promising for the in vivo delivery of CRISPR machinery, which includes a guide RNA (gRNA) or CRISPR RNA (crRNA), a Cas variant (Cas9 or Cas12 for example), due to the natural specialization of viruses in delivering genetic material to target cells. Therefore, viral vectors designed for this purpose can leverage more effective delivery compared to other methods.

[0016] Adenovirus-based viral vectors (AdVs), herpesviruses, and adenovirus-associated vectors (AAVs) and lentiviruses (LVs) are commonly employed as gene therapy delivery methods. However, AAV-based viral vectors, which have FDA-approved products, have low gene payload delivery capacity, low tropism for the CNS, adverse effects, and make genomic transgene integration viable, causing deleterious effects on the host genome. Consequently, it is desirable to use a viral vector capable of delivering large nucleotide sequences of transgenes or CRISPR machinery to CNS cells with high efficiency and selectivity, high gene payload delivery capacity, and Petition 870250015956, dated 26 / 02 / 2025, page 11 / 88 6 / 62 without genomic integration. The present invention provides a solution to these problems addressed.

[0017] The ZIKV-based viral vector platform includes an mRNA sequence encapsulated by viral structural proteins. Because it is an RNA genetic payload, the use of the Cas9 protein would not be suitable, since it would be necessary to deliver the Cas9 coding sequence and another sequence for the gRNA separately.

[0018] One solution to this problem is the use of the Cas12 system, since this protein processes its own pre-crRNA into mature crRNAs without the need for a tracRNA, being a unique effector protein with endoribonuclease and endonuclease activity. In this way, the Cas12 coding region and the pre-crRNA itself can be included in the viral vector described here in a single RNA sequence, enabling the use of viral RNA vectors. CRISPR arrays associated with Cas12 are transcribed into a long RNA, called pre-crRNA, which contains a succession of spacer sequences of ~30 nucleotides, separated by Direct Repeat (DR) sequences of 36 nucleotides. The processing of the pre-crRNA mediated by Cas12 into mature crRNAs, as well as the recognition and cleavage of the target DNA, is controlled by base pairing between the spacer and target sequences.

[0019] In mammalian cells, Cas12 has already been used for editing transcriptional control genes, as well as in single or multiplex gene knockout studies, where several genes are switched off using only one RNA sequence containing multiple crRNAs, which direct Cas12 to the simultaneous editing of several genes.

[0020] In the case of oncolytic virotherapy, two therapies have been approved by regulatory agencies: (i) Imlygic®, a modified Herpes virus, approved by the regulatory agencies of the United States of America - FDA (Food and Drug Administration) and Europe - EMA (European Medicines Agency) for the treatment of melanoma, and (ii) Delytact® (G47Δ; teserpaturev), a virus of Petition 870250015956, dated 26 / 02 / 2025, page 12 / 88 7 / 62 HERPES, modified by the company Daiichi-Sankyo, is approved in Japan for the treatment of glioblastoma. However, none of these oncolytic therapies exhibit natural tropism for the CNS and oncolysis.

[0021] Regarding the viral vector, five therapies have been approved by regulatory agencies: (i) ELEVIDYS, a viral vector based on adeno-associated virus (AAV), approved by the United States regulatory agency - FDA (Food and Drug Administration), for the treatment of Duchenne muscular dystrophy; (ii) LUXTURNA, a viral vector based on adeno-associated virus (AAV), approved by the regulatory agencies of the United States - FDA (Food and Drug Administration), Europe - EMA (European Medicines Agency) and Brazil - ANVISA (National Health Surveillance Agency), for the treatment of retinal dystrophy; (iii) ROCTAVIAN, a viral vector based on adeno-associated virus (AAV), approved by the FDA, EMA and ANVISA, for the treatment of severe hemophilia A; (iv) VYJUVEK, a viral vector based on replication-deficient herpes simplex virus type 1 (HSV-1), approved by the FDA, for the treatment of epidermolysis bullosa;(v) ZOLGENSMA, a viral vector based on adeno-associated virus (AAV), approved by the FDA, EMA and ANVISA, for the treatment of spinal muscular atrophy.

[0022] Gene therapy is becoming a viable option for clinical intervention due to the approval of some therapies for human use. However, concerns related to the lack of safety and specificity of current therapies suggest the need to generate new virus-based delivery vectors. The viral vectors predominantly used in in vivo trials for the administration of gene therapy targeted to the CNS are based on AAVs. This is due to the potential of some AAV serotypes to cross the BBB, or the use of modified AAVs designed with enhanced tropism in the CNS.

[0023] The use of AAV-based viral vectors is limited by their lower capacity to transport genetic material compared to other viral vectors and by the high inflammatory process triggered after local administration. Petition 870250015956, dated 26 / 02 / 2025, page 13 / 88 8 / 62 or systemic (immunogenicity). The maximum length of the nucleotide sequence supported by AAVs is approximately 4.7 kb, including the two regions of inverted terminal repeats that contain the viral origins of replication and the packaging signal. This capacity is insufficient for gene therapy, where the replacement of large proteins is necessary. This would be insufficient, for example, for gene therapy for SHANK3 disease, since the SHANK3 gene alone, whose coding sequence (CDS) is approximately 5.4 kb, already exceeds the genetic payload limit of AAVs.

[0024] Furthermore, AAV-based viral vectors have low efficiency in delivering genes of interest to the CNS and exhibit tropism for the liver, which can lead to hepatotoxicity, compromising the effectiveness of gene therapy. The need for high doses to achieve the desired therapeutic effect exacerbates these problems, increasing hepatotoxicity, acute liver damage, and even death.

[0025] The ZIKV genome, on the other hand, has two untranslated regions (UTRs) containing regulatory nucleotide sequences, approximately 500 bp long, and a nucleotide sequence encoding a polyprotein, approximately 10 kb long, enabling ZIKV to carry therapeutic genes of up to 10 kb, and thus be advantageous compared to the use of AAVs for CNS disorders.

[0026] ZIKV has the ability to cross the blood-brain barrier and selectively infect cells of neural origin. ZIKV has a tropism for the CNS, making it an attractive tool for the development of a viral vector for gene delivery in the treatment of various diseases, including genetic syndromes and neurodevelopmental disorders that still lack precise treatments on the market, being drug orphan diseases, such as Phelan-McDermid Syndrome (PMS).

[0027] ZIKV generated global attention when, in 2016, researchers discovered that ZIKV infections during pregnancy were linked to cases of microcephaly and a range of other birth defects. Petition 870250015956, dated 26 / 02 / 2025, page 14 / 88 9 / 62 congenital cases occur in newborns, while ZIKV infection in children and adults is associated with Guillain-Barré syndrome, neuropathy, and myelitis.

[0028] When the ZIKV epidemic emerged in Brazil and other countries in the Americas between April 2015 and November 2016, the World Health Organization (WHO) declared ZIKV infection a Public Health Emergency and in 2018 it was listed as a priority disease in the Vaccine Research and Development Plan. Despite the unprecedented speed of development of countermeasures for ZIKV, there is still no approved vaccine against ZIKV. In these countermeasures to prevent infection and disease caused by ZIKV, vaccines remain a public health priority, particularly for people who may become pregnant and who live in or travel to ZIKV-endemic regions.

[0029] The development of an attenuated or inactivated vaccine for ZIKV is a process that can be relatively quick, safe and efficient, supported by the availability on the market of other attenuated or inactivated flavivirus vaccines licensed and approved for human use, such as vaccines for yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and dengue virus (DENV).

[0030] mRNA-based vaccines have proven to be a viable alternative to conventional vaccines. This is due to their high efficiency in promoting an immune response, safety, and potential for rapid clinical trials and cell-free manufacturing. During the SARS-CoV-2 pandemic, mRNA vaccines were developed and administered at an unprecedented rate to millions of people to combat COVID-19. These vaccines, initially developed by Pfizer-BioNTech and Moderna, validated the vaccine platform and stimulated interest in the application of mRNA for both prophylactic and therapeutic indications.

[0031] mRNA vaccines are based on a synthetic mRNA molecule a Petition 870250015956, dated 26 / 02 / 2025, p. 15 / 88 10 / 62 which encodes and expresses an antigen that will trigger an immune response. This mRNA transcribed in vitro mimics the structure of an endogenous mRNA, containing five components; 1) 5' cap, 2) 5' and 3' UTR (untranslated region), 3) modified pseudouridine, 4) one or more open reading frames encoding the antigen, and, 5) poly-A tail.

[0032] The production steps for an mRNA vaccine, through synthetic mRNA production and formulation, include the following steps: 1) determination and design of the target pathogen antigen sequence, and its insertion into a plasmid DNA construct containing the regulatory sequences as described above; 2) in vitro transcription of mRNA by RNA polymerases and CAP addition; 3) purification of mRNA transcripts by high-performance liquid chromatography (HPLC) techniques to remove contaminants and incomplete mRNA; (4) encapsulation of the purified mRNA in lipid nanoparticles or liposomes; 5) dialysis or filtration of the nanoparticles to remove solvents from unencapsulated mRNA. The vaccines thus produced are then stored and subjected to immunization tests in laboratory animals, evaluation of humoral, cellular and viral neutralization immune response (vaccines against viruses).

[0033] ZIKV prominently infects neural stem cells and progenitor cells (NPCs), disrupting key cellular processes, leading to the death of infected cells and impaired self-renewal, resulting in severe CNS anomalies in neonates, such as in congenital Zika virus syndrome, which includes microcephaly. Thus, the clinical application of ZIKV as virotherapy depends on genetic modification of the virus to ensure safe and effective therapy, guaranteeing selectivity and viral activity only for tumor cells.

[0034] In this way, the oncolytic therapy of the present invention provides the modification and production of a product capable of replicating in tumor cells and selectively destroying them, with better safety than its counterparts, without compromising the replicative activity of the modified virus. Petition 870250015956, dated 26 / 02 / 2025, page 16 / 88 11 / 62

[0035] In the search for the state of the art in scientific and patent literature, the following documents were found that deal with the subject:

[0036] Document WO2023164441A1 discloses nucleic acid-based compositions to enable the transport of genetic material for applications such as gene therapy, genetic modification of cells, and the development of RNA vaccines through the efficient delivery of exogenous polynucleotides to target cells. Furthermore, the document presents a nucleotide sequence that is supposedly responsible for the encapsulation of RNA by the envelope structure of the viral vector, but does not provide conclusive results to support this claim. The present invention does not use this approach, in addition to solving these and other problems. Finally, it is relevant to mention that currently, commercially used viral vectors do not have a specific tropism for cells of the central nervous system, and may replicate in the liver and / or require very high doses, which can lead to cytopathic effects.These are products that therefore still require further research to solve these problems and achieve the desired effect.

[0037] The present invention of iOZ does not use this approach, in addition to solving these and other problems.

[0038] Document WO2023164442A2 discloses technologies related to the use of genetically modified viruses as therapeutic tools in cancer treatment. However, the nucleotide sequence disclosed in said document is not identical to the nucleotide sequence of the strain of the present invention, and furthermore, it has low selectivity, being poorly specific for expression in brain tumor cells or normal brain cells. In addition, said document discloses a nucleotide sequence that is susceptible to genetic alteration during the production process and / or when administered to the patient, potentially resulting in safety and / or efficacy problems.

[0039] Based on what can be inferred from the literature reviewed, no documents were found anticipating or suggesting the teachings of the present invention, Petition 870250015956, dated 26 / 02 / 2025, page 17 / 88 12 / 62 so that the solution proposed here has novelty and inventive activity compared to the state of the art. Summary of the Invention

[0040] The present invention solves the problems of the prior art by providing a modified and improved nucleotide sequence, therapeutic agent, a viral vector and its production process, and a treatment method that utilize information derived from the genomic sequence of flaviviruses, in particular ZIKV.

[0041] In one embodiment, Figure 1A illustrates the topology of the three functionally connected sequences (NS4A-2K-NS4B) (SEQ ID No. 23) after the translation process, highlighting which regions are exposed in the cytoplasm, which are transmembrane, and finally, which are exposed in the lumen of the endoplasmic reticulum (ER).

[0042] Thus, one of the objects of the invention is a non-natural, genetically modified flavivirus nucleotide sequence, preferably from ZIKV. The modified nucleotide sequence of the invention was specifically designed to solve prior art problems. In a preferred embodiment, said nucleotide sequence comprises at least three nucleotide sequences functionally linked to each other or not in any order of linkage thereof. The combination may vary depending on the specific application of the technology, preferably as presented in the linkage order of Figure 1, which corresponds to (i) the nucleotide sequence of part of the coding sequence of the ZIKV NS4A protein (Seq ID No. 1); (ii) the complete nucleotide sequence of the ZIKV 2K protein (Seq ID No. 2); (iii) the nucleotide sequence of part of the coding sequence of the ZIKV NS4B proteins (Seq ID No. 3).

[0043] In one embodiment, the modified flavivirus nucleotide sequence additionally comprises the nucleotide sequence of part of the coding sequence of the Zika virus Capsid C protein (Seq ID No. 4). Petition 870250015956, dated 26 / 02 / 2025, page 18 / 88 13 / 62

[0044] In one embodiment, the invention includes the introduction of microRNA Response Elements (MREs) into the viral genome for selective replication control. These MREs allow the modified virus to replicate only in cells where specific miRNAs are poorly expressed, promoting greater safety and specificity. In this embodiment, said nucleotide sequence further comprises a linkage order corresponding to: (i) the MRE nucleotide sequence (5' CGUGCCUUA 3' - Seq ID No. 12, as a miR-124-3p target) functionally linked to 5' UTR (Seq ID No. 14) between nucleotides 112 and 113; (ii) the complete nucleotide sequence of the ZIKV NS1 protein (Seq ID No. 10), functionally linked to 3' of the E protein nucleotide sequence (Seq ID No. 8); (iii) the complete nucleotide sequence of the ZIKV NS4A protein (Seq ID No. 27), functionally linked to 3' of the complete nucleotide sequence of the NS3 protein (Seq ID No.6); (iv) the MRE nucleotide sequence (5' CCGACCUGU 3' - Seq ID No. 13, as a miR-219a-2-3p target) between nucleotides 12 and 13 of the 2K protein nucleotide sequence (Seq ID No. 2) via 5' and 3' flanking restriction enzyme sites; and (v) the complete NS4B protein nucleotide sequence of ZIKV (Seq ID No. 28), functionally linked to 5' of the complete NS5 nucleotide sequence (Seq ID No. 5) described in Figure 3.

[0045] In one embodiment, the modified flavivirus nucleotide sequence further comprises: - the nucleotide sequence encoding the Zika virus NS5 protein (Seq ID No. 5) functionally linked to 3'; - the nucleotide sequence encoding the Zika virus NS3 protein (Seq ID No. 6) functionally linked to 5'; - the nucleotide sequences that encode the ZIKV proteins C, M, E, NS1, NS2A, NS2B (Seq ID Nos. 29, 7, 8, 10, 11, 12, respectively), functionally linked to each other from 5' to 3'; - the nucleotide sequences 5'UTR (Seq ID No. 14) and MRE (5' CGUGCCUUA 3' - Seq ID No. 12, as a target of miR-124-3p) functionally Petition 870250015956, dated 26 / 02 / 2025, p. 19 / 88 14 / 62 connected to each other and at 5'; - the nucleotide sequences MRE (5' CCGACCUGU 3' - Seq ID No. 13, as a target of miR-219a-2-3p) and 3'UTR (Seq ID 15) functionally linked to each other and to 3'; or combinations thereof.

[0046] In one embodiment, the nucleotide sequence additionally comprises another nucleotide sequence(s) of up to 10 kb, encoding(s) functional proteins.

[0047] In one embodiment, in Figure 1, B) shows the same sequence as in A) additionally comprising an example of the use of nucleotide sequences encoding reporter proteins functionally linked to the 5' of the NS4A-2K-NS4B sequence.

[0048] Thus, in one embodiment, the modified flavivirus nucleotide sequence further comprises: - the nucleotide sequences of part of the coding sequences of the GFP and Luciferase proteins (SEQ ID NO 17 and 18, respectively), functionally linked to each other from 5' to 3'; or combinations thereof.

[0049] In one embodiment, in Figure 1, C) shows the same sequence as A) additionally comprising an example of the use of the SHANK 3 protein coding sequence functionally linked to the IRES nucleotide sequence and the ZIKV NS5 protein coding sequence (Seq ID No. 5), both SHANK 3, IRES and NS5 sequences are functionally linked to the 5' of the NS4A-2K-NS4B sequence.

[0050] Thus, in one embodiment, the modified flavivirus nucleotide sequence further comprises: - the nucleotide sequences of part of the coding sequences of the Shank 3, IRES and NS5 proteins (SEQ ID NO 19, 20 and 5 respectively), functionally linked to each other from 5' to 3'; or combinations thereof.

[0051] In one embodiment, in Figure 1, D) shows the same sequence as in A), additionally comprising an example of the use of the Cas12 protein coding sequence. This sequence is functionally Petition 870250015956, dated 26 / 02 / 2025, p. 20 / 88 15 / 62 is linked to the nucleotide sequence of the cell localization signal, both being functionally linked to the 5' end of the NS4A-2K-NS4B sequence. The 3' end of the NS4A-2K-NS4B sequence is functionally linked to the nucleotide sequence of a non-protein-coding region and the nucleotide sequence of the Pre-crRNA.

[0052] Thus, in one embodiment, the modified flavivirus nucleotide sequence further comprises: - the nucleotide sequences of part of the coding sequences of Cas 12 proteins (Seq ID No. 21) functionally linked to 5'; - the nucleotide sequences of part of the coding sequences of the Pre crRNA proteins (Seq ID No. 22) functionally linked to 3'; or combinations thereof.

[0053] In one embodiment, the said nucleotide sequence further comprises the complete or partial nucleotide sequence of the functionally 3'-linked ZIKV NS5 protein (Seq ID No. 5).

[0054] In one embodiment, the sequence described in Figure 6, called EviOZ, provides encapsulation of iOZ for the efficient delivery of genetic material and CRISPR machinery to various tissues with cells susceptible to the viral vector for the treatment of rare genetic diseases, neurodegenerative diseases, and tumors.

[0055] In one embodiment, the said nucleotide sequence further comprises the nucleotide sequences encoding the ZIKV proteins NS1, NS2A, NS2B, NS3, NS4A and NS4B (Seq ID Nos. 10, 11, 9, 6, 27, 28 respectively), NS1, NS2A, NS2B, NS3, NS4A and NS4B are functionally linked to each other from 5' to 3' and to the sequence described in C to 5'.

[0056] In one embodiment, said nucleotide sequence further comprises the complete or partial nucleotide sequence of the functionally 3'-linked ZIKV NS5 protein (Seq ID No. 5).

[0057] Another object of the invention is the use of the nucleotide sequence to prepare a medicament or therapeutic agent for the treatment or prevention of genetic and degenerative conditions, including Syndrome of Petition 870250015956, dated 26 / 02 / 2025, page 21 / 88 16 / 62 Phelan-McDermid, Autism Spectrum Disorders, Alzheimer's and Parkinson's disease, Tourette Syndrome, syndromic autism, various genetic diseases, or for the treatment or prevention of flaviviruses, yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), and dengue virus (DENV). In one embodiment, the therapeutic agent comprises said non-natural and modified nucleotide sequence.

[0058] In one embodiment, the aforementioned viral vector is called EviOZ and provides efficient mRNA delivery to correct disorders in EviOZ-susceptible cells, primarily brain cells, through protein replacement. It may also deliver the complete CRISPR machinery for highly precise gene editing. EviOZ has the potential to treat neurological diseases, including neurodevelopmental and neurodegenerative conditions, with unprecedented precision, correcting mutations and restoring lost brain functions.

[0059] Thus, another object of the invention is the therapeutic agent comprising an excipient and / or adjuvant and the nucleotide sequence as defined herein. In one embodiment, the therapeutic agent comprises said nucleotide sequence encapsulated in a lysosome or nanoparticle, for use as a vaccine. In another embodiment, said nucleotide sequence further comprises the CMV and / or T7 promoter and poly A tail, for use as a viral vector. In yet another embodiment, the therapeutic agent comprises a modified Zika virus oncolytic virus, wherein the genome comprises: the hsa-miR-124-3p nucleotide sequence between the 5'UTR regions and the capsid protein coding region (C); and the hsa-miR-219a-2-3p nucleotide sequence within the 2K protein sequence.

[0060] In one embodiment, the aforementioned therapeutic agent, named SoziOZ, consists of an oncolytic virus for the treatment of malignant and other types of tumors. In one embodiment, the therapeutic agent is an oncolytic virus containing a modified nucleotide sequence of ZIKV, having a natural tropism for the central nervous system, acting specifically on Petition 870250015956, dated 26 / 02 / 2025, page 22 / 88 17 / 62 tumor cells, promoting their selective destruction, and stimulating the host's immune response. In one embodiment, the therapeutic agent of the invention is a viral vector for gene therapy.

[0061] In one embodiment, the aforementioned viral vector contains a modified nucleotide sequence of ZIKV and provides the delivery of therapeutic genes for replacement and expression of correct proteins in the host, in cases of diseases where genetic mutations lead to the expression of non-functional proteins or proteins with deleterious effects, or absent, or low dosage and / or gene editing machinery to cells susceptible to the viral vector, mainly CNS cells, with high efficiency and selectivity. The aforementioned vector has a high genetic load capacity and does not promote genomic integration because it is an RNA viral vector, providing a viable and safer therapeutic solution to correct mutations and neurological diseases than congeneric approaches.

[0062] In one embodiment, the therapeutic agent of the invention is a non-natural RNA sequence called ZiOZ, useful for vaccines and / or gene therapy. This embodiment, called ZiOZ, provides delivery of vaccine antigens or nucleotide sequences or CRISPR machinery for gene therapies to cells in the body, without the need for viral vectors (“naked”). ZiOZ can act both in the prevention of diseases and in the treatment of genetic and degenerative conditions. This approach avoids immunogenicity, in addition to expanding and facilitating distribution throughout the body, even in regions that the viral vector cannot reach, being especially promising for the treatment of complex diseases of the nervous, cardiovascular, digestive, respiratory, endocrine, sensory, excretory, urinary, reproductive, skeletal, muscular, immune, lymphatic and / or integumentary systems.

[0063] In one embodiment, the aforementioned therapeutic method is an oncolytic therapy. In one embodiment, the aforementioned oncolytic therapy is called SoziOZ, and is preferentially intended for tumors of brain origin or metastatic tumors located in the CNS, its action being specific to Petition 870250015956, dated 26 / 02 / 2025, page 23 / 88 18 / 62 tumor stem cells, metastatic cells, and tumor cells of neural origin, promoting their selective destruction, as well as stimulating the host's immune response. This embodiment of the invention is particularly useful for the treatment of brain tumors such as glioblastoma, providing an innovative solution that crosses the blood-brain barrier and directly targets brain tumor tissue.

[0064] Thus, another object of the invention is the use of oncolytic virus to prepare a medicament or therapeutic agent for treating cancer, including cancer selected from the group comprising tumors of brain origin, Glioblastoma, Medulloblastoma, Atypical teratoid rhabdoid tumor (ATT), Embryonic CNS tumor (pediatric), CNS tumor (adult), Breast cancer, Luminal cancer, Triple-negative tumor, Colon tumor, Colorectal tumor, Colorectal carcinoma, Mucoepidermoid lung carcinoma, Lung carcinoma (activated fatty acid metabolic pathways), Malignant melanoma, Ovarian cancer, Sarcoma-resistant tumor, ER+PR+ adenocarcinoma, Colorectal adenocarcinoma, Pancreatic adenocarcinoma, Thyroid carcinoma and sarcoma, Prostate carcinoma, Prostate adenocarcinoma, Pulmonary metastasis, Metastasis Cervical cancer, Brain metastasis, Bone metastasis, Primary tumor, Cisplatin-resistant tumor, Uterine tumor, Uterine / endometrial tumorGlioblastoma multiforme and tumors with high expression of the ITGAV membrane protein.

[0065] Another object of the invention is the process for obtaining the therapeutic agent comprising the steps of: - to obtain a modified nucleotide sequence; and - Add an excipient, adjuvant, nanoparticles and / or liposome.

[0066] In one embodiment of the process, the step of obtaining the aforementioned nucleotide sequence comprises the construction of two plasmid DNAs.

[0067] Another object of the invention is a therapeutic method that makes use of the nucleotide sequence and / or the therapeutic agent of the invention and includes the administration of an effective dose thereof. Petition 870250015956, dated 26 / 02 / 2025, page 24 / 88 19 / 62

[0068] In one embodiment, said therapeutic method is a gene therapy based on the use of the viral vector and / or modified RNA of the invention. In one embodiment, said therapeutic method provides the delivery of therapeutic genes for replacement and expression of correct proteins in the host, in cases of diseases where genetic mutations lead to the expression of non-functional proteins or proteins with deleterious effects, or absent, or low dosage and / or gene editing machinery to cells susceptible to the viral vector, mainly CNS cells, with high efficiency and selectivity. In one embodiment, said therapeutic method does not promote genomic integration, providing a viable and safer therapeutic solution to correct mutations and neurological diseases than congeneric approaches.

[0069] The iOZ Platform's gene therapy makes it possible to treat various genetic diseases through in vivo genetic modification of cells susceptible to the viral vector, such as CNS cells, tumor cells, and others. The FDA-approved technology has a more restricted application to blood diseases. This FDA-approved technology is based on ex vivo gene editing of hematopoietic stem cells; however, the application of this approach to other diseases is more complex due to the need to identify and access specific target cells in different tissues. This is overcome by the iOZ Platform's viral vector, which allows gene therapy to be delivered to cells that are difficult to access with drugs, such as CNS cells.

[0070] The Cas12 coding region and the pre-crRNA itself can be included in a single RNA sequence, enabling the use of viral RNA vectors. Furthermore, the present invention allows the production of a viral vector that encapsulates all the genetic material of this CRISPR platform, which, due to its extensive nucleotide sequence, would not be possible in viral vectors such as AAVs.

[0071] The invention in question presents an mRNA vaccine that may or may not be delivered in naked form (encased in lipid nanoparticles or liposomes) or with the ZIKV envelope. Furthermore, the mRNA described herein does not Petition 870250015956, dated 26 / 02 / 2025, page 25 / 88 The 20 / 62 vaccine requires the addition of CAP, an advantage over current mRNA vaccines, making the production process more efficient and less costly, and the treatment more effective. It enables a rapid and efficient approach to the production of vaccines against parasites and pathogens such as viruses, viroids, bacteria, fungi, protozoa, and prions.

[0072] When used without the envelope and embedded in lipid nanoparticles or liposomes, mRNA technology can also be used as gene therapy to be delivered throughout the body, since the tropism for the CNS is switched off, making it feasible to extend the tropism to other cells and tissues of the human body.

[0073] These and other objects of the invention will be immediately appreciated by those skilled in the art and will be described in more detail below. Brief Description of the Figures

[0074] The following figures are presented:

[0075] Figure 1 shows a schematic representation of the modified nucleotide sequence of the invention in some of its embodiments. In A), the modified nucleotide sequence, called the iOZ Platform, is shown, composed of three functionally linked nucleotide sequences: (i) the nucleotide sequence encoding part of the NS4A protein of the Zika virus (Seq ID No. 1); (ii) the complete nucleotide sequence encoding the 2K protein of the Zika virus (Seq ID No. 2); and (iii) the nucleotide sequence encoding part of the NS4B protein of the Zika virus (Seq ID No. 3).Furthermore, the topology of the three functionally connected sequences (NS4A-2K-NS4B) after the translation process is illustrated, highlighting which regions are exposed in the cytoplasm, which are transmembrane, and finally, which are exposed in the lumen of the endoplasmic reticulum (ER); In B) the same sequence as in A) is additionally shown, comprising an example of the use of nucleotide sequences encoding reporter proteins (Seq ID Nos. 17 and 18) functionally linked to the 5' of the NS4A-2K-NS4B sequence. Petition 870250015956, dated 26 / 02 / 2025, p. 26 / 88 In C), the same sequence as in A) is shown, comprising an example of the use of the SHANK3 protein coding sequence functionally linked to the IRES nucleotide sequence and the ZIKV NS5 protein coding sequence (Seq ID No. 5). Both SHANK3, IRES, and NS5 sequences are functionally linked to the 5' end of the NS4A2K-NS4B sequence. The 5' end of the SHANK3, IRES, and NS5 sequences is functionally linked to the nucleotide sequence of part of the C protein coding sequence (Seq ID No. 4). In D), the same sequence as in A) is shown, comprising an example of the use of the Cas12 protein coding sequence. This sequence is functionally linked to the nucleotide sequence of the cell localization signal, with both being functionally linked to the 5' end of the NS4A-2K-NS4B sequence.The 5' end of the Cas12 protein-coding nucleotide sequence is functionally linked to the nucleotide sequence of part of the C protein-coding sequence (Seq ID No. 4). The 3' end of the NS4A-2KNS4B sequence is functionally linked to the nucleotide sequences of a non-coding region of protein and the nucleotide sequence of Pre-crRNA; In E), the same sequence as in A) is shown additionally comprising the nucleotide sequences coding for the ZIKV proteins C, M, E, NS1, NS2A, NS2B, NS3, and NS4A (Seq ID Nos. 29, 7, 8, 10, 11, 9, 27 respectively), functionally linked to each other from 5' to 3' and linked to the 5' end of the NS4A-2KNS4B sequence, with overlap of the complete nucleotide sequence of the NS4A protein with the NS4A-2KNS4B sequence. The 3' end of the NS4A-2KNS4B sequence is functionally linked to the complete nucleotide sequence of the NS4B protein (Seq ID No. 28) and the nucleotide sequence coding for the NS5 protein (Seq ID No. 29).5), with overlapping of the complete nucleotide sequence of the NS4B protein with the NS4A-2K-NS4B sequence; In F) the same sequence as in E) is shown additionally comprising the nucleotide sequences of the MRE (5' CGUGCCUUA 3' - Seq ID No. 12, as a target of miR-124-3p) functionally. Petition 870250015956, dated 26 / 02 / 2025, p. 27 / 88 22 / 62 is linked to the 5' UTR (Seq ID No. 14) between nucleotides 112 and 113; both sequences are functionally linked to the 5' end of the complete coding nucleotide sequence of protein C (Seq ID No. 29). Between nucleotides 12 and 13 of the nucleotide sequence of protein 2K (Seq ID No. 2) is the nucleotide sequence of the MRE (5' CCGACCUGU 3' - Seq ID No. 13, as a target of miR219a-2-3p) functionally linked to each other. The 3' end of the nucleotide sequence encoding protein NS5 (Seq ID No. 5) is functionally linked to the nucleotide sequence of the 3' UTR (Seq ID No. 14).

[0076] Figure 2 shows a schematic illustration of some embodiments of the invention (SoziOZ, EviOZ, and ZiOZ) and their relationship to the iOZ platform, which uses information derived from the viral genomic sequence.

[0077] Figure 3 shows a diagram with the location of the miRNA targets in the genome of an oncolytic virus embodiment of the invention. The first target, located in the 5'UTR, corresponds to the MRE of miRNA 124-3p. The second target, located within the 2K protein sequence, corresponds to the MRE of miRNA 219a-2-3p.

[0078] Figure 4 shows the expression level of the aforementioned miRNA in non-tumor human tissue, from the database “The Human and Mouse Small Noncoding RNA Tissue Atlas” (https: / / ccb-web.cs.uni-saarland.de / tissueatlas2). Additionally, the expression of miRNA in some types of tumors, from the miRCancer: microRNA Cancer Association Database (http: / / mircancer.ecu.edu / index.jsp). A - miR-124-3p shows the highest expression level in the brain, as shown on the X-axis in number 6; B - miR-219a-2-3p shows the highest expression level in the brain, as shown on the X-axis in number 6; C - Expression level of miR-124-3p in tumors; D - Expression level of miR-219a-2-3p in tumors.

[0079] Figure 5 shows the strategy of replacing structural or non-structural protein sequences in the genome with those from another ZIKV strain. The figure uses the exchange of the coding sequence of the NS5 protein as an example. Petition 870250015956, dated 26 / 02 / 2025, page 28 / 88 23 / 62

[0080] Figure 6 shows an illustrative scheme presenting an embodiment of the viral vector of the present invention, EviOZ. The image also includes a schematic representation of the ZiOZ nucleotide sequence within the modified virus.

[0081] Figure 7 shows a schematic illustration of the methodology of the present invention for producing the viral vector (EviOZ), in which 2 plasmids are used.

[0082] Figure 8 shows the two plasmids used for the production of the viral vector (EviOZ).

[0083] Figure 9 shows an interactive body map relating to the average expression of the Integrin alpha-V receptor (ITGAV) in a patient with a tumor (left), when compared with samples in healthy individuals (right), with the color with the strongest intensity related to the highest expression of the transcript.

[0084] Figure 10 shows the transcript expression profile (RNAseq) across tumor samples compared to normal tissues. Each point represents the expression of the samples. The analysis of ITGAV protein transcript overexpression in tumor samples, when compared to their corresponding normal tissue, was based on a public RNA-seq database (GEPIA). Tang, Z. et al. (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res, 10.1093 / nar / gkx247.

[0085] Figure 11 shows the expression profile of the ITGAV protein transcript across tumor samples compared to normal tissues. The height of the bars represents the average expression in certain types of tumor or normal tissue.

[0086] Figure 12 shows a schematic of the oncolytic virus (SoziOZ) production process from two plasmids as templates for amplification. Initially, a fusion PCR is performed to combine the genetic information contained in the two plasmids, essential for SoziOZ production. After the fusion PCR, the resulting DNA fragment can be directly electroporated. Petition 870250015956, dated 26 / 02 / 2025, page 29 / 88 24 / 62 in cells or, alternatively, the PCR product can be transcribed in vitro to generate RNA, followed by electroporation of this RNA. Regardless of the method chosen (DNA or RNA electroporation), the end result is the production of the oncolytic virus SoziOZ, without the need for intermediate DNA cloning steps in bacteria.

[0087] Figure 13 A and B illustrate two plasmids used for the production of the oncolytic virus (SoziOZ): pSoziOZ 1, which contains the CMV promoter information up to the nucleotide sequence encoding the NS4A protein, and pSoziOZ 2, which contains the nucleotide sequence encoding the 2K protein up to the nucleotide sequence of the polyadenylation signal.

[0088] Figure 14 shows the agarose gel electrophoresis of the fusion PCR product, aiming to obtain a single DNA fragment containing the CMV and T7 promoters, functionally linked to the 5' nucleotide sequence of the SoziOZ genome. The fusion PCR was performed using amplified DNA fragments from the pSoziOZ 1 and pSoziOZ 2 plasmids. A - DNA molecular weight standard 1 kb Plus, B - Overlapping PCR product (11.8 kb).

[0089] Figure 15 shows non-denaturing agarose gel electrophoresis of RNA, the product of in vitro transcription performed from the single DNA fragment generated after fusion PCR of DNA fragments amplified from pSoziOZ 1 and pSoziOZ 2 plasmids. A - DNA molecular weight standard 1kb Plus, B - RNA, product of in vitro transcription (11 kb).

[0090] Figure 16 shows the sequence alignment of the hsa-miR-1243p miRNA with the complementary sequence of the MRE of an embodiment of the invention. A favorable alignment between the MRE and the hsa-miR-124-3 miRNA is observed. The software used to verify the homology was the EMBOSS Needle server (https: / / www.ebi.ac.uk / jdispatcher / psa / emboss_needle). Detailed Description of the Invention

[0091] The present invention provides a modified flavivirus nucleotide sequence, a therapeutic agent, a viral vector and its process of Petition 870250015956, dated 26 / 02 / 2025, page 30 / 88 25 / 62 obtaining, its use, and a treatment method that utilizes said sequence, agent, or vector.

[0092] The invention is associated with the so-called iOZ Platform, which leverages the potential of the viral genome of flaviviruses, especially ZIKV, to provide advanced therapeutic approaches, also offering targeted and effective treatments for complex diseases, including the treatment of malignant and other types of tumors. Definitions

[0093] For the purposes of the present invention, the following expressions are used, with their respective meanings: ZiOZ (Modified flavivirus nucleotide sequence); pZiOZ (plasmid used for the production of ZiOZ), pVLP (plasmid used for the production of viral vector envelope proteins), EviOZ (viral vector), SoziOZ (oncolytic virus), pSoziOZ 1 (plasmid used for the production of the oncolytic virus), pSoziOZ 2 (plasmid used for the production of the oncolytic virus).

[0094] ZIKV (Zika Virus) belongs to the Flaviviridae family, the same as other known viruses such as dengue and yellow fever. Like these, ZIKV is an arbovirus, that is, a type of virus transmitted by arthropods, especially insects. The origin of ZIKV is linked to its first identification in a Rhesus monkey in the Zika forest in Uganda in 1947, which gave the virus its name. In humans, the virus was first detected in 1952. The first significant outbreak of Zika infections occurred in 2007 on Yap Island in Micronesia. Currently, several countries are facing active outbreaks of the Zika virus.

[0095] NS4A is a membrane-associated protein of the endoplasmic reticulum (ER). Structurally, it is composed of an extramembrane N-terminal domain and three transmembrane (TM) segments, designated TM1, TM2, and TM3. Biochemical studies indicate that only TM1 and TM3 completely span the membrane, while TM2 is embedded but does not cross the lipid bilayer. Furthermore, a fourth segment, known as Petition 870250015956, dated 26 / 02 / 2025, page 31 / 88 26 / 62 fragment 2K is cleaved from the NS4A-2K-NS4B polypeptide, resulting in the release of NS4A in its mature form. This cleavage releases the NS4A protein to rearrange in the ER membrane, which is sufficient to induce structural changes similar to the highly curved membranes observed in replication complexes (RC).

[0096] The NS4B protein is characterized by having five transmembrane domains (TM1, TM2, TM3, TM4, and TM5) that span the endoplasmic reticulum (ER) membrane. This protein accumulates in the ER membrane or in ER-derived membranes, locations where non-structural viral proteins and viral RNA are concentrated, thus playing an essential role in viral replication. NS4B's functions in the replication complex are mediated by protein-protein interactions, which facilitate the formation and maintenance of the environment necessary for virus replication. Furthermore, both NS4A and NS4B are directly involved in membrane rearrangements and the viral RNA replication process, highlighting their importance in the virus's life cycle. Comparison with the State of the Art

[0097] The therapeutic agent referred to as SoziOZ, used in this invention, is capable of causing direct lysis of tumor cells, in addition to activating a specific antitumor immune response in the host. This mechanism differentiates it from technologies based on herpes simplex virus type 1 (HSV-1), such as Imlygic® and Delytact®, which rely exclusively on immune activation after infecting tumor cells. The combination of direct lysis of tumor cells and immunomodulation reduces dependence on pre-existing immune responses in the patient and increases its effectiveness in highly resistant tumors, such as glioblastoma.

[0098] The genetic modifications in the SoziOZ of this invention are minimal and highly precise, guaranteeing its replicative capacity and greater safety without losing efficacy. Unlike other extensively modified oncolytic viruses, such as Imlygic® which attenuates the virus, the solution proposed here... Petition 870250015956, dated 26 / 02 / 2025, page 32 / 88 27 / 62 maintains the natural tropism of SoziOZ for the CNS, ensuring superior selectivity and safety. Furthermore, this approach minimizes the risks of virulent reversion and promotes the genetic stability of SoziOZ.

[0099] In one embodiment, a combination of reference sequences found in the genetic database, found in the accession codes NC_012532 (https: / / www.ncbi.nlm.nih.gov / nuccore / NC_012532.1), KX197192 (https: / / www.ncbi.nlm.nih.gov / nuccore / KX197192 / ), KU509998 (https: / / www.ncbi.nlm.nih.gov / nuccore / KU509998) is used to construct the invention. Other patent documents, such as the one described in WO2023164442A2, which propose the use of ZIKV for vaccines and / or virotherapy, use a different strain from the accession code provided (MH882527.1, https: / / www.ncbi.nlm.nih.gov / nuccore / MH882527.1 / ), therefore using the HAITIAN strain, which presents significant differences in the genomic sequence, especially in the UTR regions, compared to the MH882527.1 strain.

[0100] The use of the CMV (Cytomegalovirus) promoter in the plasmid, unique to SoziOZ technology, allows for the direct production of viral vector RNA within the host cell, eliminating additional in vitro transcription steps. This simplifies the production process, reduces manufacturing costs, and increases efficiency in viral vector generation.

[0101] The technology employs the use of only two plasmids, allowing the production of the entire therapeutic system in an integrated and simplified manner, and makes it feasible to produce the SoziOZ system free of intermediate DNA cloning steps in bacteria. This approach is unprecedented in the context of oncolytic virotherapy and ZIKV-based gene therapy, representing a significant advance in terms of practicality, industrial scalability, and feasibility of obtaining the therapeutic product. Unlike other ZIKV-based patents, this architecture reduces manufacturing complexity and costs while increasing the reliability and safety of the system.

[0102] For the first time, the target sequence (MRE) drawn for the hsa-miR-124 was Petition 870250015956, dated 26 / 02 / 2025, page 33 / 88 28 / 62 incorporated into the 5'UTR region of the modified ZIKV genome, creating highly specific expression control. This innovation increases safety by allowing viral replication in tumor cells, where miR-124 is poorly expressed, while preventing replication in healthy cells, where this microRNA is highly expressed. Thus, the target miRNA, the designed sequence, and the position within ZIKV are all novel and not present in any previous document, including WO2023164442A2.

[0103] For the first time, two target sequences (MREs) were simultaneously placed into the modified ZIKV genome. One target for the microRNA hsa-miR-124 was incorporated into the 5'UTR region and the other target for the microRNA hsa-miR-219a-2-3p was added between the nucleotide sequence encoding the 2K protein, in the same reading frame as the protein. This allows for a dual mechanism of regulation of oncolytic therapy replication that increases the safety of SoziOZ not present in any previous document, including WO2023164442A2.

[0104] The present invention provides for the replacement of structural or non-structural protein sequences in the oncolytic therapy genome with those of another ZIKV lineage. The use of a chimeric sequence provides technological advantages compared to the state of the art, allowing the invention to be improved for optimization of its use as an oncolytic therapy for tumors of brain origin, metastatic tumors located in the CNS, tumor stem cells, metastatic cells, tumor cells of neural origin, and tumor cells of other origins in the body that are susceptible to oncolytic therapy.

[0105] This invention would be the first RNA-based viral vector with a high genetic payload capacity for gene therapy (up to 10 kb). Unlike DNA-based vectors, such as AAVs and Herpes with gene therapies already approved by the FDA, which have structural limitations such as low tropism for CNS cells and risks of genomic integration, the viral vector that is the subject of the invention eliminates these problems because it exhibits greater tropism for Petition 870250015956, dated 26 / 02 / 2025, page 34 / 88 29 / 62 CNS cells. Furthermore, the fact that it is a viral vector that delivers RNA that undergoes reverse transcription, and not DNA like AAV, eliminates the risk of integration of DNA sequences into the genome of the patients' target cells, making it safer and more functional for therapeutic applications.

[0106] The present invention represents a significant advance in viral vector technology, with the development of EviOZ, carrying ZiOZ which may or may not be replicative. Unlike other patents, the present invention, unlike patent WO2023164441A1, employs a different strategy in the ZiOZ sequence (SEQ ID NO 23), using a minimal nucleotide sequence of the NS4A and NS4B proteins (Seq ID Nos. 1 and 3 respectively), in addition to possessing the complete nucleotide sequence of the 2K protein (Seq ID No. 2), which are important for adequate encapsulation of ZiOZ by EviOZ. This strategy overcomes the limitations of conventional systems, such as that described in patent WO2023164441A1, which uses a sequence of the C protein for encapsulation.

[0107] The choice of the minimum nucleotide sequence coding for the NS4A and NS4B proteins (Seq ID Nos. 1 and 3 respectively), and the complete nucleotide sequence for the 2K protein (Seq ID Nos. 2), were made based on the transmembrane characteristics of these proteins, which are important during replication. In the present invention, the nucleotide sequences are essential to help ZiOZ be located in the ZIKV-like envelope structure assembly environment, allowing its encapsulation by EviOZ. This strategic modification improves safety and reduces potential adverse effects and immune reactions related to capsid expression in host cells.

[0108] Unlike other approaches, the invention of ZiOZ allows for the insertion or exclusion of the complete nucleotide sequence or part of the NS5 protein (Seq ID Nos. 5) depending on the therapeutic application of ZiOZ or the viral vector that encapsulates it. The inclusion of the complete nucleotide sequence or part of the NS5 protein (Seq ID Nos. 5) is responsible for the replication of genomic RNA. Petition 870250015956, dated 26 / 02 / 2025, page 35 / 88 30 / 62 in the ZIKV viral cycle. In ZiOZ, this sequence will be used to enable RNA replication, ensuring that the level of protein expression persists for longer.

[0109] A major difference from other gene therapies is the absence of hepatotoxicity, frequently observed in AAV vectors, which require high doses to achieve clinical efficacy. The invention allows dose / effect control with the administration of multiple doses, increasing therapeutic efficacy without compromising patient safety.

[0110] Compared to AAV (Adenovirus-associated) based vectors, the ZIKV of this invention offers a genetic payload capacity of up to 10 kb, in contrast to the 4.7 kb of AAVs. Furthermore, ZIKV crosses the blood-brain barrier more efficiently, while AAVs have lower tropism for the CNS and an affinity for the liver, requiring high doses for clinical manifestation of the therapeutic effect, and this is frequently associated with adverse effects such as hepatotoxicity. The present solution also avoids genomic integration, providing greater safety and clinical viability.

[0111] The invention in question presents an mRNA vaccine that may or may not be delivered in naked form (encased in lipid nanoparticles or liposomes) or with the ZIKV envelope. A distinguishing feature is the absence of the need for CAP addition, as occurs in current mRNA vaccines. This approach simplifies the production process, reduces costs, making it more accessible and effective.

[0112] When used without the envelope and embedded in lipid nanoparticles or liposomes, the proposed mRNA technology can also be used as gene therapy for delivery to different body tissues, since the tropism for the CNS from the envelope is switched off and redirected to other cell types. This allows application in a wide range of conditions, such as genetic diseases and metabolic disorders, significantly expanding the therapeutic scope of the technology.

[0113] One of the objects of the invention is the use comprising at least Petition 870250015956, dated 26 / 02 / 2025, page 36 / 88 31 / 62 three nucleotide sequences functionally linked together in any order of linkage. Preferably the linkage order corresponds to: (i) the nucleotide sequence of part of the coding sequence of the ZIKV NS4A proteins (Seq ID No. 1); (ii) the complete sequence of the Zika virus 2K protein (Seq ID No. 2); and (iii) the nucleotide sequence of part of the coding sequence of the Zika virus NS4B proteins (Seq ID No. 3). In one embodiment, the aforementioned set of nucleotide sequences encodes fragments of the NS4A and NS4B proteins that do not have the same protein function compared to the proteins with complete sequences of flaviviruses, since the absence of the complete sequence does not allow for the functionalization of the ER membrane rearrangement nor does it aid in replication.

[0114] In one specific embodiment, the nucleotide sequence encoding part of the NS4A and NS4B proteins, along with the nucleotide sequence encoding the 2K protein, plays the role of directing the ZiOZ RNA during the translation process to the endoplasmic reticulum. There, the EviOZ envelope structure is assembled, allowing ZiOZ to be positioned in the appropriate environment for viral vector assembly and, consequently, to be encapsulated by it. These functions are distinct from the natural functions performed by the NS4A, 2K, and NS4B proteins, highlighting a specific and targeted role in this context.

[0115] In one particular embodiment, the nucleotide sequence that encodes part of the NS4A and NS4B proteins, together with the nucleotide sequence that encodes the 2K protein, plays the role of directing or maintaining the heterologous protein in intracellular or extracellular regions, necessary for the heterologous protein to perform its function. Modified flavivirus nucleotide sequences.

[0116] The nucleotide sequences of the present invention have been modified to solve one or more of the technical problems mentioned in this patent application. Said nucleotide sequence comprises at least three nucleotide sequences functionally linked together in any order of Petition 870250015956, dated 26 / 02 / 2025, page 37 / 88 32 / 62 linkage of these. Preferably, the linkage order corresponds to: (i) the nucleotide sequence of part of the coding sequence of ZIKV NS4A proteins (Seq ID No. 1); (ii) the complete sequence of ZIKV 2K protein (Seq ID No. 2); and (iii) the nucleotide sequence of part of the coding sequence of ZIKV NS4B proteins (Seq ID No. 3).

[0117] In one embodiment, the said nucleotide sequence further comprises the complete coding nucleotide sequence of the ZIKV NS4A protein (Seq ID No. 27) functionally linked to 5', with overlap of the complete nucleotide sequence of the NS4A protein with the NS4A-2KNS4B sequence.

[0118] In one embodiment, the said nucleotide sequence further comprises the complete coding nucleotide sequence of the ZIKV NS4B protein (Seq ID No. 28) functionally linked to 3', with overlap of the complete nucleotide sequence of the NS4B protein with the NS4A-2KNS4B sequence.

[0119] In one embodiment, the said nucleotide sequence further comprises the nucleotide sequence of part of the coding sequence of the ZIKV NS5 protein (Seq ID No. 5) functionally linked to 3'.

[0120] In one embodiment, the said nucleotide sequence further comprises the nucleotide sequence of part of the coding sequence of the ZIKV NS3 protein (Seq ID No. 6) functionally linked to 5'.

[0121] In one embodiment, the said nucleotide sequence further comprises the nucleotide sequences of part of the coding sequences of the ZIKV M, E, NS1, NS2A, NS2B proteins (Seq ID Nos. 7, 8, 10, 11, 9, respectively), functionally linked to each other from 5' to 3' and to the sequence of the ZIKV NS3 protein coding sequence at 5'.

[0122] In one embodiment, the said nucleotide sequence further comprises the nucleotide sequence of part of the coding sequence of the ZIKV Capsid C protein (Seq ID No. 4), functionally linked to the 5' of the coding sequence of M (Seq ID No. 7). Petition 870250015956, dated 26 / 02 / 2025, page 38 / 88 33 / 62

[0123] In one embodiment, the said nucleotide sequence further comprises the replacement of the nucleotide sequence of part of the coding sequence of the ZIKV Capsid C protein (Seq ID No. 4), by the complete coding nucleotide sequence of the ZIKV Capsid C protein (Seq ID No. 29), functionally linked to 5' of the coding sequence of M (Seq ID No. 7).

[0124] In one embodiment, the said nucleotide sequence further comprises: at 5' the nucleotide sequences 5'UTR (Seq ID No. 14) and MRE (5' CGUGCCUUA 3' - Seq ID No. 12, as a target of miR-124-3p) functionally linked to each other; and at 3' the nucleotide sequences MRE (5' CCGACCUGU 3' Seq ID No. 13, as a target of miR-219a-2-3p) and 3'UTR (Seq ID. 15) functionally linked to each other.

[0125] The present invention also encompasses an oncolytic virotherapy based on ZIKV with natural tropism for the central nervous system that not only specifically targets tumor cells, promoting their selective destruction, but also stimulates the host's immune response. In addition to presenting a ZIKV-based viral vector capable of delivering therapeutic genes for the replacement of correct or absent proteins in individuals with genetic diseases, it can also deliver gene editing machinery to CNS cells or other susceptible cells of the organism with high efficiency and selectivity, with high genetic load capacity and without genomic integration, providing a viable and therapeutic solution for correcting genetic mutations and treating neurological diseases.

[0126] The present invention relates to methods for the production and purification of flavivirus-based virotherapy, in particular ZIKV, produced for the treatment of glioblastoma multiforme (GBM) and other metastatic tumors with the presence of tumor stem cells, and when inactivated, for use in a vaccine, as well as the vaccine composition and administration of the compound for the generation and evaluation of an anti-Zika virus immune response. Oncolytic virotherapy Petition 870250015956, dated 26 / 02 / 2025, page 39 / 88 34 / 62

[0127] The oncolytic virotherapy of the present invention uses a flavivirus-based therapy, in particular the African strain of ZIKV, which has a natural tropism for the central nervous system. This technology not only specifically targets tumor cells, promoting their selective destruction, but also stimulates the host's immune response. For CNS tumors, such as glioblastoma, this approach offers an innovative therapy solution that crosses the blood-brain barrier and directly targets tumor tissue. It can also be used for tumors of neural origin, such as embryonic CNS tumors, pediatric tumors such as ependymoma and DIPG. It also shows potential for metastatic tumors of diverse origins such as breast cancer, prostate cancer and lung cancer, but which are located in the CNS.Similarly, for any tumor that has a population of stem cells, the so-called tumor stem cells, which are responsible for resistance to current therapies and the generation of metastases and tumor recurrence.

[0128] The oncolytic virotherapy of the present invention utilizes modified ZIKV to replicate and selectively destroy tumor cells by means of cell lysis and stimulation of the host's antitumor immune response. The modification was designed to ensure product safety, given that administering an active virus with a genome and characteristics similar to the wild-type virus (wild strain) may present risks to the patient and to biosafety, potentially infecting both tumor cells and non-tumor cells. To improve safety without compromising replicative activity, miRNA response elements (MREs) were incorporated. Among these, hsa-miR-124-3p was inserted into the 5'UTR region, while hsa-miR-219a-2-3p was added to the coding region of the 2K protein, between nucleotides 12 and 13.The goal of inserting MREs is to prevent the replication of the oncolytic virus in non-tumor cells without losing its oncolytic effect; that is, without interrupting viral replication and the oncolytic effect in tumor cells. The MREs inserted into the ZIKV genome are recognized by miRNAs expressed in non-tumor cells, causing the degradation of the oncolytic virus genome and, consequently... Petition 870250015956, dated 26 / 02 / 2025, page 40 / 88 35 / 62 inhibiting replication in these cells. In contrast, in tumor cells, miRNAs are poorly expressed or absent, allowing the virus to continue replicating and being oncolytic (Figure 2 - Oncolytic virus).

[0129] Additionally, the invention eliminates or minimizes the problem of the risk of reversion of the virulence capacity of the modified virus. That is, during replication in hosts, mutations can occur in the viral genome, reversing the genetic modifications and restoring virulence, leading to the manifestation of disease in the individual undergoing therapy. Furthermore, a virus that has undergone reversion to virulence can be transmitted to other people, leading to outbreaks in communities, especially those with low vaccination coverage. This type of undesirable event has already occurred, for example, with the Oral Polio Vaccine (OPV): OPV contains attenuated poliovirus, but there have been cases of reversion to virulent forms, resulting in outbreaks of vaccine-derived poliomyelitis (cVDPV).

[0130] A distinguishing feature of the present invention is the insertion of miRNA targets in two concomitant regions. This approach allows for more refined control of viral replication, since non-tumor cells can degrade viral RNA through two independent mechanisms. This eliminates or minimizes the reversion of the virulence capacity of the modified virus. Both miRNAs (hsa-miR-1243p and hsa-miR-219a-2-3p) are predominantly expressed in non-tumor brain cells and have low expression in tumor cells, making the insertion of these MREs, complementary targets to the miRNAs, a safety-oriented strategy for the oncolytic virotherapy of the present invention applied to the treatment of brain cancer (Figure 4).

[0131] In one embodiment, the construction of the oncolytic virus of the present invention utilizes a combination of reference sequences found in the access codes: KU509998 (https: / / www.ncbi.nlm.nih.gov / nuccore / KU509998) KX197192 (https: / / www.ncbi.nlm.nih.gov / nuccore / KX197192 / ) NC_012532 (https: / / www.ncbi.nlm.nih.gov / nuccore / NC_012532.1). Petition 870250015956, dated 26 / 02 / 2025, page 41 / 88 36 / 62 Viral vector

[0132] The EviOZ viral vector is particularly useful for delivering therapeutic genes to replace correct or missing proteins, as well as providing the delivery of CRISPR machinery to cells to correct mutations, providing a viable therapeutic solution.

[0133] The EviOZ vector uses information derived from flavivirus genomic sequences, especially ZIKV, to create a versatile and efficient vector. It provides mRNA delivery to various cells, including those located in the CNS and other susceptible cells, to correct their disorders through protein replacement (mRNA-based gene replacement) and can deliver the complete CRISPR machinery for gene editing with high precision.

[0134] This innovative technology utilizes the properties of ZIKV to precisely target the delivery of therapeutic genes, offering an effective tool for both protein replacement and genetic modification through the knockout of deleterious genes (Figure 2). EviOZ is particularly useful for treating neurological diseases, including neurodevelopmental and neurodegenerative conditions, with unprecedented precision, correcting mutations and restoring lost brain functions.

[0135] EviOZ crosses the blood-brain barrier, making it an attractive tool for the development of a viral vector intended for gene delivery in the treatment of various diseases, including brain health and genetic diseases. This includes genetic syndromes and neurodevelopmental disorders, which currently lack precise treatments on the market and are considered drug orphan diseases.

[0136] EviOZ encapsulates an mRNA called ZiOZ that contains sequences derived from ZIKV that confer stability, encapsulation, and replication to the RNA. The mRNA contains sequences important for its stability due to the presence of 5'UTR and 3'UTR regions similar to those of the African strain of ZIKV. The 5'UTR region of ZIKV offers another technological advantage, allowing CAP-independent translation, which reduces the need for Petition 870250015956, dated 26 / 02 / 2025, page 42 / 88 37 / 62 enzymes are used during its production, as the addition of a CAP-analogous structure is not necessary. This characteristic is an important factor in ZiOZ's suitability for use as an RNA vaccine.

[0137] In addition, ZiOZ has two internal sequences. One of them is part of the capsid protein sequence, crucial for the formation of the secondary RNA structure, favoring its replication by the non-structural protein 5 (NS5). The other is composed of part of NS4A, the complete sequence of the 2K region and part of the NS4B sequence, necessary for its encapsulation by the ZIKV structural proteins (SEQ ID No. 23).

[0138] The structure of EviOZ, which corresponds to a virus-like particle (VLP), ensures the protection of ZiOZ during delivery and facilitates its entry into target cells, where the genetic material can be effectively translated, promoting the desired therapeutic expression. This innovative approach enhances the effectiveness of gene therapy by using ZIKV biology as a vehicle for the delivery of therapeutic genes. EviOZ is capable of delivering genes of interest up to 10 kb, therefore, the viral vector encapsulating ZiOZ can be used to treat genetic diseases that require the replacement of large proteins or protein groups, such as genetic, neurodegenerative and neurodevelopmental diseases, in addition to making it feasible to deliver the entire complete CRISPR machinery for gene editing. This expands the therapeutic application of the technology compared to approved viral vectors and ongoing clinical trials.

[0139] The invention introduces a new approach to the production of viral vectors, an optimized and efficient VLP, simplifying the process with the use of only two plasmids (Figures 7 and 8), diverging from the methodologies employed in other patents to produce AAVs or other methodologies for the production of other types of viral vectors, mainly those based on flaviviruses. This strategic modification reduces complexity and allows for increased efficiency and scalability of production. Petition 870250015956, dated 26 / 02 / 2025, page 43 / 88 38 / 62

[0140] Furthermore, the vector has tropism for cells of the nervous system. Because of this tropism for the nervous system, the therapy reduces the amount of drug needed and the potential toxicological problems.

[0141] Furthermore, EviOZ is able to cross the BBB with an efficiency of up to 10%. This delivery capability represents a major advance over the ability of current drugs on the market to reach the nervous system, which is approximately 0.01% to 0.1%.

[0142] It is worth noting that ZiOZ alone can also be used for "naked" gene therapy if encapsulated in another structure, for example lipid nanoparticles or liposomes. In addition to having the potential to be an mRNA vaccine platform.

[0143] Compared to AAV-based viral vectors, EviOZ crosses the BBB and reaches the CNS more efficiently than AAV, and does not cause hepatotoxicity as seen in AAV, allowing for greater control of the dose / effect phenomenon and avoiding severe side effects.

[0144] Because EviOZ can carry ZiOZ, which is capable of transporting genes up to 10 kb, it expands therapeutic possibilities compared to viral vectors approved for human use. This is especially relevant for the treatment of genetic diseases that require the replacement of large proteins or protein complexes, as well as allowing the complete delivery of the CRISPR machinery for gene editing. Furthermore, the fact that it is a viral vector that delivers RNA, and not DNA like AAV, presents an additional advantage, as it eliminates the risk of integration of DNA sequences into the genome of the patients' target cells.

[0145] It has been found that part of the capsid protein sequence is important for the formation of the secondary structure of viral RNA, essential for the replication process. Because of this, it is suggested that the sequence be maintained, along with the insertion of the NS5 protein sequence into ZiOZ. It is recommended to use a nucleotide for ATA, so that the sequence ceases to be coding, but still retains most of its sequence for Petition 870250015956, dated 26 / 02 / 2025, page 44 / 88 39 / 62 preserve this crucial characteristic for replication (Figure 6). In addition, the third A of the ATA codon (SEQ ID No. 4) can be replaced by G.

[0146] In one embodiment, ZiOZ features the inclusion of the NS5 protein sequence, responsible for genomic RNA replication in the ZIKV viral cycle. In ZiOZ, this sequence is used to enable RNA replication, ensuring that the level of protein expression persists for longer.

[0147] Another significant modification is in the viral vector production methodology. In the present methodology, only two plasmids are used for viral vector production. In addition, the 5' and 3' UTR regulatory regions of the Xenopus globin gene can be replaced by another UTR. mRNA (ZiOZ)

[0148] In one embodiment, ZiOZ is an mRNA that utilizes information derived from the flavivirus genomic sequence, especially ZIKV. This results in a stable mRNA due to the presence of the 5'UTR and 3'UTR regions similar to those of the African ZIKV strain.

[0149] Using mRNA to deliver vaccines or gene therapies directly into the body, without the need for viral vectors (“naked”). In this context, ZiOZ acts both in the prevention of diseases and in the treatment of genetic and degenerative conditions. This approach avoids excessive immunogenicity and facilitates distribution throughout the body, being especially promising for the treatment of complex neurological conditions.

[0150] EviOZ encapsulates mRNA called ZiOZ (SEQ ID No. 16) which contains sequences important for its stability, due to the presence of 5'UTR and 3'UTR regions similar to those of the African strain of ZIKV.

[0151] Due to the properties of ZiOZ in the embodiment that has in its structure the 5'UTR of the African ZIKV strain, allowing CAP-independent translation, which reduces the need for enzymes during its production, since the addition of a CAP-analogous structure is not necessary. This opens up possibilities for the production of an mRNA vaccine with simplified manufacturing, optimized processes, greater production efficiency and scalability, making its Petition 870250015956, dated 26 / 02 / 2025, page 45 / 88 40 / 62 allows for faster manufacturing, adaptable to different demands, and reduces production costs. Furthermore, the genetic material loading capacity of ZiOZ (~10 Kb) makes it feasible to insert long and complex antigens into the mRNA vaccine, including the addition of multiple antigens, aiming to accommodate more than one antigenic target and / or a vaccine for multiple viral strains.

[0152] Furthermore, in one embodiment, iOZ has two internal sequences. One of them is part of the capsid protein sequence, crucial for the formation of the RNA secondary structure, favoring its replication by the non-structural protein 5 (NS5). The other is composed of part of NS4A, the complete sequence of the 2K region and part of the NS4B sequence, necessary for its encapsulation by the ZIKV structural proteins (SEQ ID No 23).

[0153] In one embodiment, the structure of EviOZ, which corresponds to a VLP, ensures the protection of ZiOZ during delivery and facilitates its entry into target cells, where the genetic material can be effectively translated, promoting the desired therapeutic expression. This innovative approach enhances the effectiveness of gene therapy by using ZIKV biology as a vehicle for the delivery of therapeutic genes. EviOZ provides delivery of genes of interest up to 10 kb, in addition to having tropism for cells of the nervous system. Currently, commercially used viral vectors do not have this specific tropism, and may accumulate in the liver, requiring very high doses that can lead to cytopathic effects. The therapy of the present invention, by having this tropism for the nervous system, reduces the amount of drug needed and potential toxicological problems, as well as ensuring effective delivery of the therapy only to target cells. Derived products

[0154] Through downstream viral inactivation processing, SoziOZ is useful as an inactivated vaccine against ZIKV. Furthermore, mRNA can additionally comprise a pathogen antigen as a gene of interest, and thus, combined with the lipid nanoparticle formulation, provide a novel mRNA vaccine strategy with unique forms. Petition 870250015956, dated 26 / 02 / 2025, page 46 / 88 41 / 62 dosage and / or dosage regimen of the product.

[0155] Additionally, SoziOZ is useful for concomitant use with EviOZ, as well as other immunotherapies in the fight against tumors, including, but not limited to, checkpoint inhibitors (e.g., anti-PD-1, anti-PD-L1, anti-CTLA-4), therapies based on genetically modified cells, such as CAR-T, CAR macrophages and CAR-NK, or other immunological approaches. The combination of these therapies potentiates the immunomodulatory effects of the oncolytic virus, promoting a more robust and effective immune response against tumor cells. The integration of these approaches offers a versatile therapeutic solution, adaptable to different tumor profiles and with the potential to improve clinical outcomes. Method for detecting / locating genes of interest

[0156] The present invention also provides a kit and in vitro process for evaluating the expression of a gene of interest. Said process comprises at least one step of contacting a biological sample containing a gene of interest with at least one modified flavivirus nucleotide sequence of the present invention.

[0157] Preferably, the flavivirus nucleotide sequence of the kit or process of the invention is chemically modified to facilitate the detection / location of genes of interest. In a preferred embodiment, said chemical modification involves the inclusion, in a region of the sequence, of one or more chromophores and / or radioactive element(s).

[0158] The aforementioned in vitro kit or process provides selective ligation of genes of interest, quantification of the expression level of such genes and / or quantification of the binding intensity of the nucleotide sequence to the gene of interest. The aforementioned in vitro kit or process is particularly useful for the detection of other molecular entities binding to the gene of interest, or even molecular entities that affect the selective or non-selective binding of the nucleotide sequences of the invention to the genes of interest.

[0159] The aforementioned kit or in vitro process is also particularly useful for Petition 870250015956, dated 26 / 02 / 2025, p. 47 / 88 42 / 62 subsequent customization of therapeutic treatments based on pharmaceutical compositions comprising the modified flavivirus nucleotide sequence of the invention. The kit or process of the invention is therefore applicable to the therapeutic prediction / prognosis of genetic and degenerative conditions, or even the potential therapeutic success in the treatment of: various genetic diseases, flaviviruses and tumors.

[0160] The invention can also be defined by the following clauses:

[0161] Modified flavivirus nucleotide sequence comprising at least three functionally linked nucleotide sequences in any order of linkage thereof, selected from: (i) the nucleotide sequence of part of the coding sequence of ZIKV NS4A proteins (Seq ID No. 1); (ii) the partial, modified or complete sequence of ZIKV 2K protein (Seq ID No. 2); and (iii) the nucleotide sequence of part of the coding sequence of ZIKV NS4B proteins (Seq ID No. 3).

[0162] Use of the nucleotide sequence as defined above, to prepare a medicament or therapeutic agent for the treatment or prevention of genetic and degenerative conditions, including Phelan-McDermid Syndrome, Autism Spectrum Disorder, Alzheimer's, Parkinson's, Tourette Syndrome, various genetic diseases or for the treatment or prevention against flaviviruses, yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and dengue virus (DENV).

[0163] Therapeutic agent comprising an excipient and / or adjuvant and the nucleotide sequence as defined above.

[0164] Use of oncolytic virus as defined above, to prepare a medicament or therapeutic agent for treating cancer, including selected cancer from the group comprising tumors of brain origin, Glioblastoma, Medulloblastoma, Atypical teratoid rhabdoid tumor (ATRT), Embryonic CNS tumor (pediatric), CNS tumor (adult), breast cancer, Luminal cancer, Triple-negative tumor, Colon tumor, Colorectal tumor, Colorectal carcinoma, Mucoepidermoid lung carcinoma, Carcinoma of Petition 870250015956, dated 26 / 02 / 2025, page 48 / 88 43 / 62 Lung (activated fatty acid metabolic pathways), Malignant melanoma, Ovarian cancer, Sarcoma-resistant tumor, ER+PR+ adenocarcinoma, Colorectal adenocarcinoma, Pancreatic adenocarcinoma, Thyroid carcinoma and sarcoma, Prostate carcinoma, Prostate adenocarcinoma, Lung metastasis, Cervical metastasis, Brain metastasis, Bone metastasis, Primary focus tumor, Cisplatin-resistant tumor, Uterine tumor, Uterine / Endometrial tumor, and glioblastoma multiforme. In particular, the therapeutic agent for treating tumors with high expression of the ITGAV membrane protein (Ensembl ID: ENSG00000138448.11), when compared to their corresponding non-tumor tissue.According to the GEPIA (Gene Expression Profiling Interactive Analysis) database, these tumors would be lymphoid neoplasm, diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, low-grade brain glioma, pancreatic adenocarcinoma, stomach adenocarcinoma, and thymoma, as shown in figures 9 to 11.

[0165] Process for obtaining the therapeutic agent defined above comprising the following steps: - obtain a modified nucleotide sequence as defined above; and - Add an excipient, adjuvant, and / or nanoparticles.

[0166] Method of treatment or prevention of genetic and degenerative conditions, cancer or infections or diseases caused by viruses, comprising administering to a subject in need thereof an effective amount of a therapeutic agent as defined above. Examples

[0167] The examples shown here are intended only to illustrate one of the numerous ways of carrying out the invention, however without limiting its scope. Example 1 - Production of the oncolytic virus

[0168] For the production of the oncolytic virus, the genome was divided into two Petition 870250015956, dated 26 / 02 / 2025, page 49 / 88 44 / 62 distinct plasmids (Figures 12 and 13). After obtaining the plasmids, the genomic information is fused using molecular biology techniques for subsequent sequencing of the constructs (Figures 12 and 14). In addition to the oncolytic virus RNA genome, the final construct has CMV and T7 promoters, as well as a poly-A tail.

[0169] The generation of a DNA construct containing the CMV promoter results in the feasibility of reducing steps in the production process. From the use of the CMV promoter in the production of the oncolytic virus, in vitro transcription to generate RNA is not necessary (Figure 12). Additionally, in vitro transcription and viral RNA generation were performed to generate active virus through electroporation (Figure 15).

[0170] The genetic material, obtained after combining the genome information, is transfected into Vero or HEK-293 cells using electroporation and / or lipofectamine methods (Figure 12). After transfection, the cells were incubated and monitored for a period of five days to observe cytopathic effects. After detecting cytopathic effects, the supernatant is collected and purified (Figure 12).

[0171] After the production and purification of the oncolytic virus, a serial dilution infection is performed in cells to select viruses with genetic homogeneity (clones). Five days after infection, the clones are selected, collected, and subjected to two rounds of infection to expand the clonal population. Five days after the last infection, the supernatant is collected for PCR, PFU, and RNA extraction for sequencing.

[0172] In one embodiment, the production process of SoziOZ is carried out using the product of fusion PCR, allowing for both DNA and RNA electroporation. This versatility demonstrates that the production method, using the product of fusion PCR, eliminates the need for bacteria in intermediate DNA cloning steps in the process, highlighting its difference and improved productivity compared to other methods. Petition 870250015956, dated 26 / 02 / 2025, page 50 / 88 45 / 62 traditional models described in the state of the art. Plasmid diagrams.

[0173] The plasmid design was performed using the complete ZIKV genome, divided into two regions: The first part contains the 5'UTR region up to the nucleotide sequence encoding the NS4A protein, cloned into a low-copy plasmid; and the second, encompassing the 2k region up to the 3'UTR of SoziOZ, cloned into a high-copy plasmid (Figure 13).

[0174] The pSoziOZ 1 plasmid contains not only the information necessary for the production of SoziOZ, but also the ribozyme, the MRE (miRNA Response Element) directed to the hsa-miR-124-3p miRNA, flanked by restriction enzyme sites in the 5'UTR region. Additionally, the plasmid includes the CMV and T7 promoters (Figure 13).

[0175] The pSoziOZ 2 plasmid contains not only the information necessary for the production of SoziOZ, but also the MRE (miRNA Response Element) for the miRNA hsa-miR-219a-2-3p, located within the nucleotide sequence encoding the 2K protein, as well as a ribozyme and a polyadenylation signal (Figure 13). To reconstruct the genome of the oncolytic virus (SoziOZ) after obtaining the plasmids, the genetic information contained in the pSoziOZ 1 and pSoziOZ 2 plasmids was joined by means of a fusion PCR (overlapping PCR). This process allowed the creation of a single DNA fragment containing the complete SoziOZ sequence, containing the CMV and T7 promoters (Figure 14). After the fusion PCR, the PCR product was purified and quantified. Next, agarose gel electrophoresis was performed to confirm amplification and generation of the single fragment of interest (Figure 14).

[0176] In agarose gel electrophoresis, a band was detected between the 15 kb and 10 kb standards, which is in accordance with the expected size of 11.8 kb for the single fragment (Figure 14). This result confirms that the fragment was successfully obtained after fusion PCR.

[0177] Immediately afterwards, in vitro transcription of the single fragment was performed to Petition 870250015956, dated 26 / 02 / 2025, page 51 / 88 46 / 62 generate RNA containing the information necessary for the production of SoziOZ (Figure 15). As observed in Figure 15, using non-denaturing agarose gel electrophoresis, it was possible to qualitatively verify the presence of RNA in the gel. Since the gel is non-denaturing, the band size is not expected to correspond exactly to the expected RNA size, as the RNA is folded into its secondary structure. However, it was possible to identify a mass concentration of RNA folded into its secondary structure between sizes of 4 and 3 kb. This result indicates that the single fragment, containing the complete SoziOZ sequence, was transcribed in a way that generated the expected product.

[0178] The final step consists of transfecting the RNA by electroporation into Vero or HEK-293 cells. This procedure allows the expression of the SoziOZ genetic sequence and, consequently, the production of the oncolytic virus (Figure 12).

[0179] The following describes the steps in the production of the oncolytic virus:

[0180] Candidate miRNA targets: a) In silico analysis of miRNA expression profile in normal tissue and tumor cells (focus on glioblastoma) (Figure 4). b) Design of the target MRE sequences of the selected miRNA and for the development of an insertion strategy into the iOZ Platform plasmids (SEQ ID No. 12 - 5' CGUGCCUUA 3' and SEQ ID No. 13 - 5' CCGACCUGU 3'). c) In silico analysis of pairing, binding strength, and annealing score of the MRE region drawn with the miRNA (Figure 16).

[0181] Plasmid drawings a) The plasmid design is based on the complete ZIKV genome divided into two regions: one from the 5'UTR to NS4A in a low-copy plasmid, and another from the 2k to 3'UTR region in a high-copy plasmid. b) pSoziOZ 1 with BsiWI insertion site flanking the MRE for the hsa-miR-124-3p miRNA in the 5'UTR region, containing the CMV and T7 promoters. Petition 870250015956, dated 26 / 02 / 2025, page 52 / 88 47 / 62 c) pSoziOZ 2 encoding from the 2k to the 3'UTR region, ribozyme and a polyadenylation signal.

[0182] Reconstruction of the modified virus genome: a) After obtaining the plasmids, immediate molecular biology is performed to join the fragments by overlapping PCR – a strategy to generate a single fragment containing the entire ZIKV sequence, optionally including T7 and / or CMV as promoters and / or a polyA tail. If necessary, after obtaining the plasmids, they undergo expansion, transformation, culture, and midi prep processes before the overlapping PCR step. Plasmid expansion Transformation of dH5-alpha bacteria with plasmids using a heat shock technique. Selection of transformants in selective solid culture medium (antibiotic). Expansion of colonies transformed into a liquid medium. Mini prep, mid prep, or max prep of the grow. DNA quantification by microdroplet spectrophotometry. Store the permanent colony in a -80°C freezer (Golden Clone). Evaluation of intact plasmids by agarose gel electrophoresis. Evaluation of plasmids by enzymatic restriction in agarose gel electrophoresis. Storage of selected plasmids. Disposal of unapproved permanent colonies. b) Purification of the PCR product by precipitation after electrophoresis on agarose gel to verify amplification. c) Quantification of genetic material. Petition 870250015956, dated 26 / 02 / 2025, page 53 / 88 48 / 62 d) Sequencing of constructions. Transfection of DNA material by electroporation and / or lipofectamine (Figure 12). (e) As an alternative, in vitro transcription is performed to generate and purify mRNA. f) Transfection by electroporation of RNA material (Figures 12 and 15).

[0183] Transfection of VERO and HEK cells: a) Cell culture of VERO and HEK according to the established protocol. b) On the day of the experiment, trypsinization and cell counting are performed. c) Aliquots of the required number of cells are made for each group. d) Complex DNA with lipofectamine, incubate. (e) Mix the DNA+lipofectamine complex, culture medium, and cells corresponding to each group. f) In the case of transfection of genetic material (DNA or RNA) by electroporation, mix the genetic material, cells, and specific buffer, and perform the shock following the instructions of the electroporation equipment manufacturer. g) Transfer the mixture to T25 bottles.

[0184] Cloning of the virus and limiting dilution to ensure the genetic homogeneity of the viral stock. a) Cell plating: b) Plate Vero cells in 96-well plates. c) Serial infection and dilution for clone selection: i. Addition of infection wire containing heterogeneous virus to the monolayer of previously plated cells, in a serial dilution format. Add the virus to the first eight wells, homogenize, and transfer the stipulated amount to the next eight wells, repeating the process successively. Petition 870250015956, dated 26 / 02 / 2025, page 54 / 88 49 / 62 d) Selection of clones and first reinfection: i. 5 days after infection, select the clones, collect them, and add them, according to the experimental design, to the monolayer of cells previously plated in a 12-well plate. e) Second reinfection: i. After 5 days, collect the clones, according to the experimental design, and add them to the monolayer of Vero cells previously plated in T25 flasks. ii. Collection for PCR, PFU, and RNA extraction for sequencing. iii. Storage of the produced clone stock. Example 2 - Passage effect for analysis of the reversal of the oncolytic effect.

[0185] After the production of the oncolytic virus clone, several consecutive passages of the virus are performed in order to analyze whether the passage effect causes a reversal of the virus's safety in relation to miRNA activity against targets present in the ZIKV genome.

[0186] For the experiment, Vero cells are cultured in 6-well plates. Infection with the oncolytic virus is performed in T25 culture flasks and, after 5 days, the supernatant is collected for re-infection in Vero cells. This process is repeated for 5 to 10 passages. At the end of the last passage, the supernatant is collected, and in-process control analytical tests are performed, in which viral titration / viral viability (PCR - need for RNA extraction in the sample - and TCID50) and viral modification by sequencing are performed, which comprises the extraction and sequencing of viral RNA from Sanger, Minion or NGS sequencing.

[0187] In addition, viral efficiency is analyzed by cytotoxicity assay, both in tumor and non-tumor cells, and by measuring the area and perimeter of spheres in NPCs. Example 3 - Viral efficiency test by analyzing cell viability and cytotoxicity: Petition 870250015956, dated 26 / 02 / 2025, page 55 / 88 50 / 62

[0188] At the end of the last pass, cell viability analysis is performed using a luminescence assay on the LN-18 tumor cell line.

[0189] An 8-point infection curve with a 1:3 dilution factor of the oncolytic virus is performed. Each point on the viral dilution curve has at least 6 LN-18 infection replicates in 96-well plates. Example 4 - In vitro assays with cell lines. Oncolytic selectivity (in vitro effectiveness assay)

[0190] The main objective of the in vitro study is to understand tumor selectivity and safety in non-tumor cell lines. For this, cytotoxicity / lysis and viral replication assays should be performed in both permissive and non-permissive cell lines to viral infection.

[0191] The main objective of the effectiveness study is to evaluate the selectivity of oncolytic therapy, that is, to detect viral preference for certain tumor subtypes in different cell lines, through the analysis of oncolytic potential — replication of its genetic material at high levels, which leads to lysis of the cell membrane.

[0192] For the experiment, commercial tumor and non-tumor cell cultures (Neural progenitor cells - NPC; Human cerebral microvessel as an endothelial cell model - hCMEC / D3; Human microglia - HMC3) are performed with plating of cells in 96-well plates. Infection with wild-type and modified virus is performed in a serial dilution, and after 5 days of infection, cell viability is analyzed by ATP quantification (luminescence assay) and cytotoxicity by neutral red lysosomal capture, both in tumor cells of different origins and stages. Example 5 - Selectivity of viral replication (in vitro cytotoxicity assays)

[0193] Validation of cytotoxicity includes confirmation of non-replication of the oncolytic virus in non-tumor cells, primarily neural progenitor cells (NPCs), a well-known target of ZIKV, and in non-tumor CNS cells. Non-tumor cell lines are cultured from plating of Petition 870250015956, dated 26 / 02 / 2025, page 56 / 88 51 / 62 cells in 96-well plates, infected in a serial dilution with wild-type and modified oncolytic virus, five days after infection the supernatant is collected in order to confirm viral replication by PFU and RT-PCR. Cell viability and viral cytotoxicity are also evaluated, analyzed by ATP quantification (luminescence assay) and neutral red lysosomal uptake in the 3 non-tumor cell lines mentioned. Example 6 - In vivo pharmacodynamic study

[0194] Female BALB / c nude mice are used for the study. Tumor cells are then injected into the right cerebral ventricle of the mice to induce tumor mass formation. Tumor formation is assessed within one week using an orthotopic metastatic model to ensure accurate tumor development.

[0195] After confirmation of brain tumor formation, mice are divided into separate groups for treatment administration. Wild-type ZIKV, SoziOZ, and a saline negative control are administered. Administration is performed via intraperitoneal (3 doses) and intracranial (1 dose) routes in distinct groups (Table 1). Table 1 Treatment Group | Number of Animals | 1- Negative Control | Saline | 5-15 | 2- Wild-type ZIKV Treatment | 5-15 | 3- SoziOZ Treatment | 5-15

[0196] Analyses are performed three weeks after treatment. Clinical symptoms, such as weight loss, ataxia, altered behavior, and signs of pain, are observed to monitor the health of the mice. Tumor progression is assessed weekly using IVIS (Small Animal Luminescence Imaging System) imaging, providing a clear view of tumor growth or remission of the tumor mass in animals that received virotherapy. Petition 870250015956, dated 26 / 02 / 2025, pp. 57 / 88 52 / 62

[0197] The survival of the mice is monitored throughout the experiment. Euthanasia is performed if there is confirmation of a 30% loss of initial body weight, the presence of an apparent tumor, or severe ataxia. After euthanasia, the brains of the mice are collected for histological analysis. The samples are stained with hematoxylin and eosin (H&E) to assess tissue structure and tumor extent, ensuring a detailed analysis of the treatment response. Example 7 - Obtaining the vaccine compound and testing

[0198] Virus production is preferably carried out in a way that takes advantage of the production steps of viral candidates and virus cloning and limiting dilution to ensure the genetic homogeneity of the viral stock, since these steps are similar for both strategies (whether oncolytic virus or vaccine).

[0199] Product purification processes a) Clarification by centrifugation and filtration (0.45 μm - 0.2 μm) b) Concentration in an ultrafilter for centrifuge. c) Purification with resin or chromatography filter. d) Analysis of material purity by chromatographic profile. e) Analysis of material purity by SDS-PAGE electrophoresis.

[0200] Viral inactivation with Formalin (0.05% at 22°C for 7 days): a) Alternatively, other viral inactivation conditions are also tested, including beta-prolactone, and other variables such as temperature and time. b) Removal of formalin by dialysis. Other methods for removing the viral inactivating chemical compound are optionally evaluated depending on the chemical product used. c) PFU inactivation analysis.

[0201] Preparation of the formulation containing adjuvant compound: Preferably, adjuvants based on aluminum salts are tested. Petition 870250015956, dated 26 / 02 / 2025, pp. 58 / 88 53 / 62 (aluminum hydroxide, aluminum phosphate or aluminum sulfate and phosphate) authorized for human use, or others authorized for human use, such as Monophosphoryl Lipid A (MPLA), saponins and others.

[0202] Immunization tests in a model animal: Administration of the vaccine compound. a) Maintaining animals in a vivarium. b) Collection of pre-immune serum from animals. c) Administration of the vaccine compound in various dose titers (e.g., 12, 3, 1 pg) via intraperitoneal or intramuscular injection. d) Clinical symptoms (weight loss, ataxia, behavioral changes, and pain). (e) Serum collection after 15 days and administration of the second dose. f) Serum collection 15 days after administration of the third dose.

[0203] Plate reduction neutralization test a) Culture of Vero cells in 12-well plates. b) Incubation of serum from tested animals with the target wild-type ZIKV. c) Addition of serum + wild-type ZIKV to the cell culture and incubation for 5 days. d) Staining with crystal violet and counting of cell plates. Example 8 - pZiOZ Drawing

[0204] For the design of pZiOZ, the pcDNA 3.1 (+ or -) plasmid containing the CMV and T7 promoters is created. pZiOZ comprises the 5' and 3' UTR of ZIKV. One of them is part of the capsid protein sequence, crucial for the formation of the secondary RNA structure, favoring its replication by the non-structural protein 5 (NS5). The other is composed of part of NS4A, the complete sequence of the 2K region, and part of the NS4B sequence, necessary for its encapsulation by the structural proteins of ZIKV. Example 9 - Production of the vaccine compound from pZiOZ

[0205] For antigen insertion into pZiOZ (pZiOZ-Ant), PCR amplification of the prME protein gene (as an example) is performed from the modified synthetic viral genome and the amplified DNA is purified. Optionally, the prME gene is modified to alter the coding sequence. Petition 870250015956, dated 26 / 02 / 2025, pp. 59 / 88 54 / 62

[0206] Next, the prME nucleotide sequence is inserted into pZiOZ using enzymatic restriction and T4 DNA ligase ligation techniques, the pZiOZ-prME plasmid is cloned into bacteria, and the plasmid is then sequenced.

[0207] The following steps were then followed:

[0208] Gene expression assessment: a) Cell culture. b) Obtaining mRNA in vitro. c) Transfection of cells with DNA using lipofectamine. d) Transfection of cells with mRNA using lipofectamine. e) Evaluation of prME expression by Western blot

[0209] Encapsulation of mRNA (ZiOZ) in lipid nanoparticles and evaluation of gene expression (nano-ZiOZ-Anti): a) Obtaining nanoparticles containing encapsulated mRNA. a. In vitro transcription with pseudouridine b) Cell culture. c) Obtaining mRNA in vitro. d) Transfection of cells with DNA. e) Transfection of cells with mRNA. f) Evaluation of prME expression by Western blot and ELISA.

[0210] Preparation of the formulation containing mRNA nanoparticles and adjuvant compound: Preferably, adjuvants based on aluminum salts (aluminum hydroxide, aluminum phosphate or aluminum sulfate and phosphate) authorized for human use, or others authorized for human use, such as Monophosphoryl Lipid A (MPLA), saponins and others, are tested.

[0211] Immunization tests in animal models: a) Maintaining animals in a vivarium. b) Administration of the vaccine compound: c) Collection of pre-immune serum from animals. d) Administration of the vaccine compound at various dose titers (e.g. Petition 870250015956, dated 26 / 02 / 2025, pp. 60 / 88 55 / 62 pg, 3 pg, 1 pg) via intraperitoneal or intramuscular injection. e) Clinical symptoms (weight loss, ataxia, behavioral changes, and pain). f) Serum collection after 15 days and administration of the second dose. g) Serum collection 15 days after administration of the third dose.

[0212] Plate reduction neutralization test: a) Culture of Vero cells in 12-well plates. b) Incubation of serum from tested animals with the target wild-type ZIKV. c) Addition of serum + wild-type ZIKV to the cell culture and incubation for 5 days. d) Staining with crystal violet and counting of cell plates. Example 10 - ZiOZ Experiments - Evaluation of CAP activity independently

[0213] From the plasmid (pZiOZ) containing the ZiOZ information, RNA is produced through in vitro transcription using a kit that does not add a CAP-like structure during the transcription process. The RNA is then transfected into Vero and HEK-293 cells. After 48 hours of transfection, the transfected cells are lysed to check the activity of the firefly luciferase enzyme.

[0214] Another group of cells transfected with ZiOZ is kept in culture for up to 7 days after transfection to monitor whether the firefly luciferase activity signal is maintained over time. At the end of the 7 days, the enzymatic activity is checked. In this way, it is possible to evaluate the effectiveness of the transfection and the stability of protein expression over different time intervals, providing valuable data for the development of future biotechnological applications. Example 11 - Production of the viral vector (EviOZ) - Plasmid design containing structural proteins and some non-structural proteins.

[0215] EviOZ production is achieved through the transfection of two plasmids (Figure 8). One is used for the production of structural proteins in a high-copy plasmid (pcDNA 3.1 as an example) containing the CMV and T7 promoters. This plasmid has 5' and 3' UTR regions that do not Petition 870250015956, dated 26 / 02 / 2025, pp. 61 / 88 56 / 62 correspond to those of ZIKV (beta-globin, for example) as one of the ways to avoid encapsulation of this RNA by EviOZ. Among the ZIKV sequences necessary for the production of EviOZ, those of the capsid, membrane and envelope proteins were included, crucial for the formation of VLPs (virus-like particles), in addition to some non-structural proteins that assist in the production of EviOZ.

[0216] EviOZ production involves: a) Co-transfection of HEK-293 cells using the plasmid required for production of the EviOZ VLP structure along with the pZiOZ containing luciferase as the gene of interest (pZiOZ-LUC). b) Collection of the supernatant 72 hours after transfection. c) Centrifugation to remove cellular debris. Example 12 - Characterization of the viral vector (EviOZ)

[0217] Quantification of genetic material encapsulated by EviOZ by ddPCR: a) Design of primers and probes for pZiOZ-LUC b) Preparation of the viral vector sample. c) Preparation of the master mix. d) Preparation of the reaction. e) Quantification by ddPCR. f) Data analysis and plotting.

[0218] Structural characterization of EviOZ by transmission electron microscopy (TEM): a) Addition of viral vector sample and wild-type ZIKV onto carbon-coated TEM grid. b) Addition of uranyl acetate dye. c) Observation of the samples under an electron microscope. d) Analysis of the morphology and size of viral vector particles compared to wild-type ZIKV. Example 13 - Validation of viral vector production

[0219] First, co-transfection of HEK-293 cells is performed using two Petition 870250015956, dated 26 / 02 / 2025, pp. 62 / 88 57 / 62 plasmids: one containing the sequence of structural proteins along with some non-structural proteins, and the other containing the pZiOZ containing the luciferase reporter gene (ZiOZ-GFP / LUC), as an example of a gene of interest. After 72 hours, the supernatant is collected from the transfected cells. The supernatant is then analyzed by RT-qPCR or digital PCR to detect the presence of ZiOZ-GFP / LUC in the produced viral vector.

[0220] To verify the ability of the viral vector to deliver RNA (ZiOZGFP / LUC), the supernatant containing the EviOZ viral vector encapsulating ZiOZGFP / LUC is incubated with Vero and HEK-293 cells. After 24 and / or 48 hours of viral vector delivery to the target cells, luminescence is analyzed. A luciferase assay is performed, as ZiOZ-GFP / LUC contains the coding sequence for the Firefly luciferase enzyme. Therefore, when delivery and expression of the enzyme are observed in these cells after exposure to the viral vector, it confirms that the viral vector was produced and was able to encapsulate and deliver ZiOZ-GFP / LUC to host cells. In addition, cell lysate from Vero cells is analyzed to verify ZiOZ-GFP / LUC delivery using RT-qPCR or digital PCR. This methodology allows validating the efficiency of therapeutic RNA delivery to target cells. Example 14 - Evaluation of the minimum sequence required to encapsulate ZiOZ

[0221] To assess the possible presence of other sequences in the ZIKV genome that may assist in the encapsulation of ZiOZ-GFP / LUC, overlapping 1000bp segments of the ZIKV genome are generated and individually inserted into ZiOZ-GFP / LUC. To generate the viral vector, HEK-293 cells are co-transfected using two plasmids: one containing the structural sequences and the other containing ZiOZ-GFP / LUC. After 72 hours of incubation, the supernatant from the transfected cells is collected.

[0222] To verify if the generated viral vector is capable of encapsulating ZiOZGFP / LUC with ZIKV sequence segments, the supernatant is added to Vero cells. This is done 24 hours and / or 48 hours after vector delivery. Petition 870250015956, dated 26 / 02 / 2025, pp. 63 / 88 58 / 62 viral in target cells, the luminescence of the cells is analyzed, since ZiOZ-GFP / LUC contains the coding sequence of the Firefly luciferase enzyme. The presence and expression of this enzyme in Vero cells indicate that the viral vector was produced correctly and is able to encapsulate and deliver ZiOZ-GFP / LUC efficiently. Example 15 - Evaluation of the in vitro tropism of the viral vector

[0223] Tests are conducted on various cell lines in cell culture to determine which ones are susceptible to EviOZ.

[0224] The viral vector is added to the cells and incubated at 37°C in an atmosphere with 5% CO2. If the viral vector has the ability to enter the cell lines, the RNA with the reporter gene encoding the Luciferase enzyme is delivered and translated, being detected by luminescence assay after 24 and / or 48 hours of incubation. In addition to luminescence analysis, an RT-qPCR analysis is performed to verify the presence of ZiOZGFP / LUC RNA after contact with the viral vector. Example 16 - Evaluation of the in vivo tropism of the viral vector

[0225] For the evaluation of in vivo delivery of the viral vector, in vivo tracking is performed using bioluminescence imaging. This assay shows the biodistribution of EviOZ after its administration by different routes of administration and verifies the tropism of the viral vector for the central nervous system and other tissues compared with a saline group.

[0226] In the assay, BALB / c or C57BL / 6 hSTAT2 mice (7 weeks old, 18g to 25g) are used, with one group receiving EviOZ and the other receiving saline solution. The routes of administration vary between intraperitoneal and intravenous for both groups. Bioluminescence activity is evaluated at 0 days, 1 day, 2 days, and 7 days after EviOZ administration. Luciferase activity is measured after luciferin injection, with luminescence analysis performed using the IVIS®-Spectrum system. Quantification of luminescence over time allows observation of viral vector distribution and EviOZ's preference for specific organs. Petition 870250015956, dated 26 / 02 / 2025, pp. 64 / 88 59 / 62 based on photon emission in different regions of the animals. In addition, organs are collected for RNA extraction and quantification by RT-qPCR. Example 17 - Production of Viral Vector for Phelan-McDermid Syndrome Therapy

[0227] Insertion of the SHANK3 gene into pZiOZ: a) Cloning of the plasmid containing the SHANK3 gene. b) Insertion of the SHANK3 gene into pZiOZ using enzymatic restriction and T4 DNA ligase. c) Cloning of the pZiOZ-SHANK3 plasmid in bacteria. d) Plasmid sequencing.

[0228] Gene expression assessment: a) Cell culture. b) Transfection of cells with DNA. c) Transfection of cells with mRNA. d) Evaluation of SHANK3 expression by Western blot and ELISA.

[0229] Development of a viral vector (EviOZ) that encapsulates ZiOZ-SHANK3: a) Co-transfection of plasmids for VLP and pZiOZ-SHANK3. b) Collection of the supernatant. c) Vero cell transduction. d) Evaluation of SHANK3 expression by Western blot and ELISA.

[0230] In vivo validation of SHANK3 expression in mouse brains: a) Administration of the viral vector for SHANK3 in mice. b) Brain collection. c) Immunofluorescence analysis with anti-SHANK3 antibodies. d) Analysis of total soluble protein expression obtained from mouse brains. a. Analysis by Western blot and quantitative ELISA.

[0231] Obtaining and maintaining a model animal: Shank animal model 3fx / fx:CreER+ / - a) Obtaining the SHANK3fx / fxe CAGGs-CreER + strains Petition 870250015956, dated 26 / 02 / 2025, pp. 65 / 88 60 / 62 b) Embryo recovery. c) Crossing to obtain SHANK3fx / fx:CreER+ / - d) Maintaining animals in a vivarium.

[0232] Gene Therapy in animal models: 1. Maintenance in animal vivarium SHANK3fx / fx:CreER+ / - 2. Administration of the ZiOZ-SHANK3 encapsulating viral vector in 3-week-old (young) and / or 3.5-month-old (adult) animal models. 3. Behavioral tests: a) Elevated labyrinth. b) Locomotor activity for creation. c) Dark / light test. d) Rotary bar test. e) Open field test. f) Social interaction tests. g) Test for social behavior. 3-chamber test. h) Morris Water Maze Spatial Learning and Memory Test Example 18 - Production of a Viral Vector for gene editing based on CRISPR technology

[0233] Insertion of the CRISPR Cas12 machinery into pZiOZ: The basic composition of the CRISPR Cas12 machinery contains the pZiOZ backbone, including sequences necessary for Cas12 expression, a region for stabilizing mRNA after cleavage by Cas12 (triplex or ZIKV 3'UTR), a region containing single or multiplex crRNA flanked by the Direct Repeat (DR) sequence. • CMV Promoter / Enhancer • T7 Promoter • Hammerhead ribozyme • Cap15 • P2A Petition 870250015956, dated 26 / 02 / 2025, pp. 66 / 88 61 / 62 • AsCpfl (Cas-12 gene) • Nucleoplasmin (NLS) • Triplex region or ZIKV 3'UTR • Direct repeat sequence (DR) • crRNA sequence (Luciferase target) • Direct repeat sequence (DR) • 3'UTR • HDV Ribozyme • BGH poly(A) signal

[0234] Obtaining the pZiOZ-CRISPR construct: a) Synthesis of the pZiOZ-CRISPR plasmid. b) Plasmid cloning. c) Plasmid sequencing.

[0235] Evaluation of Cas12 gene expression: a) Cell culture. b) In vitro transcription of mRNA. c) Transfection of cells with DNA. d) Transfection of cells with mRNA. e) Evaluation of Cas12 expression by Western blot.

[0236] Evaluation of luciferase knockout in cells with constitutive luciferase expression: a) Cell culture. b) Transfection of cells with DNA and lipofectamine, and negative control. c) Transfection of cells with mRNA and lipofectamine, and negative control. d) Luciferase assay after 24 and / or 48 hours of incubation. e) RT-PCR or sequencing of the edited region in the cellular genome.

[0237] Development of a viral vector (EviOZ) that encapsulates ZiOZ-CRISPR a) Co-transfection of plasmids for VLP and pZiOZ-CRISPR. b) Collection of the supernatant. c) Transduction of constitutive luciferase cells. Petition 870250015956, dated 26 / 02 / 2025, pp. 67 / 88 62 / 62 d) Evaluation of Cas12 expression by Western blot. e) Luciferase assay after 24 and / or 48 hours of incubation. f) RT-PCR or sequencing of the edited region in the cellular genome.

[0238] Those skilled in the art will appreciate the knowledge presented here and may reproduce the invention in the forms presented and in other variants and alternatives, covered by the scope of the following claims. Petition 870250015956, dated 26 / 02 / 2025, pages 68 / 88

Claims

1 / 4 Claims 1. Modified flavivirus nucleotide sequence characterized by comprising at least three nucleotide sequences functionally linked together in any order of linkage thereof, selected from: (i) the nucleotide sequence of part of the coding sequence of the Zika virus NS4A protein (Seq ID No. 1); (ii) the complete sequence of the Zika virus 2K protein (Seq ID No. 2); and (iii) the nucleotide sequence of part of the coding sequence of the Zika virus NS4B proteins (Seq ID No. 3).

2. Modified flavivirus nucleotide sequence according to claim 1, characterized by additionally comprising the nucleotide sequence of part of the coding sequence of the Zika virus Capsid C protein (Seq ID No. 4).

3. Modified flavivirus nucleotide sequence according to claim 1, characterized by further comprising: - the nucleotide sequence of part of the coding sequence of the Zika virus NS5 protein (Seq ID No. 5) functionally linked to 3'; - the nucleotide sequence of part of the coding sequence of the Zika virus NS3 protein (Seq ID No. 6) functionally linked to 5'; - the nucleotide sequences of part of the coding sequences of the ZIKV C, M, E, NS1, NS2A, NS2B proteins (Seq ID Nos. 29, 7, 8, 10, 11 and 12 respectively), functionally linked to each other from 5' to 3'; - optionally, it comprises the nucleotide sequences 5'UTR (Seq ID No. 14) and MRE (Seq ID No. 12, as a target of miR-124-3p) functionally linked to each other and to 5'; - optionally, it comprises the nucleotide sequences MRE (Seq ID No. 13, as a target of miR-219a-2-3p) in the 2K protein (Seq ID No. 2).

4. Nucleotide sequence according to any of the preceding claims characterized by additionally comprising other nucleotide sequence(s) of up to 10 kb, encoding functional proteins. Petition 870250015956, dated 26 / 02 / 2025, p. 69 / 88 2 / 4 5. Modified flavivirus nucleotide sequence according to claim 4, characterized by further comprising: - the nucleotide sequences of part of the coding sequences of the GFP and Luciferase proteins (SEQ ID NO 17 and 18, respectively), functionally linked to each other from 5' to 3'; or combinations thereof.

6. Modified flavivirus nucleotide sequence according to claim 4, characterized by further comprising: - the nucleotide sequences of part of the coding sequences of the Shank3, IRES and NS5 proteins (SEQ ID No. 19, 20 and 5 respectively), functionally linked together from 5' to 3'; or combinations thereof.

7. Modified flavivirus nucleotide sequence according to claim 4, characterized by further comprising: - the nucleotide sequences of part of the coding sequences of Cas 12 proteins (Seq ID No. 21) functionally linked to 5'; - the nucleotide sequences of part of the coding sequences of Pré crRNA proteins (Seq ID No. 22) functionally linked to 3'; or combinations thereof.

8. Use of the nucleotide sequence as defined in any of claims 1-7 characterized in being for preparing a medicament or therapeutic agent for the treatment or prevention of genetic and degenerative conditions, including Phelan-McDermid Syndrome, Autism Spectrum Disorders, Alzheimer's and Parkinson's Disease, Tourette Syndrome, syndromic autism, various genetic diseases, or for the treatment or prevention of flaviviruses, yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), and dengue virus (DENV).

9. Therapeutic agent characterized by comprising an excipient and / or adjuvant and the nucleotide sequence as defined in any of claims 1-7.

10. Therapeutic agent according to claim 9 characterized in that said nucleotide sequence is encapsulated in a lysosome or nanoparticle, for use as a vaccine.

11. Therapeutic agent according to claim 9 characterized in that said nucleotide sequence additionally comprises the CMV and / or T7 promoter and poly A tail, for use as a viral vector.

12. Therapeutic agent according to claim 9 characterized in that it comprises a modified Zika virus oncolytic virus, wherein the genome comprises: the hsa-miR-124-3p nucleotide sequence between the 5'UTR regions and the capsid protein coding region (C); and the hsa-miR-219a-2-3p nucleotide sequence within the 2K protein sequence.

13. Use of oncolytic virus as defined in claim 12 characterized in being for preparing a medicament or therapeutic agent for treating cancer, including cancer selected from the group comprising tumors of brain origin, Glioblastoma, Medulloblastoma, Atypical teratoid rhabdoid tumor (TTRA), Embryonic CNS tumor (pediatric), CNS tumor (adult), Breast cancer, Luminal cancer, Triple-negative tumor, Colon tumor, Colorectal tumor, Colorectal carcinoma, Mucoepidermoid lung carcinoma, Lung carcinoma (activated fatty acid metabolic pathways), Malignant melanoma, Ovarian cancer, Sarcoma-resistant tumor, ER+PR+ adenocarcinoma, Colorectal adenocarcinoma, Pancreatic adenocarcinoma, Thyroid carcinoma and sarcoma, Prostate carcinoma, Prostate adenocarcinoma, Pulmonary metastasis, Cervical metastasis, Brain metastasis, Bone metastasis, Primary focus tumor, Cisplatin-resistant tumor, Uterine tumor, Uterine / Endometrial tumorGlioblastoma multiforme and tumors with high expression of the ITGAV membrane protein.

14. Process for obtaining the therapeutic agent defined in any one of claims 9-12 characterized by comprising the steps of: Petition 870250015956, dated 26 / 02 / 2025, page 71 / 88 4 / 4 - obtaining a modified nucleotide sequence as defined in one of claims 1-7; and - adding an excipient, adjuvant, nanoparticles and / or liposome.

15. Process according to claim 14 characterized in that the step of obtaining said nucleotide sequence comprises the construction of two plasmid DNAs.

16. A method for treating or preventing genetic and degenerative conditions, cancer, or infections or diseases caused by viruses, characterized by comprising administering to a subject in need thereof an effective amount of a therapeutic agent as defined in any of claims 9-12. Petition 870250015956, dated 26 / 02 / 2025, pp. 72 / 88

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