Modified flavivirus nucleotide sequence, use of the nucleotide sequence, therapeutic agent, use of an oncolytic virus, viral vector, process for obtaining same and method of treatment

Abstract

The present invention relates to the fields of synthetic biology, molecular biology, biotechnology, nanotechnology, pharmacology, vaccinology, medicine, virology and oncology. More specifically, the invention provides a modified flavivirus nucleotide sequence, a therapeutic agent, a viral vector and a process for obtaining same, and a method of treatment using said sequence, agent or vector. The invention is associated with the "iOZ platform", which harnesses the potential of the viral genome of flaviviruses – in particular the African strain of the Zika virus (ZIKV) – to provide advanced therapeutic approaches, whilst also providing targeted and effective treatments for complex diseases, including the treatment of malignant tumours and other types. In one embodiment, the therapeutic agent of the invention is a viral vector for gene therapy. In one embodiment, the therapeutic agent of the invention is a non-natural RNA sequence, suitable for use in vaccines and / or gene therapy. One embodiment of the therapeutic method of the invention is oncolytic therapy. Another embodiment of the therapeutic method of the invention is gene therapy based on use of the modified virus, the viral vector or the modified RNA of the invention.

Description

[0001] Nucleotide Sequence of Modified Flavivirus, Use of the Nucleotide Sequence, Therapeutic Agent, Use of Oncolytic Virus, Viral Vector, Obtaining Process and Treatment Method

[0002] Field of Invention

[0003] [1] 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 chimeric 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 invented therapeutic method is an oncolytic therapy. Another embodiment of the invented therapeutic method 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.

[0004] Background of the Invention

[0005] [2] 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

[0006] REPLACEMENT SHEET (RULE 26) for conventional medications to cross the blood-brain barrier (BBB).

[0007] [3] 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.

[0008] [4] 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 BBB limits the effective reach of most traditional and modern drugs and therapies available to patients.

[0009] [5] 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. There is therefore a need in the field for new, more effective and less aggressive therapies. The present invention provides an alternative to these problems of current therapies, meeting the needs mentioned above.

[0010] [6] Glioma stem cells (GSCs) have self-renewal, tumorigenic and differentiation potential, and are therefore similar to cells

[0011] REPLACEMENT SHEET (RULE 26) 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.

[0012] [7] 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). In addition, clinical studies in patients with PMS 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.

[0013] [8] Autism Spectrum Disorders (ASD) have been associated with several mutations in the DEAF1 gene, including missense, frameshift, and indel variants. These mutations give rise to two distinct intellectual disability (ID) syndromes: the autosomal dominant Vulto-van Silfhout-de Vries (VSVS) syndrome and the neurodevelopmental recessive condition (NEDHELS). Both conditions are clinically classified as DEAF1-associated neurodevelopmental disorders (DAND), caused by loss of function or dominant-negative effects of the DEAF1 transcription factor. Currently, there are no effective treatments available for DAND.

[0014] [9] 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 concept

[0015] The fundamental aspect of oncolytic virotherapy is treating 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.

[0016]

[0010] Viral organisms (VOs) 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 VOs 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 VOs.

[0017]

[0011] 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.

[0018]

[0012] 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.

[0019] REPLACEMENT SHEET (RULE 26)

[0013] 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.

[0020]

[0014] 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. Furthermore, viral vectors can also be used to assist in the treatment of tumors. In the context of rare diseases, viral vectors offer new hope for patients who often lack effective treatment options. These diseases are generally 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.

[0021]

[0015] 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.

[0022]

[0016] 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.

[0023] REPLACEMENT SHEET (RULE 26) Therefore, viral vectors designed for this purpose can take advantage of greater effectiveness delivery when compared to other methods.

[0024]

[0017] 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 genetic payload delivery capacity, low tropism for the CNS, adverse effects, and make genomic transgene integration unfeasible, 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 genetic payload delivery capacity, and without genomic integration. The present invention provides a solution to these problems.

[0025]

[0018] The ZIKV-based viral vector platform features 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.

[0026]

[0019] 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 pre-crRNA mediated by Cas12 into mature crRNAs, as well as the recognition and cleavage of

[0027] SUBSTITUTION SHEET (RULE 26) Target DNA is controlled by base pairing between spacer and target sequences.

[0028]

[0020] 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.

[0029]

[0021] 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® (G47A; teserpaturev), a modified Herpes virus by the company Daiichi-Sankyo, approved in Japan for the treatment of glioblastoma. However, neither of these oncolytic therapies exhibits natural tropism for the CNS and oncolysis.

[0030]

[0022] Regarding viral vectors, 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.

[0031] REPLACEMENT SHEET (RULE 26)

[0023] 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.

[0032]

[0024] The use of AAV-based viral vectors is limited by their lower genetic material transport capacity compared to other viral vectors and by the high inflammatory process triggered after local or systemic administration (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.

[0033]

[0025] 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.

[0034]

[0026] 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...

[0035] REPLACEMENT SHEET (RULE 26) a polyprotein, approximately 10 kb, enabling ZIKV to carry therapeutic genes of up to 10 kb, and thus be advantageous compared to the use of AAVs for CNS disorders.

[0036]

[0027] 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).

[0037]

[0028] 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 congenital malformations in newborns, while ZIKV infection in children and adults is linked to Guillain-Barré syndrome, neuropathy, and myelitis.

[0038]

[0029] 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.

[0039]

[0030] 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) and the yellow fever-transmitted encephalitis virus.

[0040] REPLACEMENT LEAF (RULE 26) for ticks (TBEV), Japanese encephalitis virus (JEV) and dengue virus (DENV).

[0041]

[0031] 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.

[0042]

[0032] mRNA vaccines are based on a synthetic mRNA molecule that 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.

[0043]

[0033] 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

[0044] REPLACEMENT SHEET (RULE 26) humoral, cellular and viral neutralization immune response (virus vaccines).

[0045]

[0034] 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.

[0046]

[0035] 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.

[0047]

[0036] In the search for the state of the art in scientific and patent literature, the following documents were found that deal with the subject:

[0048]

[0037] Document WO2023164441 A1 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 encapsulating RNA within the envelope structure of the viral vector, but does not provide conclusive results to support this claim. The present invention does not utilize 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 central nervous system cells, and may replicate in the liver and / or require very high doses, which can lead to cytopathic effects.These products, therefore, still require further research to solve these problems and achieve the desired effect.

[0049] REPLACEMENT SHEET (RULE 26)

[0038] The present invention by iOZ does not make use of this approach, in addition to solving these and other problems.

[0050]

[0039] 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.

[0051]

[0040] Based on what can be inferred from the literature reviewed, no documents were found anticipating or suggesting the teachings of the present invention, so the solution proposed here has novelty and inventive activity compared to the state of the art.

[0052] Summary of the Invention

[0053]

[0041] The present invention solves the problems of the prior art by providing a chimeric 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.

[0054]

[0042] In a concrete example, Figure 1A illustrates the topology of the three functionally connected sequences (NS4A-2K-NS4B) (SEQ ID Nos. 1, 2 and 3) 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).

[0055]

[0043] Thus, one of the objects of the invention is a non-natural and chimeric nucleotide sequence, genetically modified from flaviviruses, preferably

[0056] ZIKV SUBSTITUTION SHEET (RULE 26). The chimeric 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 shown 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).

[0057]

[0044] In one embodiment, the chimeric flavivirus nucleotide sequence additionally comprises the nucleotide sequence of part of the modified sequence encoding the Zika virus Capsid C protein (Seq ID No. 4).

[0058]

[0045] 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 more efficiently only in cells where specific miRNAs are poorly expressed, i.e., tumor cells, promoting greater safety, specificity, and oncolytic power. In this embodiment, said nucleotide sequence further comprises a linkage order corresponding to: (i) the MRE nucleotide sequence (5' CGUGCCUUA 3', 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 the 3' nucleotide sequence of the E protein (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 nucleotide sequence of the MRE (5' CCGACCUGU 3', as a target of miR-219a-2-3p) between nucleotides 12 and 13 of the nucleotide sequence of the 2K protein.

[0059] (v) the complete nucleotide sequence of the ZIKV NS4B protein (Seq ID No. 62) through 5' and 3' flanking restriction enzyme sites; and (v) the complete nucleotide sequence of the ZIKV NS4B protein (Seq ID No.

[0060] 28), functionally linked to the 5' end of the complete NS5 nucleotide sequence (Seq ID No. 5) described in Figure 3.

[0061]

[0046] In one embodiment, the chimeric flavivirus nucleotide sequence additionally comprises:

[0062] - the nucleotide sequence encoding the Zika virus NS5 protein (Seq ID No. 5) functionally linked to 3';

[0063] - the nucleotide sequence encoding the Zika virus NS3 protein (Seq ID No. 6) functionally linked to 5';

[0064] - 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';

[0065] - the nucleotide sequences 5'UTR (Seq ID No. 14) and MRE (5' CGUGCCUUA 3', as a target of miR-124-3p) functionally linked to each other and to 5';

[0066] - the nucleotide sequences MRE (5' CCGACCUGU 3', 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.

[0067]

[0047] In one embodiment, the nucleotide sequence additionally comprises another nucleotide sequence(s) of up to 10 kb, encoding(s) functional proteins.

[0068]

[0048] 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.

[0069]

[0049] Thus, in one embodiment, the chimeric flavivirus nucleotide sequence additionally comprises:

[0070] - the nucleotide sequences of part of the coding sequences of the GFP and Luciferase proteins (SEQ ID NO 17 and 18, respectively),

[0071] REPLACEMENT SHEET (RULE 26) functionally linked to each other from 5' to 3'; or combinations thereof.

[0072]

[0050] In one embodiment, in Figure 1, in C) the same sequence as in A) is shown 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 SHANK3, IRES and NS5 sequences are functionally linked to the 5' of the NS4A-2K-NS4B sequence.

[0073]

[0051] In one embodiment, Figure 1 shows the same sequence as A) additionally comprising an example of the use of the DEAF1 monomer transcription factor coding sequence (Seq ID No. 52) (Figure 1, in D) and DEAF1 dimer (Seq ID No. 54) (Figure 1, in E) functionally linked to the IRES nucleotide sequence and the ZIKV NS5 protein coding sequence (Seq ID No. 5), both DEAF1 monomer and DEAF1 dimer sequences, IRES and NS5 are functionally linked to the 5' of the NS4A-2K-NS4B sequence.

[0074]

[0052] Thus, in one embodiment, the chimeric flavivirus nucleotide sequence additionally comprises:

[0075] - 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 to each other from 5' to 3'; or combinations thereof.

[0076] - the nucleotide sequences of part of the coding sequences of the DEAF1 monomer and DEAF1 dimer transcription factor, IRES and NS5 (SEQ ID NO 19, 20 and 5 respectively), functionally linked to each other from 5' to 3'; or combinations thereof.

[0077]

[0053] In one embodiment, in Figure 1, F) shows the same sequence as in A), additionally 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, both being functionally linked to the 5' of the NS4A-2K-NS4B sequence. The 3' of the NS4A-2K-NS4B sequence is functionally linked to the nucleotide sequence of a

[0078] SUBSTITUTION SHEET (RULE 26) non-coding region of protein and the nucleotide sequence of Pre-crRNA.

[0079]

[0054] Thus, in one embodiment, the chimeric flavivirus nucleotide sequence additionally comprises:

[0080] - the nucleotide sequences of part of the coding sequences of Cas 12 proteins (Seq ID No. 21) functionally linked to 5';

[0081] - 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.

[0082]

[0055] 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).

[0083]

[0056] 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.

[0084]

[0057] 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'.

[0085]

[0058] 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).

[0086]

[0059] 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 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),

[0087] SUBSTITUTION SHEET (RULE 26) tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and dengue virus (DENV). In one embodiment, the therapeutic agent comprises said unnatural and chimeric nucleotide sequence.

[0088]

[0060] 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.

[0089]

[0061] 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 complementary nucleotide sequence of hsa-miR-124-3p between the 5'UTR regions and the capsid protein coding region (C); and the complementary nucleotide sequence of hsa-miR-219a-2-3p within the 2K protein sequence.

[0090]

[0062] 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 chimeric nucleotide sequence of ZIKV, having a natural tropism for the central nervous system, acting specifically on tumor cells, promoting their selective destruction, and stimulating the

[0091] REPLACEMENT SHEET (RULE 26) host immune response. In one embodiment, the therapeutic agent of the invention is a viral vector for gene therapy.

[0092]

[0063] In one embodiment, the aforementioned viral vector contains a chimeric 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.

[0093]

[0064] 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.

[0094]

[0065] 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

[0095] REPLACEMENT SHEET (RULE 26) targets 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.

[0096]

[0066] 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.

[0097]

[0067] Another object of the invention is the process for obtaining the therapeutic agent comprising the steps of:

[0098] - to obtain a chimeric nucleotide sequence; and

[0099] - Add an excipient, adjuvant, nanoparticles and / or liposome.

[0100]

[0068] In a concrete implementation of the process, the step of obtaining the aforementioned nucleotide sequence comprises the construction of two plasmid DNAs.

[0101]

[0069] 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.

[0102] REPLACEMENT SHEET (RULE 26)

[0070] 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 neurological mutations and diseases than congeneric approaches.

[0103]

[0071] 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.

[0104]

[0072] 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.

[0105]

[0073] The invention in question presents an mRNA vaccine that can or

[0106] The replacement sheet (Rule 26) must not be delivered in "naked" form (encased in lipid nanoparticles or liposomes) or with the ZIKV envelope. Furthermore, the mRNA described here does not require the addition of a CAP, an advantage over current mRNA vaccines, making the production process more efficient and less costly, and the treatment more effective. This makes a rapid and efficient approach viable for the production of vaccines against parasites and pathogens such as viruses, viroids, bacteria, fungi, protozoa, and prions.

[0107]

[0074] 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.

[0108]

[0075] 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.

[0109] Brief Description of the Figures

[0110]

[0076] The following figures are presented:

[0111]

[0077] Figure 1 shows a schematic representation of the chimeric 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). In addition, 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 shown, additionally comprising a

[0112] SUBSTITUTION SHEET (RULE 26) 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. The 5' of the nucleotide sequences encoding reporter proteins is functionally linked to the nucleotide sequence of part of the coding sequence of protein C (Seq ID No. 4); In C) the same sequence as in A) is shown additionally 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' of the NS4A-2K-NS4B sequence. The 5' end of the SHANK3, IRES, and NS5 sequences is functionally linked to the nucleotide sequence of part of the coding sequence of protein C (Seq ID No.4); In D) the same sequence as in A) is shown additionally 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, both being functionally linked to the 5' of the NS4A-2K-NS4B sequence. The 5' 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' of the NS4A-2K-NS4B sequence are functionally linked to the nucleotide sequences of a non-protein coding region and the nucleotide sequence of the Pre-crRNA; In E) the same sequence as in A) is shown additionally comprising the nucleotide sequences encoding the C, M, E, NS1, NS2A, NS2B, NS3 and NS4A proteins of ZIKV (Seq ID Nos.29, 7, 8, 10, 11, 9, 27 respectively), functionally linked to each other from 5' to 3' and linked to 5' of the NS4A-2K-NS4B sequence, with overlap of the complete nucleotide sequence of the NS4A protein with the NS4A-2K-NS4B sequence. At 3' of the NS4A-2K-NS4B sequence, there are functional links 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. 5), with overlap of the...

[0113] SUBSTITUTION SHEET (RULE 26) complete nucleotide sequence of the NS4B protein with the sequence NS4A-2K-NS4B; Figure 2 shows a schematic illustration of some of the embodiments of the invention (SoziOZ, EviOZ and ZiOZ) and their relationship to the iOZ platform, which uses information derived from the viral genomic sequence.

[0114]

[0078] Figure 3 shows a schematic representation of the chimeric nucleotide sequence of the invention in one of its oncolytic virus embodiments. Highlighted is the first exogenous sequence, located in the 5'UTR, corresponding to the MRE of miRNA 124-3p. The second exogenous sequence is located within the 2K protein sequence and corresponds to the MRE of miRNA 219a-2-3p. Both exogenous sequences together confer greater specificity, safety, and greater oncolytic power in tumor cells, with high replication and release of active virus after infection.

[0115]

[0079] 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.

[0116]

[0080] 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.

[0117]

[0081] 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 virus.

[0118] REPLACEMENT SHEET (RULE 26) chimeric.

[0119]

[0082] 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.

[0120]

[0083] Figure 8 shows the pZiOZ plasmid used for the production of the viral vector (EviOZ).

[0121]

[0084] 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.

[0122]

[0085] 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.

[0123]

[0086] 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 of certain types of tumor or normal tissue.

[0124]

[0087] Figure 12 shows the schematic of the production process of the oncolytic virus (SoziOZ) 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 the production of SoziOZ. After the fusion PCR, the resulting DNA fragment can be directly electroporated into cells or, alternatively, the PCR product can be transcribed in vitro to generate RNA, followed by electroporation of this RNA.

[0125] REPLACEMENT SHEET (RULE 26) 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.

[0126]

[0088] 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.

[0127]

[0089] 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).

[0128]

[0090] 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).

[0129]

[0091] Figure 16 shows the sequence alignment of the hsa-miR-124-3p 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).

[0130]

[0092] Figure 17 shows photographic records (10x) of all wells with USP07 cell line, three replicates / well per group, 3 days post-infection (dpi) at MOI 0.1, with Bar: 100 px = 100 pm.

[0131]

[0093] Figure 18 shows the result of the titration of the potency test samples, by RT-PCR, from the number of copies / pL using primer of

[0132] REPLACEMENT SHEET (RULE 26) region of the Envelope, beginning of the sequence. Samples are presented as biological replicates, with each bar originating from each well of a 6-well plate. Where * = p<0.1, ** = p<0.01, *** = p<0.001 and **** = p<0.0001.

[0133]

[0094] Figure 19 shows the result of titration of the potency test samples by RT-PCR, based on the number of copies / pL using primer from the NS5 region, at the end of the sequence. The samples are presented as biological replicates, with each bar originating from each well of a 6-well plate. Where * = p<0.1, ** = p<0.01, *** = p<0.001 and **** = p<0.0001.

[0134]

[0095] Figure 20 shows the restriction site of the BamHI, Xbal, and EcoRI enzymes in the pCME, pZiOZ, pZiOZ_CRISPR, and pZiOZ EcoRI plasmids (without NS4 and NS5). Enzymes that are not annotated in each plasmid do not recognize the plasmid DNA sequence.

[0135]

[0096] Figure 21 refers to the Gel showing the enzymatic restriction of pCME and pZiOZ plasmids with the enzymes BamHI and Xbal (channels 1 to 13), 1 Kb Plus DNA Ladder (channel 14).

[0136]

[0097] Figure 22 refers to the gel showing the enzymatic restriction of the pCME, pZiOZ and pZiOZ_CRISPR plasmids with the BamHI, Xbal and EcoRI enzymes (lanes 1 to 13), 1 Kb Plus DNA Ladder (lane 14).

[0137]

[0098] Figure 23 refers to the gel showing the enzymatic restriction of the pZiOZ_CRISPR and pZiOZ EcoRI plasmids (without NS4 and NS5) with the BamHI, Xbal and EcoRI enzymes (lanes 1 to 12), 1 Kb Plus DNA Ladder (lane 13).

[0138]

[0099] Figure 24 shows the TCID Board50 / mL for titration of candidates VSZ018-P6 (left) and VSZ018-P11. Assay performed with 8 biological replicates for VSZ018-P6, and 8 biological replicates for VSZ018-P11 (lines G and H were used as negative control), each column representing a point in the serial dilution starting at dilution 10e0 (Column 1) up to dilution 10e11 (Column 12). Yellow color of the culture medium in the wells indicates positive infection (CPE +); pink color of the wells indicates negative infection (CPE -).

[0139]

[0100] Figure 25 shows the TCID Board 50 mL for candidate titration

[0140] REPLACEMENT SHEET (RULE 26) VSZ020-P6 (left) and VSZ020-P11. Assay performed with 8 biological replicates for VSZ018-P6, and 7 biological replicates for VSZ018-P11 (line H was used as a negative control), each column being a point of the serial dilution starting from dilution 10e0 (Column 1) to dilution 10e11 (Column 12). The yellow color of the culture medium in the wells indicates positive infection (CPE +); pink color of the wells indicates negative infection (CPE -).

[0141]

[0101] Figure 26 shows photographic records (10x) of the Daoy cell line (medulloblastoma / pediatric CNS tumor), two replicates / wells per group, 3 days post-infection (dpi) with candidates VSZ018, passages 2 and 10, and VSZ020, passages 2 and 10, in MOI 1 and MOI 2. Bar: 200 pm.

[0142]

[0102] Figure 27 shows the photographic records (10x) of the ONS-76 cell line (medulloblastoma / pediatric CNS tumor), two replicates / wells per group, 3 days post-infection (dpi) with candidates VSZ018, passages 2 and 10, and VSZ020, passages 2 and 10, in MOI 1 and MOI 2. Bar: 200 pm.

[0143]

[0103] Figure 28 shows photographic records (10x) of the LN-18 cell line (glioblastoma / adult CNS tumor), two replicates / wells per group, 3 days post-infection (dpi) with candidates VSZ018, passages 2 and 10, and VSZ020, passages 2 and 10, in MOI 1 and MOI 2. Bar: 200 pm.

[0144]

[0104] Figure 29 shows the luciferase quantification assay in a neural cell line (Daoy) 24 hours after exposure to the transfection supernatant of the following groups: Lipofectamina (absence of genetic material); PMD-005 (presence of plasmids with structural proteins only); PMD-006 (presence of pZiOZ plasmid only); EVZ-002 (presence of both plasmids in transfection with HEK-293 cells in adhesion).

[0145]

[0105] Figure 30 shows the titration of samples from the potency test performed on medulloblastoma (Daoy and ONS-76) and glioblastoma (LN18) cell lines, based on the number of copies / pL using primer from the Envelope region, beginning of the sequence. Samples shown as replicates.

[0146] REPLACEMENT SHEET (RULE 26) biological, with each bar originating from each well of a 12-well plate subjected to infection with the viral candidates VSZ-018 and VSZ-020, from passages 2 and 10, in MOI 1 and MOI 2.

[0147]

[0106] Figure 31 shows Sanger sequencing performed from amplicons generated after RT-qPCR of the viral genome after a transfection and reinfection cycle (VSZ-018 / P1) and the positive control, Synthzika, generated and sequenced by NGS in Example 1. (A) Alignment of the VSZ-018 / P1 amplicon with the complete Synthzika sequence; (B) Alignment of the VSZ-018 / P1 amplicon with the complete wild-type virus sequence (KX197192.1); (C) Alignment of the Synthzika amplicon with the complete Synthzika sequence; (D) Alignment of the Synthzika amplicon with the complete wild-type virus sequence (KX197192.1).

[0148]

[0107] Figure 32 shows the RT-PCR quantification of delivered ZiOZ RNA copies by the viral vector in Vero cells, 24 hours after transduction of the following groups: CTR (-) (absence of genetic material in the first transfection); Viral Vector (EVZ-005) produced from the transfection of pCME and pZiOZ (containing the reduced fragment of NS4A-B).

[0149]

[0108] Figure 33 shows the luciferase quantification assay in a cell line (HEK) 24 hours after the second transfection (delivery of pZiOZ_CRISPR) of the following groups: CTR (-) (absence of genetic material in the first and second transfection); Luciferase black bar (presence of only the Luciferase plasmid in the first transfection); Luciferase gray bar (presence of the pZiOZ_CRISPR plasmid. p-value***<0.001.

[0150]

[0109] Figure 17: Analysis of miR-124 expression profile in non-tumor cell lines: hESC, NPC and neuron (derived from the Brazilian embryonic cell line BR6), adult mesenchymal stem cells (M10 and ABC); and in tumor cell lines: commercial medulloblastoma (Daoy and D283) and glioblastoma (HDMB03) cell lines, non-commercial (isolated from Brazilian patients) medulloblastoma (USP13), ATRT (USP7) and glioblastoma (G7436) cell lines; synthetic virus-producing cell

[0151] REPLACEMENT SHEET (RULE 26)(Vero).

[0152]

[0110] Figure 18: Fusion PCR under conditions recommended by the DNA polymerase manufacturer (lane 1) and changes in primer annealing temperature (lanes 2 to 6). DNA size pattern (lane 7).

[0153]

[0111] Figure 19: Fusion PCR reaction with changes in primer annealing temperature, containing 0.5 pM primers (lanes 1 to 6) and 0.4 pM primers (lanes 8 to 12). DNA size standard (lane 7).

[0154]

[0112] Figure 20: Fusion PCR reaction with changes in primer annealing temperature, containing 0.4 pM primers (channels 1 to 3) and 0.3 pM primers (channels 4 to 6). DNA size standard (channel 7).

[0155]

[0113] Figure 21: Fusion PCR reaction under ideal conditions established after a single PCR reaction purification process (channel 1). DNA size standard (channel 2).

[0156]

[0114] Figure 22: Next-generation sequencing (NGS) performed from the DNA fragment generated after fusion PCR. (A) An image of the mapping, using Shovill, which resulted in the generation of a consensus region with a contig of 11,790 bases. (B) The nucleotide sequence extracted from the mapping of this consensus region.

[0157]

[0115] Figure 23: Agarose gel with in vitro transcription and purification products with 40% (v / v) isopropanol, containing Molecular Weight Marker (Channel 1), RNA transcript from the positive control of the in vitro transcription kit (Channel 2) and RNA-3 transcript with the new RNA purification conditions (Channels 3).

[0158]

[0116] Figure 24: Number of viral RNA copies per pL when analyzing the beginning of the sequence (Envelope primers). Data are presented as means. P-values ​​were calculated by two-way ANOVA followed by Bonferroni post-hoc test for multiple comparisons (GSIS).

[0159]

[0117] Figure 25: Vero cells after being transfected with RNA or DNA (P0) with a positive cytopathic effect. 10X magnification, where 100 px equals 100 pm.

[0160]

[0118] Figure 26: Number of viral RNA copies per pL when analyzing the start of

[0161] SHEET REPLACEMENT (RULE 26) sequence (Envelope primers) of viruses with promising results during electroporation in Vero cells. Data are presented as means. P-values ​​were calculated by two-way ANOVA followed by Bonferroni post-hoc test for multiple comparisons (GSIS).

[0162]

[0119] Figure 27: Photographic records (10x) of T25 bottles, representing the cytopathic effect observed in different infections. Scale bar: 100 px = 100 pm.

[0163]

[0120] Figure 28: Titration of synthetic viruses VSZ-018 / BT and VSZ-020 / BT in different passages (P2 -P11), based on the number of copies / pL using primer from the Envelope region, beginning of the sequence.

[0164]

[0121] Figure 29: TCID plate 50 / mL for titration of candidates VSZ018-P6 (left) and VSZ018-P11. Assay performed with 8 biological replicates for VSZ018-P6, and 8 biological replicates for VSZ018-P11 (lines G and H were used as negative control), each column representing a point in the serial dilution starting at dilution 10e0 (Column 1) up to dilution 10e11 (Column 12). Yellow color of the culture medium in the wells indicates positive infection (CPE +); pink color of the wells indicates negative infection (CPE -).

[0165]

[0122] Figure 30: TCID plate 50 / mL for titration of candidates VSZ020-P6 (left) and VSZ020-P11. Assay performed with 8 biological replicates for VSZ018-P6, and 7 biological replicates for VSZ018-P11 (line H was used as a negative control), each column representing a point in the serial dilution starting at dilution 10e0 (Column 1) up to dilution 10e11 (Column 12). Yellow color of the culture medium in the wells indicates positive infection (CPE +); pink color of the wells indicates negative infection (CPE -).

[0166]

[0123] Figure 31: Photographic records (10x) of the power test of candidates VSZ-018 and VSZ-020 in passage 6. Each replica is a well of the 6-well plate. Bar: 100 px = 100 pm.

[0167]

[0124] Figure 32: Supernatant titration in the potency assay of candidates VSZ-018 and VSZ-020 at passage 6 and reinfection, determined from the number of copies / pL using primer from the initial region of the Envelope gene. *Bar with blue asterisk indicates the use of supernatant containing

[0168] REPLACEMENT SHEET (RULE 26) VSZ-018 / BT-P6 2 dpi for reinfection. Bar with red asterisk indicates the use of supernatant containing VSZ-018 / BT-P6 3 dpi for reinfection.

[0169]

[0125] Figure 33: Photographic records (10x) of the potency test of candidates VSZ-018 and VSZ-020 at passage 10, at 2 and 3 days post-infection (dpi). Each replicate is a well of the 6-well plate. Bar: 100 px = 100 pm.

[0170]

[0126] Figure 34: Supernatant titration in the potency assay of candidates VSZ-018 and VSZ-020 at passage 10, determined from the number of copies / pL using primer from the initial region of the Envelope gene.

[0171]

[0127] Figure 35: Photographic records (10x) of reinfection of tumor cells using supernatant from potency testing of VSZ-018 candidates collected at 2 and 3 dpi on passage 6. Scale bar: 100 px = 100 pm.

[0172]

[0128] Figure 36: Photographic records (10x) of all wells, three replicates / wells per group, 3 days post-infection (dpi) at MOI 0.1. Scale bar: 100 px = 100 pm.

[0173]

[0129] Figure 37: Photographic records (10x) of the Daoy cell line (medulloblastoma / pediatric CNS tumor), two replicates / wells per group, 3 days post-infection (dpi) with candidates VSZ018, passages 2 and 10, and VSZ020, passages 2 and 10, in MOI 1 and MOI 2. Bar: 200 pm.

[0174]

[0130] Figure 38: Photographic records (10x) of the ONS-76 cell line (medulloblastoma / pediatric CNS tumor), two replicates / wells per group, 3 days post-infection (dpi) with candidates VSZ018, passages 2 and 10, and VSZ020, passages 2 and 10, in MOI 1 and MOI 2. Bar: 200 pm.

[0175]

[0131] Figure 39: Photographic records (10x) of the LN18 cell line (glioblastoma / adult CNS tumor), two replicates / wells per group, 3 days post-infection (dpi) with candidates VSZ018, passages 2 and 10, and VSZ020, passages 2 and 10, in MOI 1 and MOI 2. Bar: 200 pm.

[0176]

[0132] Figure 40: Titration of potency test samples, based on the number of copies / pL using primer from the Envelope region, beginning of the sequence. Samples presented as biological replicates, with each bar originating from each well of a 6-well plate. *p<0.1; **p<0.01; ***p<0.001;

[0177] REPLACEMENT SHEET (RULE 26)****p<0.0001.

[0178]

[0133] Figure 41: Titration of potency test samples, based on the number of copies / pL using primer from the NS5 region, end of the sequence. Samples presented as biological replicates, with each bar originating from each well of a 6-well plate. *p<0.1; **p<0.01; ***p<0.001; ****p<0.0001.

[0179]

[0134] Figure 42: Titration of samples from the potency test performed on medulloblastoma (Daoy and ONS-76) and glioblastoma (LN18) cell lines, based on the number of copies / pL using primer from the Envelope region, beginning of the sequence. Samples presented as biological replicates, with each bar originating from each well of a 12-well plate subjected to infection with the viral candidates VSZ-018 and VSZ-020, from passages 2 and 10, in MOI 1 and MOI 2.

[0180]

[0135] Figure 43: Figures 43A and B illustrate the two plasmids essential for the production of the viral vector (EviOZ): pZiOZ and pCME. The pZiOZ plasmid carries the information to be encapsulated by the viral vector and delivered to the target cells. The pCME plasmid contains the instructions for the formation of VLPs (Virus-Like Particles) that will encapsulate ZiOZ, in addition to encoding non-structural proteins that assist in the production of EviOZ.

[0181]

[0136] Figure 44: Restriction site of the BamHI, Xbal, and EcoRI enzymes in the pCME, pZiOZ, pZiOZ_CRISPR, and pZiOZ EcoRI plasmids (without NS4 and NS5). Enzymes that are not annotated in each plasmid do not recognize the plasmid DNA sequence.

[0182]

[0137] Figure 45: Gel showing the enzymatic restriction of pCME and pZiOZ plasmids with BamHI and Xbal enzymes (lanes 1 to 13), DNA Ladder 1 Kb Plus (lane 14).

[0183]

[0138] Figure 46: Gel showing the enzymatic restriction of pCME, pZiOZ and pZiOZ_CRISPR plasmids with BamHI, Xbal and EcoRI enzymes (lanes 1 to 13), DNA Ladder 1 Kb Plus (lane 14).

[0184]

[0139] Figure 47: Gel showing the enzymatic restriction of the pZiOZ_CRISPR and pZiOZ EcoRI plasmids (without NS4 and NS5) with the BamHI enzymes,

[0185] REPLACEMENT SHEET (RULE 26) Xbal and EcoRI (channels 1 to 12), 1 Kb Plus DNA Ladder (channel 13).

[0186]

[0140] Figure 48 shows the luciferase quantification assay in a neural cell line (Daoy) 24 hours after exposure to the transfection supernatant of the following groups: Lipofectamina (absence of genetic material); PMD-005 (presence of plasmids with structural proteins only); PMD-006 (presence of pZiOZ plasmid only); EVZ-002 (presence of both plasmids in transfection with HEK-293 cells in adhesion).

[0187]

[0141] Figure 49: Luciferase quantification assay in a cell line (HEK) 24 hours after the second transfection (delivery of pZiOZ_CRISPR) of the following groups: CTR (-) (absence of genetic material in the first and second transfection); Luciferase black bar (presence of the Luciferase plasmid only in the first transfection); Luciferase gray bar (presence of the pZiOZ_CRISPR plasmid. p-value***<0.001.

[0188] Detailed Description of the Invention

[0189]

[0142] The present invention provides a chimeric flavivirus nucleotide sequence, a therapeutic agent, a viral vector and its process for obtaining it, its use, and a treatment method that utilizes said sequence, agent or vector.

[0190]

[0143] 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.

[0191] Definitions

[0192]

[0144] For the purposes of the present invention, the following expressions are used, with their respective meanings: ZiOZ (Chimeric flavivirus nucleotide sequence); pZiOZ (plasmid used for the production of ZiOZ), pVLP (plasmid used for the production of viral vector envelope proteins),

[0193] REPLACEMENT SHEET (RULE 26) 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).

[0194]

[0145] 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.

[0195]

[0146] 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 traverse the membrane, while TM2 is embedded but does not cross the lipid bilayer. Furthermore, a fourth segment, known as the 2K fragment, 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).

[0196]

[0147] 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, sites where non-structural viral proteins and viral RNA are concentrated, thus playing an essential role in viral replication. The functions of NS4B in the replication complex are mediated by protein-protein interactions, which facilitate the formation and

[0197] REPLACEMENT SHEET (RULE 26) maintains 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 life cycle.

[0198] Comparison with the State of the Art

[0199]

[0148] 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 host antitumor immune response. 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.

[0200]

[0149] The genetic modifications to SoziOZ in this invention are minimal and highly precise, ensuring its replicative capacity and greater safety without compromising efficacy. Unlike other extensively modified oncolytic viruses, such as Imlygic® which attenuates the virus, the solution proposed here maintains the natural tropism of SoziOZ for the CNS, ensuring superior selectivity and safety. Furthermore, the approach minimizes the risks of virulent reversion and promotes the genetic stability of SoziOZ.

[0201]

[0150] 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

[0202] REPLACEMENT LEAF (RULE 26) HAITIAN strain, which presents significant differences in the genomic sequence, especially in the UTR regions, compared to the MH882527.1 strain.

[0203]

[0151] 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.

[0204]

[0152] 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 system reliability and safety.

[0205]

[0153] For the first time, a designed target sequence (MRE) of hsa-miR-124 has been 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.

[0206]

[0154] For the first time, two target sequences (MREs) were simultaneously placed in the chimeric 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

[0207] The substitution sheet (rule 26) encoding the 2K protein is located in the same reading frame as the protein. This allows for a dual mechanism of replication regulation of the oncolytic therapy, increasing the safety and oncolytic potential of SoziOZ, as demonstrated by the unpublished data presented in Table 4, showing the release of high levels of active virus after infection in tumor cells, from SoziOZ not present in any previous document, including WO2023164442A2.

[0208]

[0155] 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.

[0209]

[0156] 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 CNS cells. In addition, the fact that it is a viral vector that delivers RNA that undergoes reverse transcription, and not DNA like AAVs, 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.

[0210]

[0157] 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 WO2023164441 A1, employs a different strategy.

[0211] The ZiOZ substitution sheet (rule 26) utilizes 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 WO2023164441 A1, which uses a sequence of the C protein for encapsulation.

[0212]

[0158] 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.

[0213]

[0159] Unlike other approaches, the invention of ZiOZ allows for the inclusion 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), responsible for the replication of genomic RNA in the ZIKV viral cycle. In ZiOZ, this sequence will be used to allow RNA replication, ensuring that the level of protein expression persists for longer.

[0214]

[0160] 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.

[0215] SUBSTITUTION SHEET (RULE 26)

[0161] Compared to AAV (Adenovirus-associated virus) 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 BBB 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.

[0216]

[0162] 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.

[0217]

[0163] 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.

[0218]

[0164] One of the objects of the invention is the use comprising at least three nucleotide sequences functionally linked together in any order of linkage thereof. 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, said set of nucleotide sequences encodes

[0219] SUBSTITUTION SHEET (RULE 26) fragments of NS4A and NS4B proteins that do not have the same protein function compared to the complete sequence proteins of flaviviruses, because the absence of the complete sequence does not allow for the functionalization of the ER membrane rearrangement nor does it aid in replication.

[0220]

[0165] In a 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. In this location, 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.

[0221]

[0166] 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.

[0222] Nucleotide sequences of chimeric flaviviruses.

[0223]

[0167] 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 linkage thereof. 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 ZIKV 2K protein (Seq ID No. 2); and (iii) the nucleotide sequence of part of the coding sequence of the ZIKV NS4B proteins (Seq ID No. 3).

[0224]

[0168] In one embodiment, the aforementioned nucleotide sequence comprises

[0225] SUBSTITUTION SHEET (RULE 26) additionally 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-2K-NS4B sequence.

[0226]

[0169] 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-2K-NS4B sequence.

[0227]

[0170] 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'.

[0228]

[0171] 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'.

[0229]

[0172] In one embodiment, the said nucleotide sequence further comprises the nucleotide sequences of part of the coding sequences of the ZIKV proteins M, E, NS1, NS2A, NS2B (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'.

[0230]

[0173] 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 5' of the coding sequence of M (Seq ID No. 7).

[0231]

[0174] 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).

[0232] SUBSTITUTION SHEET (RULE 26)

[0175] In one embodiment, the said nucleotide sequence further comprises: the 5'UTR (Seq ID No. 14) and 3'UTR (Seq ID. 15) nucleotide sequences functionally linked together.

[0233]

[0176] 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.

[0234]

[0177] 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 vaccines, as well as the vaccine composition and administration of the compound for the generation and evaluation of an anti-Zika virus immune response.

[0235] Oncolytic virotherapy

[0236]

[0178] 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. Also

[0237] Replacement leaflet (Rule 26) can 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 various origins such as breast cancer, prostate cancer, and lung cancer, but located in the CNS. Likewise, it is suitable 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.

[0238]

[0179] 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 increasing its 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, 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).

[0239]

[0180] 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,

[0240] REPLACEMENT SHEET (RULE 26) reversing genetic modifications and restoring virulence leads 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).

[0241]

[0181] 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 and increased oncolytic potential, 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-124-3p 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 strategy aimed at safety and increased oncolytic potential for the oncolytic virotherapy of the present invention applied to the treatment of brain cancer (Figure 4).

[0242]

[0182] In one embodiment, the construction of the oncolytic virus of the present invention utilizes a combination of reference sequences found in the access codes:

[0243] KU509998 (https: / / www.ncbi.nlm.nih.gov / nuccore / KU509998)

[0244] KX197192 (https: / / www.ncbi.nlm.nih.gov / nuccore / KX197192 / )

[0245] NC_012532 (https: / / www.ncbi.nlm.nih.gOv / nuccore / NC_012532.1).

[0246] viral vector

[0247]

[0183] 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.

[0248] REPLACEMENT SHEET (RULE 26)

[0184] 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 highly precise gene editing.

[0249]

[0185] 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.

[0250]

[0186] 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.

[0251]

[0187] 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 enzymes during its production, since the addition of a CAP-analogous structure is not necessary. This characteristic is an important factor for ZiOZ to be used as an RNA vaccine.

[0252]

[0188] In addition, ZiOZ has two internal sequences. One of them is part

[0253] The substitution sheet (rule 26) of the capsid protein sequence is 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 Nos. 1, 2, and 3).

[0254]

[0189] 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 CRISPR machinery for gene editing. This expands the therapeutic application of the technology compared to approved viral vectors and ongoing clinical trials.

[0255]

[0190] 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.

[0256]

[0191] 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 potential toxicological problems.

[0257]

[0192] In addition, EviOZ is able to cross the BBB with efficiency

[0258] REPLACEMENT SHEET (RULE 26) 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%.

[0259]

[0193] 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, it has the potential to be an mRNA vaccine platform.

[0260]

[0194] 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.

[0261]

[0195] 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.

[0262]

[0196] 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 to 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.

[0263]

[0197] In one embodiment, ZiOZ features the inclusion of the NS5 protein sequence, responsible for genomic RNA replication in the viral cycle of

[0264] ZIKV SUBSTITUTION SHEET (RULE 26). In ZiOZ, this sequence is used to allow RNA replication, ensuring that the level of protein expression persists for longer.

[0265]

[0198] 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.

[0266]

[0199] In one embodiment, 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 to 3'UTR region of SoziOZ, cloned into a high-copy plasmid (Figure 13). The pSoziOZ 1 plasmid contains not only the information necessary for the production of SoziOZ, but also the ribozyme, the MRE (miRNA Response Element) targeting 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).

[0267] mRNA (ZiOZ)

[0268]

[0200] 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.

[0269]

[0201] 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.

[0270]

[0202] EviOZ encapsulates mRNA called ZiOZ (SEQ ID No. 16) which contains sequences important for its stability, due to the presence of

[0271] REPLACEMENT LEAF (RULE 26) regions 5'UTR and 3'UTR similar to those of the African strain of ZIKV.

[0272]

[0203] Due to the properties of ZiOZ in the embodiment that has in its structure the 5'UTR of ZIKV African 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 premises in the production of an mRNA vaccine with simplified manufacturing, optimized processes, greater production efficiency and scalability, making its manufacture faster and adaptable to different demands, in addition to reducing production costs. Furthermore, the genetic material loading capacity in 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.

[0273]

[0204] 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 Nos 1, 2 and 3).

[0274]

[0205] 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, which can lead to cytopathic effects. The therapy of the present invention, by having this tropism for the nervous system,

[0275] The REPLACEMENT SHEET (RULE 26) reduces the amount of drug needed and potential toxicological problems, as well as ensuring effective delivery of the therapy only to the target cells.

[0276] Derived products

[0277]

[0206] Through downstream viral inactivation processing, SoziOZ is useful as an inactivated vaccine against ZIKV. In addition, mRNA can also 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 dosing forms and / or product dosing regimens.

[0278]

[0207] 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.

[0279] Method for detecting / locating genes of interest

[0280]

[0208] The present invention also provides an in vitro kit and 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.

[0281]

[0209] Preferably, the flavivirus nucleotide sequence of the kit or process of the invention is chemically modified to facilitate the detection / localization of genes of interest. In a preferred embodiment, said chemical modification involves the inclusion, in a region of

[0282] REPLACEMENT SHEET (RULE 26) sequence, of one or more chromophores and / or radioactive element(s).

[0283]

[0210] 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 that bind 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.

[0284]

[0211] The aforementioned in vitro kit or process is also particularly useful for the subsequent customization of therapeutic treatments based on pharmaceutical compositions comprising the chimeric 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.

[0285]

[0212] The invention can also be defined by the following clauses:

[0286]

[0213] Chimeric flavivirus nucleotide sequence 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 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).

[0287]

[0214] 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 of flaviviruses, yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and dengue virus.

[0288] REPLACEMENT SHEET (RULE 26)(DENV).

[0289]

[0215] Therapeutic agent comprising an excipient and / or adjuvant and the nucleotide sequence as defined above.

[0290]

[0216] Use of oncolytic virus as defined above, 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, Cervical metastasis, Metastasis Cerebral, Bone Metastasis, Primary Focus Tumor, Cisplatin-Resistant Tumor, Uterine Tumor, Uterine / Endometrial Tumor, and Glioblastoma Multiforme.Specifically, the therapeutic agent for treating tumors with high expression of the ITGAV membrane protein (Ensembl ID: ENSG00000138448.11), when compared to its corresponding non-tumor tissue. According to the GEPIA (Gene Expression Profiling Interactive Analysis) database, these tumors would be lymphoid neoplasm, diffuse large B-cell lymphoid 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.

[0291]

[0217] Process for obtaining the therapeutic agent defined above comprising the following steps:

[0292] - obtain a chimeric nucleotide sequence as defined above; and

[0293] REPLACEMENT SHEET (RULE 26) - add an excipient, adjuvant and / or nanoparticles.

[0294]

[0218] 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.

[0295] Examples

[0296]

[0219] The examples shown here are intended only to illustrate one of the numerous ways of carrying out the invention, however without limiting its scope.

[0297] Example 1 - Production of the oncolytic virus

[0298]

[0220] For the production of the oncolytic virus, the genome was divided into two 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.

[0299]

[0221] 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).

[0300]

[0222] 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 subjected to purification (Figure 12).

[0301]

[0223] After the production and purification of the oncolytic virus, an infection is carried out to assemble virus banks with genetic homogeneity.

[0302] REPLACEMENT SHEET (RULE 26) (clones). Three days after infection, the supernatant is collected for PCR, PFU, and RNA extraction for sequencing.

[0303]

[0224] In one embodiment, the production process of SoziOZ is carried out from the product of fusion PCR, allowing both DNA and RNA electroporation. This versatility shows that the production method, from the product of fusion PCR, eliminates the need for bacteria in intermediate DNA cloning steps in the process, highlighting its difference and productive improvement compared to traditional methods described in the state of the art.

[0304] Plasmid diagrams.

[0305]

[0225] 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).

[0306]

[0226] The pSoziOZ 1 plasmid contains not only the information necessary for the production of SoziOZ, but also the ribozyme, the miRNA Response Element (MRE) targeting the hsa-miR-124-3p miRNA, flanked by restriction enzyme sites within the 5'UTR region. Additionally, the plasmid includes the CMV and T7 promoters (Figure 13).

[0307]

[0227] 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 coding for the 2K protein, as well as a ribozyme and a polyadenylation signal (Figure 13). For in vitro validation of the miR-124 candidate selected in the in silico analyses, total miRNA-enriched RNA is extracted from the samples using the Qiagen miRNA-specific kit, following the manufacturer's instructions, ensuring the preservation and purification of small RNAs. Subsequently, the purified miRNAs are subjected to reverse transcription using reagents and conditions

[0308] Substitution sheet (rule 26) recommended for cDNA synthesis from miRNA. Expression quantification is performed by real-time RT-qPCR using Qiagen-specific commercial primers, in a system compatible with fluorescent detection, with data normalization by appropriate endogenous controls. Relative miRNA expression analysis is conducted using standard quantitative methods, allowing comparison between experimental groups (Figure 17). For the reconstruction of the oncolytic virus (SoziOZ) genome 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).

[0309]

[0228] 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.

[0310]

[0229] Subsequently, in vitro transcription of the single fragment was performed to generate RNA containing the information necessary for the production of SoziOZ (Figure 15). As observed in Figure 15, the presence of RNA in the gel was qualitatively verified by non-denaturing agarose gel electrophoresis. 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.

[0311] REPLACEMENT SHEET (RULE 26)

[0230] 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).

[0312]

[0231] The following describes the steps in the production of the oncolytic virus:

[0313]

[0232] Candidates targeted by miRNA:

[0314] a) In silico analysis of miRNA expression profile in normal tissue and tumor cells (focus on glioblastoma) (Figure 4).

[0315] 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 12 and 13).

[0316] c) In silico analysis of pairing, binding strength, and annealing score of the MRE region drawn with the miRNA (Figure 16).

[0317] d) In vitro validation of the miRNA target candidate (Figure 17). Figure 17 shows that miR-124 exhibited high expression in normal embryonic cells, in different phases of neural development, and also in adult mesenchymal cells. However, it did not show positive gene expression in patient tumor cell lines and in the Vero cell line, confirming the expression profile found in the in silico analysis, necessary for the use of this target in the viral replication inhibition mechanism of oncolytic therapy. Its high expression in commercial cells does not preclude its use as an inhibition target due to the classic lack of representativeness of commercial cell lines when compared to primary or low-passage cell lines in cell culture.

[0318]

[0233] Plasmid drawings

[0319] 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 (Figure 13 A) and another from the 2k to 3'UTR region in a high-copy plasmid (Figure 13 B).

[0320] SUBSTITUTION SHEET (RULE 26)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 (Figure 13 A).

[0321] c) pSoziOZ 2 encoding from the 2k to the 3'UTR region, ribozyme and a polyadenylation signal (Figure 13 B).

[0322]

[0234] Reconstruction of the modified virus genome:

[0323] 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.

[0324] If necessary, after obtaining the plasmids, it goes through the expansion, transformation, culture, and midi prep process before the overlapping PCR step:

[0325] Plasmid expansion

[0326] ■ Transformation of dH5-alpha bacteria with plasmids using a heat shock technique.

[0327] ■ Selection of transformants in selective solid culture medium (antibiotic).

[0328] ■ Expansion of colonies transformed into a liquid medium.

[0329] ■ Mini prep, Mid prep, or Max prep of the grow.

[0330] ■ DNA quantification by microdroplet spectrophotometry.

[0331] ■ Store the permanent colony in a -80°C freezer (Golden Clone).

[0332] ■ Evaluation of intact plasmids by agarose gel electrophoresis.

[0333] ■ Evaluation of plasmids by enzymatic restriction in agarose gel electrophoresis.

[0334] ■ Storage of selected plasmids.

[0335] REPLACEMENT SHEET (RULE 26)

[0336] b) Different amplification conditions for fusion PCR were performed to join the genetic information contained in the pSoziOZ 1 and pSoziOZ 2 plasmids (Figure 13 A and B). The conditions analyzed were the primer annealing temperature and the concentration of each primer used per reaction (Table 1, Figures 18 to 20). The DNA polymerase enzyme manufacturer indicates that 0.5 pM of each primer should be used in each reaction, and the annealing temperature to be used is 60 °C. As indicated in Table 1 (condition 1) and Figure 18 (Channel 1).

[0337] Table 1: Fusion PCR conditions tested and their respective indications in figures 18 to 21.

[0338] Condition Temperature Amount of primer Figure Groove annealing Forward and Reverse

[0339] 1 60°C 0.5 pM 18 1 2 62°C 0.5 pM 18 2 3 64°C 0.5 pM 18 3 4 66°C 0.5 pM 18 4 5 68°C 0.5 pM 18 5 6 70°C 0.5 pM 18 6 7 63°C 0.5 pM 19 1 8 63.5°C 0.5 pM 19 2 9 64°C 0.5 pM 19 3 10 64.5°C 0.5 pM 19 4

[0340]

[0341] 11 65°C 0.5 pM 19 5

[0342] REPLACEMENT SHEET (RULE 26) 12 65.5°C 0.5 pM 19 6 13 63°C 0.4 pM 19 8 14 63.5°C 0.4 pM 19 9 15 64°C 0.4 pM 19 10 16 64.5°C 0.4 pM 19 11 17 65°C 0.4 pM 19 12 18 65.5°C 0.4 pM 19 13 19 64°C 0.4 pM 20 1 20 64.5°C 0.4 pM 20 2 21 65°C 0.4 pM 20 3 22 64°C 0.3 pM 20 4 23 64.5°C 0.3 pM 20 5 24 65°C 0.3 pM 20 6

[0343]

[0344] 25 (=24) 65°C 0.3 pM 21 1 c) It is observed that under the temperature condition of 65°C and a primer quantity of 0.3 pM (Figure 20, channel 6), amplification of the specific DNA fragment between 10 and 15 kb occurs, and no nonspecific DNA amplification. This generates a substantial improvement in the PCR reaction, ensuring only the amplification of the DNA of interest without nonspecific amplification contaminants. This reduces the three steps of DNA fragment production, unifying the information from pSoziOZ 1 and pSoziOZ 2 (Figure 13) to only 1 step, the purification of the PCR reaction (Figure 21, channel 1), reducing costs and production time of base material for the production of the oncolytic virus.

[0345] SUBSTITUTION SHEET (RULE 26) d) After performing the fusion PCR reaction, three steps were carried out in the viral genome production process, which were (1) separation of the PCR amplifiers by agarose gel electrophoresis, (2) cutting out the region of the agarose gel containing the amplified DNA of interest (between 10 and 15 kb - Figure 21, channel 1) and (3) DNA purification using agarose gel DNA extraction kits. A synthetic virus production process free from the use of bacteria presents highly relevant regulatory and intellectual property advantages. From a regulatory point of view, the absence of bacteria reduces classic biosafety risks, such as contamination by endotoxins, residual bacterial DNA and antibiotic resistance genes, which simplifies impurity control requirements and makes the CMC dossier leaner and more predictable.This type of process also aligns better with current regulatory trends focused on synthetic and cell-free technologies, favoring more efficient interactions with regulatory agencies and a more suitable safety profile for repeated clinical use.

[0346] e) From an intellectual property perspective, this is an approach with a strong character of novelty and inventive activity, since it departs from traditional methods based on cloning and bacterial amplification. The protection of the process is robust, as production methods are difficult to circumvent and cannot be easily inferred from the final product. Furthermore, independence from bacterial systems reduces risks to freedom of operation and allows the construction of a patentable platform applicable to different viruses and applications. Together, we believe that these characteristics increase the strategic, regulatory, and commercial value of the technology.

[0347] f) Purification of the PCR product by precipitation after agarose gel electrophoresis to verify amplification.

[0348] g) Quantification of genetic material.

[0349] SUBSTITUTION SHEET (RULE 26) h) Sequencing of constructs: Sequencing analysis (Figure 22) confirmed that the fragment generated by fusion PCR contained all the expected information for the product. No deletions or insertions of sequences were detected, demonstrating the success of the aPCR protocol in generating a DNA fragment suitable for use in transfections and in vitro transcription.

[0350] i) In vitro transcription: The material from the fusion PCR (DNA), although suitable for transfection into animal cells since it contains the CMV promoter, was used for in vitro transcription as recommended by the supplier of the in vitro transcription kit. This allows us to generate viruses from both DNA and RNA. The transcription reaction was set up using T7 polymerase buffer, T7 polymerase, water, ribonucleotides, and the fusion PCR product. The material was incubated at 37 °C for 2-4 hours. After the incubation period, the material was treated with DNase for 15 minutes at 37 °C to remove the template DNA.

[0351] j) For RNA purification, precipitation was performed with lithium chloride (LiCl). Nuclease-free water, LiCl solution, and isopropanol were added, and the mixture was incubated at -20 °C for at least 18 hours. After the incubation time, the material was centrifuged to pellet the RNA. The pellet was washed with 70% ethanol, and the RNA was resuspended in nuclease-free water. The RNA was quantified by Nanodrop and Qubit, and aliquots were run on non-denaturing agarose gel for qualitative analysis.

[0352] k) As observed in Figure 15, using non-denaturing agarose gel electrophoresis, it was possible to qualitatively verify the presence of RNA-1 in the gel (Table 2). Since the gel is non-denaturing, the band size is not expected to correspond exactly to the expected RNA size, as the RNA is coiled in its secondary structure. However, the observed band intensity does not

[0353] The REPLACEMENT SHEET (RULE 26) proved to be strong, leading to the hypothesis of a low quantity of RNA produced.

[0354] l) The agarose gel result is consistent with the quantifications obtained by Qubit (Table 2). However, RNA-1 showed a significantly higher quantification in Nanodrop than that obtained by Qubit. Qubit is a more precise method, based on fluorimetry for quantifying RNA strands, unlike Nanodrop which uses quantification by light diffraction. By Qubit, a concentration of 30.8 ng / µL was observed, a value more than 140 times lower than that indicated by Nanodrop.

[0355] m) To improve RNA quantification and quality, the purification methodology was adjusted. Previously, an excessive volume of isopropanol was used to aid RNA precipitation. However, when used in excess, this reagent precipitates not only RNA but also other components, such as proteins. Therefore, the amount of isopropanol added was reduced to 40% (v / v). Under this new condition, a new in vitro transcription was performed for comparison: RNA-2 (Table 2) maintained the same purification conditions used for RNA-1, while RNA-3 (Table 2) used the new purification methodology with 40% (v / v) isopropanol.

[0356] n) RNA purification using 40% (v / v) isopropanol resulted in improved sample quality and purity (Table 2). A reduction in the quantification difference between the Nanodrop and Qubit methods (ng / pL) to only two times was observed. Furthermore, the 260 / 280 and 260 / 230 purity ratios showed values ​​closer to the ideal, with the 260 / 280 ratio close to 2 and the 260 / 230 ratio between 2 and 2.2, indicating better RNA quality (Table 2).

[0357] o) The result observed in the quantifications was confirmed by agarose gel analysis of RNA-3 (Figure 23 - Channel 3), which showed a clearer and more intense band when compared to the positive control of the Kit.

[0358] SUBSTITUTION SHEET (RULE 26) of in vitro transcription (Figure 23 - Channel 2). To verify reproducibility, two more in vitro transcriptions were performed (Table Y - RNAs 4 and 5). Like RNA-3, these new RNAs (4 and 5) also demonstrated high purity, with a 260 / 280 ratio close to 2 and a 260 / 230 ratio between 2 and 2.2. Furthermore, the difference between the quantifications performed by Nanodrop and Qubit remained at most two times. p) Table 2: Quantification by Nanodrop and Qubit after in vitro transcription using the fusion PCR product as a template.

[0359] Nanodrop

[0360] Qubit

[0361] RNA

[0362] (ng / pL) ng / pL 260 / 280 260 / 230

[0363] 1 4,335.70 1.97 2.43 30.8

[0364] 2,686.80 1.68 2.11 31.6

[0365] 3 46.97 2.02 2.23 37.2

[0366] 4,214,798 1,993 2,345 172

[0367] 5 424,118 2,107 2,428 312

[0368]

[0369] q)

[0370] r) Transfection by electroporation and / or lipofectamine of the DNA material (Figure 12).

[0371] s) As an alternative, in vitro transcription is performed to generate and purify mRNA.

[0372] t) Transfection by electroporation of RNA material (Figures 12 and 15). DNA obtained from fusion PCR and RNA from in vitro transcription were used in electroporation. 2.5 pg of transcribed RNA were introduced into 1 x io 6 Vero cells at T25 by electroporation, using 4 mm cuvettes in the GenePulser Xcell instrument (Bio-Rad), with parameters of 200 V, 950 pF and a single pulse.

[0373] REPLACEMENT SHEET (RULE 26)

[0235] Transfection of VERO and HEK cells:

[0374] a) VERO and HEK cell culture 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.

[0375] d) Complex DNA with lipofectamine, incubate.

[0376] (e) Mix the DNA+lipofectamine complex, culture medium, and cells corresponding to each group.

[0377] 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.

[0378] g) Transfer the mixture to T25 flasks or 6-well plates. After electroporation, the transfected cells were transferred to T25 flasks containing DMEM medium supplemented with 5% fetal bovine serum and without normocin, and incubated at 37 °C and 5% CO2. Cell cultures were monitored daily for cytopathic effect. The supernatant was collected five days after transfection, clarified by centrifugation at 3200 °C, and RT-qPCR and reinfection were performed to amplify the viral load.

[0379] h) Results: The production of synthetic oncolytic virus through RNA and / or DNA electroporation was repeated 30 times until an effective and reproducible standard process for the production of synthetic oncolytic virus was developed. During the 30 tests, the following critical parameters were varied throughout each stage of the production process:

[0380] i. Electroporation parameters: Including voltage, capacitance, and number of pulses, aiming to determine the ideal condition that maximizes the introduction of viral genetic material into the host cell without significantly compromising cell viability.

[0381] REPLACEMENT SHEET (RULE 26) ii. Host cell type and condition: Evaluation of cell density, growth phase (e.g., logarithmic), and pre-electroporation viability.

[0382] iii. Concentration of genetic material: Analysis of the impact of viral DNA or RNA concentration (plasmid or purified viral genome) on transfection efficiency and subsequent viral production rate.

[0383] iv. Electroporation medium volume: Optimization of the electroporation medium volume.

[0384] v. Culture conditions after electroporation: Establishing ideal conditions for viral recovery and proliferation after electroporation.

[0385] vi. Viral concentration: Comparison and adjustment of concentration techniques to ensure a final product with high purity and high titer.

[0386] vii. Viral infection: Adjusting the viral infection technique to better obtain viral titers.

[0387] As observed in Figures 22 and 23, in the first 14 attempts to generate the virus, a cytopathic effect was observed in Vero cells 5 days after electroporation in 5 groups (a 50% improvement compared to other techniques). Virus production was observed after electroporation with both DNA and RNA, demonstrating for the first time the generation of viruses from a plasmid with a CMV promoter (Figures 24 and 25). Following the virus generation, consensus methodological parameters were established and standardized after several optimization tests to obtain suitable parameters for the production of the oncolytic virus (Tables 3 and 4).

[0388] The viruses obtained in trials 18 to 21 demonstrated the best results in terms of genetic material propagation and cytopathic effect in Vero cells (Figure 26). Consequently, after the stage

[0389] REPLACEMENT SHEET (RULE 26) for reinfection intended for viral amplification, the viruses were quantified for infective particle size (Table 5) and selected for subsequent process steps, including the assembly of mother and working banks, safety and potency tests, and evaluation of the passage effect, even after a total of 30 trials.

[0390] Table 3 - Initial establishment of parameters and processes used in electroporation in Vero cells for the production of synthetic virus (VSZ).

[0391] Electroporation

[0392] Volume

[0393] Parameter Number

[0394] Final volume of

[0395] Bottle / cells concentrate DNA RNA from Opti- medium after electroporation plate PEG MEM electroporation

[0396] of the action

[0397] dog

[0398] Maximum

[0399] the of

[0400] Quantity plate 0.5 - 5 4x10 5 - 1 According

[0401] 6 wells x10 6 400 - pg 800 p with 1 - 5 mL Yes or T25 base (Qubit) L manufacturer

[0402] no

[0403] Qubit

[0404]

[0405] Table 4 - Initial establishment of parameters and processes used in reinfection in Vero cells for the production of synthetic virus (VSZ).

[0406] Reinfection

[0407] Final volume

[0408] Bottle / Thread Number Thread Removal Time

[0409] Medium after filtration Plate cells infection incubation of infection

[0410] reinfection

[0411] T25 1 - 30 minutes 0.22 pm 3x10 5 1 mL - 1 hour Yes 5 - 12 mL

[0412]

[0413] Table 5: Viral titer by TCID50 and RT-PCR

[0414] Sample name TCID50 / ml_ PFU / mL viral copies / uL VSZ-018 / SE-P1 3, 16x10 5 2.21x10 5 6.66x10 6 VSZ-019 / SE-P1 3, 16x10 6 2.21x10 6 1.17x10 7

[0415]

[0416] REPLACEMENT SHEET (RULE 26) VSZ-020 / SE-P1 3, 16x10 4 2.21x10 4 3.16x10 6 VSZ-021 / SE-P1 3, 16x10 6 2.21x10 6 1.96x10 7

[0417]

[0418]

[0236] Generation of a bank with a clone of the synthetic virus to guarantee the genetic homogeneity of the viral stock.

[0419] a) Cell plating:

[0420] b) Vero cells were plated in T182 flasks for the production of VSZ-018 / SE-P1, VSZ-019 / SE-P1, and VSZ-021 / SE-P1 cell banks, and in T75 flasks for the VSZ-020 / SE-P1 cell bank. At the time of plating, cell viability was 94.7%. The cells were incubated for 24 hours in DMEM medium supplemented with 10% serum and 25 mM HEPES. The following day, infection was performed with the Passage 1 seed bank, using a Multiplicity of Infection (MOI) of 0.01. Infection was performed with DMEM medium supplemented with 25 mM HEPES, and the incubation period was 60 minutes.

[0421] c) After incubation with the virus, the infection medium was removed and replaced with DMEM medium supplemented with 2% serum and 25 mM HEPES. Virus harvesting was performed 3 dpi (days after infection). Harvesting was performed on 3 dpi due to the high viral replication observed, with a high rate of cell death, no plated cells in the bottle, and a reddish-colored medium. Therefore, harvesting was brought forward to ensure greater virus viability. The supernatant was centrifuged at 3200 g 4°C for 30 min and filtered with 0.22 µm. The TCID50 service was contracted by CROP Labs, and RT-PCR was performed.

[0422] d) From the production of synthetic viruses VSZ018, VSZ019, VSZ020, VSZ021, produced by electroporation and reinfection, a stock bank and pass-through work 2 were assembled. This strategy is fundamental to guarantee the traceability, uniformity, and reproducibility of the viral batches used in future tests. The banks

[0423] The established REPLACEMENT SHEET (RULE 26) was used for a series of rigorous analytical tests and for the establishment of in-process controls. The preliminary results presented in Table 6 provide evidence of the robustness of the optimized production process. These data demonstrate highly efficient viral production, manifested by a high viral titer for all four vectors (VSZ018, VSZ019, VSZ020, and VSZ021). The high titer is a critical indicator as it allows for the use of lower doses or larger-scale production, with direct implications for the feasibility and cost-effectiveness of subsequent development.

[0424] Table 6: Viral titer by TCID50 and Frr-PCR

[0425] copies Sample name TCID50 / ml_ viral PFU / mL / uL VSZ-018 / BA-P2 1.78E+07 1.24E+07 1.95E+08 VSZ-019 / BA-P2 5.62E+07 3.94E+07 1.92E+08 VSZ-020 / BA-P2 1.00E+07 7.00E+06 2.75E+08 VSZ-021 / BA-P2 3.16E+07 2.21 E+07 3.16E+08

[0426]

[0427] e) ■

[0428] Example 2 - Passage effect for analysis of the reversal of the oncolytic effect.

[0429]

[0237] 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.

[0430]

[0238] 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

[0431] REPLACEMENT SHEET (RULE 26) of the last passage, the supernatant is collected, performing analytical control tests in process, in which viral titration / viral viability (PCR - need for RNA extraction in the sample - and TCID50) and viral modification by sequencing are performed, which includes the extraction and sequencing of viral RNA from sequencing by Sanger, Minion or NGS.

[0432]

[0239] In addition, viral efficiency is analyzed by cytotoxicity assay, both in tumor cells.

[0433]

[0240] For the experiment, VSZ-018 / SE-P1 and VSZ-020 / SE-P1 viruses were used. Vero cells are initially infected with the oncolytic virus in T25 culture flasks. After 5 days, the supernatant is collected and used to infect a new set of Vero cells. This cycle is repeated for a total of 2 to 11 passages. At each passage, the supernatant is collected for in-process control analytical testing, which includes the evaluation of viral titration and viability. This analysis is performed using PCR (which requires RNA extraction from the sample) and TCID50. Additionally, viral efficiency is determined by a USP7 oncolytic potency assay.

[0434]

[0241] The results obtained after a total of 11 in vitro passages represent a significant milestone in the evaluation of the stability and efficacy of the oncolytic viral candidates VSZ-018 and VSZ-020. This series of 11 successive viral infections demonstrated that both vectors maintained their essential characteristics: replicative stability and maintenance of the oncolytic effect throughout the entire evaluation period. The persistence of these properties is fundamental to the therapeutic viability of the candidates and was consistently confirmed by the data visualized in Figures 27 and 28.

[0435]

[0242] The infectious titer was determined by TCID assay. 50Using Vero cells seeded in 96-well plates until monolayer formation. The viral sample was initially considered 100% concentrated (10° dilution) and subjected to serial decimal dilutions in an appropriate medium. Each dilution was inoculated into multiple wells, maintaining a final volume of 100 pL per well. The plates were incubated under standard conditions (37 °C, 5% CO2).

[0436] REPLACEMENT SHEET (RULE 26) and monitored for the development of cytopathic effect (CPE). After a 6-day incubation period, wells were classified as positive or negative for CPE, and the infectious titer was calculated using the Spearman-Kärber method, with the result expressed in TCID. 50 / mL, considering the volume inoculated per well. Table 7 shows that the viruses remained active even after 11 infection passages, confirming the viral stability of the synthetic candidate. The high titer of the analyzed samples is noteworthy, especially that of VSZ020 / BT-P11, which even in the most advanced passage showed a titer greater than >3.16E+12, something extremely unprecedented for this class of viruses (flaviviruses), confirming that the genetic modifications and promoters included in the sequence intentionally increased the production efficiency of the viral candidate. The images of the TCID plates are shown. 50 / mL, confirming the high titer of viable viruses in the samples tested (Figures 29 and 30).

[0437] Table 7: Viral titration by TCID50

[0438] TCID Sample 50 / ml_

[0439] VSZ018 / BT-P6 7.50E+11

[0440] VSZ018 / BT-P11 6.81 E+07

[0441] VSZ020 / BT-P6 7.50E+09

[0442] VSZ020 / BT-P11 >3.16E+12

[0443]

[0444]

[0243] To identify the presence of the miR-124-3p target in the viral genome after a transfection and reinfection cycle, the RT-qPCR product of viral titration, the amplicon, which encompasses the insertion region of the miR-124-3p target, was used to perform Sanger sequencing. The supernatant of the synthetic passage 1 virus, its RNA, was extracted and quantified by RT-qPCR. The amplicon was purified and, together with selected primers, was used in 35 PCR cycles with the BigDye Terminator v 3.1 sequencing kit. Subsequently, the DNA was precipitated, washed, and resuspended in Hi-DiFormamide in a 96-well plate. Later, the

[0445] REPLACEMENT SHEET (RULE 26) DNA was denatured for the run, which was read on the 3500xL sequencer (Applied Biosystems) on polymer (Applied Biosystems).

[0446]

[0244] The resulting raw data were processed. As a control, Synthzika, generated and sequenced by NGS in Example 1, was used, which also underwent RT-qPCR processing. Both the PCR product of the viral genome after a transfection and reinfection cycle and that of Synthzika were aligned with the complete Synthzika sequence and the wild-type virus genome (KX197192.1) using the Smith-Waterman method.

[0447]

[0245] Result: The VSZ-018 / P1 sequence showed 85% identity with the Synthzika sequence, such that, compared to the wild-type virus genome (KX197192.1), it showed 76% identity (Figure 30B). A similar identity pattern was observed in the positive control, where the Synthzika amplicon showed 87% identity with the Synthzika sequence, such that, compared to the wild-type virus genome (KX197192.1), it showed 78% identity (Figure 30C). Additionally, sequencing of VSZ-018 / P1 reveals, through alignment, a large portion of the miR-124-3p target sequence, as well as the restriction enzymes that flank it (Figure 30).

[0448]

[0246] It was expected that the sequence analysis would present noise, since the amplicon generated by RT-qPCR is small and Sanger sequencing has low chromatogram resolution at the ends of the sequence. However, comparison with the positive control (the synthetic virus reference sequence) revealed similarities. These similarities confirm that the miR-124-3p target was identified even after a cycle of transfection and reinfection (Figure 30D and E).

[0449] Example 3 - Viral efficiency test by analyzing cell viability and cytotoxicity:

[0450]

[0247] At the end of the last passage of the virus (passage 11) and in intermediate passage (passage 6), cell viability analysis is performed by

[0451] REPLACEMENT SHEET (RULE 26) potency test, followed by viral replication (RT-PCR) in the USP07 tumor cell line.

[0452]

[0248] In a more in-depth evaluation of tumor destruction capacity, i.e., the oncolytic effect, the viral candidates VSZ-018 and VSZ-020 were subjected to specific tests at different times during the passage process. The results confirmed a high infective potential, which translated into intense viral replication and, consequently, a consistently high tumor destruction capacity. This remarkable performance was specifically documented and quantified at both passage 6 and passage 10, serving as checkpoints that endorse the functional stability of the virus. The efficacy and kinetics of the oncolytic process were captured and visualized in detail in Figures 31, 32, 33, 34.

[0453]

[0249] Additionally, the virus isolated after infection of tumor cells (Passage 6 virus) demonstrated maintenance of its replicative and functional capacity. This indicates that the oncolytic potential is preserved even after oncolysis, suggesting an effective viral spread capability (Figure 35). These results attest to the biological robustness of the candidates generated with two MREs inserted in the sequence, and reinforce their potential for use in oncolytic therapeutic applications, even after a total of 11 viral passages.

[0454] Example 4 - In vitro assays with cell lines. Oncolytic selectivity (in vitro effectiveness assay)

[0455]

[0250] 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.

[0456]

[0251] 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,

[0457] REPLACEMENT SHEET (RULE 26) that leads to cell membrane lysis.

[0458]

[0252] The USP07 cell line, a model for tumor stem cells, is plated in 6-well plates, and the virus under test (viral candidate) is placed in contact with the cells to assess whether the virus is oncolytic, i.e., capable of causing cell lysis after 72 hours of infection. The conditions of the potency test are described below:

[0459] the. Viruses used: VSZ-018 / SE-P1, VSZ-019 / SE-P1, VSZ-020 / SE-P1, VSZ-021 / SE-P1 and Wild Virus (Pernambuco strain) b. MOI: 0.1

[0460] c. Number of cells plated on day -1: 1 x 10⁵ cells / well d. Incubation time on the infection wire: 30 minutes

[0461] e. Photographic record: 3 dpi; 4 photographic records per well f. Sample collection: PCR and PFU (3 units 1 ml; 1 unit 200 pL) g. Replicates: 3 biological replicates / wells - 3 wells per candidate h. Groups:

[0462] i. Control (without infection process);

[0463] ii. MOCK (infection with a virus-free medium);

[0464] iii. Candidates (VSZ-018 / SE-P1, VSZ-019 / SE-P1, VSZ-020 / SE-P1, VSZ-021 / SE-P1);

[0465] iv. Positive Control (wild virus - VWT-001 / BT-P4).

[0466]

[0253] Oncolytic effect test, used as a process control, for the synthetic viruses VSZ-018 / SE-P1, VSZ-019 / SE-P1, VSZ-020 / SE-P1 and VSZ-021 / SE-P1. The response is qualitative, based on the observation of floating cells under a microscope after a 3-day incubation period. All candidates presented showed an oncolytic effect when compared with the negative controls (CONTROL, MOCK) and the positive control (WT) (Figure 36).

[0467]

[0254] The model cell lines for adult CNS tumor, glioblastoma (LN-18), and pediatric CNS tumor, medulloblastoma (Daoy and ONS-76), were plated in 12-well plates, and the virus under test

[0468] The REPLACEMENT SHEET (RULE 26) (viral candidate) was placed in contact with cells to assess whether the virus is oncolytic, i.e., capable of causing cell lysis 72 hours after infection. The potency test conditions are described below:

[0469] the. Viruses used: VSZ-018 / BT-P2, VSZ-018 / BT-P10, VSZ-020 / BT-P2, VSZ-020 / BT-P10 and Wild Virus (VWT-001 / BT-P4) b. MOI: 1 and 2.

[0470] c. Number of cells plated on day -1: 4 x 10⁴ cells / well d. Incubation time on the infection wire: 30 minutes

[0471] e. Photo recording: 3 dpi; 4 photographic records per well f. Replicates: 2 biological replicas / wells - 2 wells per candidate g. Groups:

[0472] I. MOCK (infection with a virus-free environment);

[0473] ii. Candidates (VSZ-018 / BT-P2, VSZ-018 / BT-P10, VSZ-020 / BT-P2, VSZ-020 / BT-P10);

[0474] iii. Positive Control (wild virus - VWT-001 / BT-P4).

[0475]

[0255] Oncolytic effect test, used as a process control, for the synthetic viruses VSZ-018 / BT-P2, VSZ-018 / BT-P10, VSZ-020 / BT-P2, VSZ-020 / BT-P10. The response is qualitative, based on the observation of floating cells under a microscope after an incubation period of 3 days.

[0476]

[0256] All candidates showed an oncolytic effect when compared with the negative controls and the positive control (Figures 37, 38, 39).

[0477]

[0257] In Daoy, passages 10 of both candidates showed more cell death when compared with their respective passages 2 and the wild-type virus, demonstrating greater efficiency of the synthetic virus in passage 10 (Figure 37).

[0478]

[0258] E m ONS, also derived from medulloblastoma like Daoy, showed an oncolytic effect similar to the wild-type virus, with the exception of the candidate VSZ020-P10, which showed more cell death (Figure 38).

[0479]

[0259] In LN-18, adult glioblastoma, only the candidate VSZ020 showed a greater oncolytic effect than the wild-type virus (Figure 39).

[0480] REPLACEMENT SHEET (RULE 26) Example 5 - Selectivity of viral replication (in vitro cytotoxicity assays)

[0481]

[0260] Validation of cytotoxicity includes confirmation of non-replication of the oncolytic virus in non-tumor cells. Tumor cell lines are cultured from plating tumor cells, infection is performed in a serial dilution with wild-type and modified oncolytic virus, and three days after infection the supernatant is collected in order to confirm viral replication by TCID50 and RT-PCR.

[0482]

[0261] Viral copy titration of the supernatant from viral infection of tumor cells was performed by RT-PCR (Figure 40). As expected, a positive viral RNA titer was found, in quantities on the order of 10e8 copies / pL, considered to be high viral replication (Figures 40, 41 and Table 8).

[0483] Table 8: Final titration (copies / pL) of the samples.

[0484] ENV Titration NS5 Titration Sample

[0485] (copies / pL) (copies / pL) Wild Virus Infection - 1 8.41 E+07 8.29E+06 Wild Virus Infection - 2 1.50E+08 1.27E+07 Wild Virus Infection - 3 1.07E+08 1.02E+07 VSZ-018 / SE-P1 Infection - 1 3.94E+07 7.92E+06 VSZ-018 / SE-P1 Infection - 2 7.09E+07 7.83E+06 VSZ-018 / SE-P1 Infection - 3 7.24E+07 7.28E+06 VSZ-019 / SE-P1 Infection - 1 8.66E+07 1.29E+07 VSZ-019 / SE-P1 infection - 2 5.73E+07 5.54E+06 VSZ-019 / SE-P1 infection - 3 8.36E+07 8.55E+06 VSZ-020 / SE-P1 infection - 1 6.42E+07 5.86E+06 VSZ-020 / SE-P1 infection - 2 3.76E+07 3.87E+06 VSZ-020 / SE-P1 infection - 3 2.30E+07 3.19E+06 VSZ-021 / SE-P1 infection - 1 6.08E+07 5.75E+06 VSZ-021 / SE-P1 infection - 2 1.47E+08 1.38E+07

[0486]

[0487] VSZ-021 / SE-P1 infection - 3 7.48E+07 7.48E+06

[0488]

[0262] Viruses released after infection of tumor cells showed high titers of active virus, all above 10 8 PFU (Table 9). This result

[0489] The REPLACEMENT SHEET (RULE 26) is noteworthy because it had not been previously described in scientific studies with wild Zika virus, in which the viral load (PFU) after tumor infection remained below 10. 3 This increased oncolytic potential is attributed to the unique characteristics of the sequence designed with two MREs. All candidates tested showed oncolytic activity, with confirmed viral replication (Table 9). Viral shedding after tumor infection was also detected, indicating the presence of active virus at high titers.

[0490]

[0263] Samples sent for TCDI50 / PFU at CROP Labs were:

[0491] a. PPA-069 - Supernatant derived from infection of USP7 with VSZ-018 / SE-P1

[0492] b. PPA-070 - Supernatant derived from infection of USP7 with VSZ-019 / SE-P1

[0493] c. PPA-071 - Supernatant derived from infection of USP7 with VSZ-020 / SE-P1

[0494] d. PPA-072 - Supernatant derived from infection of USP7 with VSZ-021 / SE-P1

[0495] Table 9: Viral titration by TCDI50 / ml_ and conversion to PFU / mL of the samples.

[0496] Sample TCDI50 / mL PFU / mL

[0497] PPA-069 5.62E+08 3.94E+08

[0498] PPA-070 3.89E+09 2.72E+09

[0499] PPA-071 5.62E+08 3.94E+08

[0500] PPA-072 5.62E+08 3.94E+08

[0501]

[0502]

[0264] Viral copy titration of the supernatant from viral infection of Daoy, ONS-76, and LN-18 tumor cells was performed by RT-PCR in order to evaluate positive viral replication in tumor cells and confirmation.

[0503] REPLACEMENT SHEET (RULE 26) of the oncolytic effect in a quantitative, and not just qualitative, manner (Figure 42).

[0504]

[0265] Positive viral replication was observed 3 days after infection in all synthetic virus candidates tested, both passage 2 and passage 10, in both MOIs (1 and 2), with viral copy numbers similar to those observed in the positive control - wild-type virus (WT) (Figure 59), confirming the findings of cell death (oncolytic effect) observed in images 2 to 4. The candidates from the more advanced passages, P10, were those that presented the highest levels of viral replication, often exceeding those of the wild-type virus. The glioblastoma cell line proved to be more resistant, but still susceptible to the oncolytic effect of the developed viral therapy.

[0505] Example 6 - pZiOZ Diagram

[0506]

[0266] 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.

[0507] Example 7 - Production of the viral vector (EviOZ) - Plasmid design containing structural proteins and some non-structural proteins

[0267] EviOZ production is carried out through the transfection of two plasmids (Figure 7). 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 correspond to those of ZIKV (beta-globin, as an example) as one of the ways to avoid encapsulation of this RNA by EviOZ. Among the ZIKV sequences necessary for the production of EviOZ, the sequence of the protein of

[0508] REPLACEMENT SHEET (RULE 26) capsid, membrane and envelope, crucial for the formation of VLPs (virus-like particles), in addition to some non-structural proteins that assist in the production of EviOZ.

[0509]

[0268] The pZiOZ design is based on an expression plasmid for eukaryotic cells. pZiOZ incorporates the 5' and 3' UTR regions of ZIKV, strain KX197192.1. The 5' UTR region includes part of the capsid protein sequence, which is essential for the formation of the RNA secondary structure. This structure favors RNA replication by the non-structural protein 5 (NS5), which, in pZiOZ, is derived from the ZIKV strain MH882527.1. The 3' UTR region is composed of a segment of NS4A, the complete sequence of the 2K region, and a segment of NS4B, these sequences being necessary for the encapsulation of RNA by the ZIKV structural proteins (Figure 23A).

[0510]

[0269] EviOZ production is carried out through the transfection of two plasmids (Figure 7). The pCME is the eukaryotic cell expression plasmid necessary for the production of structural proteins (Figure 43B). This plasmid has 5' and 3' UTR regions that do not correspond to those of ZIKV to improve RNA stability (Figure 43B) and also as one of the ways to prevent encapsulation of this RNA by EviOZ. Among the ZIKV sequences necessary for EviOZ production were those of the capsid, membrane, and envelope proteins, crucial for the formation of VLPs (virus-like particles), as well as some non-structural proteins that assist in EviOZ production.

[0511]

[0270] The pCME and pZiOZ plasmids were strategically designed in silico to obtain DNA sequences for the production of the viral vector. After obtaining the sequences in silico, they were submitted to companies in the field of DNA synthesis. After synthesis, the plasmids were sequenced by NGS and the obtained sequences were aligned with those designed in silico to observe the efficiency of gene synthesis. The correct synthesis of the pCME and pZiOZ plasmids was confirmed, obtaining 100% identity for each alignment group (Table 10).

[0512] REPLACEMENT SHEET (RULE 26) Table 10: Alignment of plasmid sequences containing the DNA regions required for the viral vector drawn in silico and their respective synthesized and sequenced DNA sequences, confirming correct DNA synthesis.

[0513] A) B) C)

[0514] Information Information Information

[0515] pCME [In silico drawing]: pZiOZ [In silico drawing): pZiZZ CRISPR pZiOZ_CRISPR [In silico drawing]:

[0516] Coverage: 7267 / 7267 | Coverage: 6779 / 6779 | Coverage: 5259 / 5259 | 100% 100% 100%

[0517] Gaps in coverage: 0 Gaps in coverage: 0 Gaps in coverage: 0 Identity: 7267 / (7267+0) | Identity: 6779 / (6779+0) | Identity: 5259 / (5259+0) | 100% 100% 100%

[0518] pCME_Synthesized pZiOZ_Synthesized pZiOZ_CRISPR Synthesized [Sequencing]: [Sequencing]: [Sequencing]:

[0519] Coverage: 7267 / 7267 | Coverage: 6779 / 6779 | Coverage: 5259 / 5259 | 100% 100% 100%

[0520] Gaps in coverage: 0 Gaps in coverage: 0 Gaps in coverage: 0 Identity: 7267 / (7267+0) | Identity: 6779 / (6779+0) | Identity: 5259 / (5259+0) | 100% 100% 100%

[0521]

[0522]

[0271] To obtain ideal quantities of plasmids for product development, the synthesized pCME and pZiOZ plasmids were cloned into chemocompetent E. coli bacteria using the heat shock technique and then seeded in LB-agar bacterial culture medium containing ampicillin for transformant selection. Two colonies of each plasmid were used for inoculation in liquid LB bacterial culture medium containing ampicillin for transformant selection and bacterial growth. These inocula were processed and plasmid extraction was performed using the MidiPrep technique.

[0523]

[0272] For each colony, the generated plasmids were evaluated for their identity by enzymatic restriction of DNA, according to the cleavage site.

[0524] The REPLACEMENT SHEET (RULE 26) of each enzyme (Fig. 44) was identified through individual digestion by the restriction enzymes BamHI, Xbal, and EcoRI (Table 11, Figs. 45-47). Correct identity was confirmed in all plasmids, as all predicted fragments were observed by agarose gel electrophoresis (Table 2, Figs. 45-47).

[0525]

[0273] The pZiOZ plasmid was subjected to enzymatic digestion with EcoRI to remove the NS4A-B, 2K and NS5 fragments. The digestion product was subjected to agarose gel electrophoresis. The fragment corresponding to the plasmid backbone was excised from the gel and purified using the agarose gel extraction kit according to the manufacturer's instructions.

[0526]

[0274] The purified DNA was quantified using a Nanodrop spectrophotometer, and the linearized plasmid was used in a ligation reaction to circularize the vector. The reaction was conducted with DNA ligase, following the protocol recommended by the manufacturer.

[0527]

[0275] Subsequently, the re-ligated plasmid (named pZiOZ Eco RI - Fig 44D) was used to transform competent bacteria. The cells were transformed by heat shock technique. The cells were seeded in LB-agar medium containing ampicillin for transformant selection. Two colonies of each plasmid were used for inoculation in liquid LB bacterial culture medium containing ampicillin for transformant selection and bacterial growth. These inocula were processed and plasmid extraction was performed using the MidiPrep technique.

[0528]

[0276] To confirm the restriction profile, the DNA from each colony was individually digested with the enzymes EcoRI, BamHI, and Xbal, and the digestion products were analyzed by agarose gel electrophoresis. The digestion patterns obtained, along with the undigested plasmids, were visualized using a transilluminator, and the fragment sizes were compared with a 1 kb ladder (Table 11, Fig. 47), confirming the removal of the NS4A-B, 2K, and NS5 regions from the pZiOZ plasmid and its correct circularization at the EcoRI site, since its digestion after re-circularization with the EcoRI enzyme only linearized the plasmid (Table 2, Fig. 47).

[0529] REPLACEMENT SHEET (RULE 26) Channels 6 and 10).

[0530]

[0277] Table 11: Enzyme restriction map of the pCME, pZiOZ, pZiOZ_CRISPR and pZiOZ EcoRI plasmids (without NS4 and NS5) showing the location of each digestion with the BamHI, Xbal and EcoRI enzymes, as well as the expected fragments for each enzyme restriction reaction.

[0531] Plasmid / Non-BamHI Xbal EcoRI Fragments Colony digested (fragments) (fragments) (Fragments) visualized (fragment)

[0532] pCME / 1 Fig. 45 Fig. 45 Fig. 46 Fig. 46 Yes Channel 1 and Channel 6 Channel 3 Channel 2

[0533] Fig. 22 (12.128 kb (12.128 kb (8.285 and 3.913 Channel 1 linearized) linearized)* kb)

[0534] (12,128 kb)

[0535] pCME / 2 Fig. 45 Fig. 45 Fig. 45 Fig. 46 Yes Channel 2 and Channel 7 Channel Channel 5

[0536] Fig. 22 (12,128 kb 12(12,128 kb (8,285 and 3,913 Channel 4 linearized) linearized)* kb)

[0537] (12,128 kb)

[0538] pZiOZ / 1 Fig. 45 Fig. 45 Fig. 45 Fig. 46 Yes Channel 3 and Channel 8 Channel 11 Channel 7

[0539] Fig. 46 (12,123 kb Fig. 47 (8,550 kb and Channel 6 linearized) Channel 13 3,773kb)

[0540] (12,123 kb) (6,942; 4,743

[0541] and 0.438 kb)

[0542] pZiOZ / 2 Fig. 45 Fig. 45 Fig. 45 Fig. 46 Yes Channel 4 and Channel 9 Channel 14 Channel 9

[0543] Fig. 22 (12.123 kb (6.942; 4.743 (8.550 kb and Channel 8 linearized) and 0.438 kb) 3.773kb)

[0544] (12,123 kb)

[0545] pZiOZ_CRI Fig. 46 Fig. 46 Fig. 46 Fig. 46 Yes SPR / 1 Channel 10 Channel 12 Channel 13 Channel 11

[0546]

[0547] REPLACEMENT SHEET (RULE 26) (10,603 kb) (10,603 kb (10,603 kb (10,159 and

[0548] (does not digest) linearized) 0.444 kb)

[0549] pZiOZ_CRI Fig. 47 Fig. 47 Fig. 47 Fig. 47 Sim SPR / 2 Channel 1 Channel 3 Channel 4 Channel 2

[0550] (10,603 kb) (10,603 kb (10,603 kb (10,159 and

[0551] (does not digest) linearized) 0.444 kb)

[0552] pZiOZ Fig. 47 Fig. 47 Fig. 47 Fig. 47 Sim EcoRI / 1 Channel 5 Channel 7 Channel 8 Channel 6

[0553] (Without NS4 (8,550 kb) (8,550 kb (8,550 kb (8,550 kb)

[0554] (and NS5) does not digest) linearized) linearized)

[0555] pZiOZ Fig. 47 Fig. 47 Fig. 47 Fig. 47 Sim EcoRI / 2 Channel 9 Channel 11 Channel Channel 10

[0556] (Without NS4 (8,550 kb) (8,550 kb 12(8,550 kb (8,550 kb)

[0557] (and NS5) does not digest) linearized) linearized)

[0558]

[0559] * Xbal digests two regions in this sequence, which would generate 2 fragments, however one region is retained and does not undergo digestion, thus the plasmid is only linearized.

[0560]

[0278] EviOZ production involves:

[0561] 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).

[0562] b) Collection of the supernatant 48 hours after transfection.

[0563] c) Centrifugation to remove cellular debris.

[0564] Example 8 - Characterization of the viral vector (EviOZ)

[0565]

[0279] Q

[0566] Example 9 - Validation of viral vector production

[0567]

[0280] First, HEK-293 cells are co-transfected using two 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. Then, the supernatant is analyzed by RT-qPCR or digital PCR to

[0568] REPLACEMENT SHEET (RULE 26) detect the presence of ZiOZ-GFP / LUC in the produced viral vector.

[0569]

[0281] To verify the ability of the viral vector to deliver RNA (ZiOZ-GFP / LUC), the supernatant containing the EviOZ viral vector encapsulating ZiOZ-GFP / 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 for the validation of the efficiency of therapeutic RNA delivery to target cells.

[0570]

[0282] For viral vector production, HEK-293 cells were co-transfected using two plasmids: one containing the sequence of structural proteins along with some non-structural proteins (pCME), and the other containing the pZiOZ containing the luciferase reporter gene (ZiOZ-GFP / LUC), as an example of a gene of interest. After 48 hours, the supernatant was collected from the transfected cells.

[0571]

[0283] To verify the ability of the viral vector to deliver RNA (ZiOZ-GFP / LUC), as well as to confirm its in vitro tropism, the supernatant containing the EviOZ viral vector encapsulating ZiOZ-GFP / LUC was incubated with neural cells (Daoy). After 24 hours of viral vector delivery to the target cells, luminescence was analyzed using the lucierase assay system kit and quantified using the GLOMAX instrument, following the manufacturer's instructions.

[0572]

[0284] Observation of luciferase enzyme expression 24 hours after exposure to the viral vector in neural cells confirmed the effective production of the vector and its ability to encapsulate and deliver ZiOZ-GFP / LUC to host cells. This result validates the efficiency of RNA delivery.

[0573] REPLACEMENT SHEET (RULE 26) therapeutic to target cells.

[0574]

[0285] The absence of luminescence in the three negative control groups (Lipofectamine, PMD-005 and PMD-006), as illustrated in Figure 48, confirms the efficiency of the viral vector production strategy using two plasmids. Adherence transfection proved to be efficient, validating the delivery and production of luciferase in target cells of neural origin. This highlights the potential of the viral vector for the delivery of genes of interest in therapies for cells of neural origin.

[0575]

[0286] To confirm the importance and novelty of the fragment of the reduced NS4A and NS4B regions, including the 2K region, in the assembly of the viral vector, an analysis of the delivery of ZiOZ RNA by the vector was performed. This analysis, in line with the methodologies described, correlates the presence of the shortened fragments of the NS4A-B regions, including the 2K region, with delivery efficiency.

[0576]

[0287] To measure the impact, cells subjected to transduction with the viral vectors produced by the transfection in question were evaluated by RT-qPCR. The objective was to quantify the level of ZiOZ RNA delivery in copy number. The results obtained were compared with two fundamental control groups:

[0577]

[0288] Positive Control: Cells that received the viral vector successfully constructed from co-transfection of pCME and complete pZiOZ plasmids (containing the NS4A-B, 2K, and NS5 regions). This group establishes the encapsulation and delivery of the expected ZiOZ RNA in the presence of crucial components.

[0578]

[0289] Negative Control: Cells transfected with transfection reagent only. This group serves to demonstrate the absence of functional vector production when the viral genome structure component (pZiOZ) is missing, validating the need for both plasmids for complete and functional vector assembly.

[0579]

[0290] Result: Comparison between negative and positive controls

[0580] The REPLACEMENT SHEET (RULE 26) demonstrated that the fragment of the shortened NS4A and NS4B regions, which includes the 2K region, contains critical sequences (Figure 48). This finding is an important novelty, as it indicates that shortening these sequences maintained their essential functions, such as effective encapsulation and subsequent delivery to target cells, as well as efficient assembly, replication, and delivery of the viral genome. Therefore, these results confirm the importance of these sequences for the functionality of the viral platform.

[0581] Example 10 - Production of Viral Vector for therapy of Phelan McDermid Syndrome and DEAF1.

[0582]

[0291] Insertion of the SHANK3 gene or DEAF1 Monomer or DEAF1 Dimer into pZiOZ: The plasmids, pZiOZ_DEAF1 Monomer, pZiOZ_DEAF1 Dimer, and pZiOZ_SHANK3 were strategically designed in silico to obtain DNA sequences for the production of the viral vector and for the delivery of the genes of therapeutic interest, DEAF1 in monomeric and dimeric versions, and the SHANK3 gene, for genetic replacement strategies for genetic diseases related to syndromic autism. After obtaining the sequences in silico, they were submitted to companies in the field of DNA synthesis. After synthesis, the plasmids were sequenced by NGS, and the obtained sequences were aligned with those designed in silico to observe the efficiency of gene synthesis. The correct synthesis of the pZiOZ_DEAF1 Monomer, pZiOZ_DEAF1 Dimer, and pZioZ_SHANK3 plasmids was confirmed, achieving 100% identity for each alignment group (Table 12).

[0583] Table 12: Alignment of plasmid sequences containing genes of therapeutic interest for gene therapy designed in silico and their respective DNA sequences synthesized and sequenced by NSG, confirming correct DNA synthesis.

[0584] Information Information Information

[0585] ABC

[0586]

[0587] REPLACEMENT SHEET (RULE 26) pZiOZ_DEAF1

[0588] pZiOZ_DEAF1 Dimer pZiOZ_SHANK3 Monomer [drawing]

[0589] [Drawing in silico]: [drawing in silico]: in silico]:

[0590] Coverage: 1712 / Coverage: 3425 / Coverage: 5435 / 1712 | 100% 3425 | 100% 5435 | 100% Coverage gaps: 0 Coverage gaps: 0 Coverage gaps: 0 Identity: 1712 / Identity: 3425 / Identity: 5435 / (1712+0) | 100% (3425+0) | 100% (5435+0) | 100%

[0591] pZiOZ_DEAF1 pZiOZ_DEAF1 Dimer pZiOZ_SHANK3 Monomer Synthesized Synthesized Synthesized [Sequencing]: [Sequencing]: [Sequencing]: Coverage: 1712 / Coverage: 3425 / Coverage: 5435 / 1712 | 100% 3425 | 100% 5435 | 100% Coverage gaps: 0 Coverage gaps: 0 Coverage gaps: 0 Identity: 1712 / Identity: 3425 / Identity: 5435 / (1712+0) | 100% (3425+0) | 100% (5435+0) | 100%

[0592]

[0593]

[0292]

[0594]

[0293]

[0595] the)

[0596] b) Synthesis of the SHANK3 gene, DEAF1 monomer, or DEAF1 dimer in pZiOZ by companies in the field.

[0597] c) Cloning of the pZiOZ-SHANK3 plasmid in bacteria.

[0598] d) Enzyme restriction map of plasmids

[0599] e) Plasmid sequencing.

[0600] the)

[0601] Example 11 - Production of a Viral Vector for chemical editing based on CRISPR technology

[0602] REPLACEMENT SHEET (RULE 26)

[0294] Due to the tropism of the Zika virus for CNS cells and its sufficient carrying capacity to carry all the CRISPR Cas12 machinery, a viral vector was developed to deliver the CRISPR Cas12 machinery capable of performing gene editing in target cells, aiming at the implementation of a CNS cell editing tool and future use for therapy of genetic and / or neurodegenerative diseases. For this purpose, the viral vector for delivering the CRISPR machinery was constructed and its ability to knock out a Luciferase reporter gene in cell culture was evaluated.

[0603]

[0295] 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, including or not the fragment of the reduced NS4A and NS4B regions, including region 2, 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.

[0604]

[0296] Obtaining the pZiOZ-CRISPR construct:

[0605]

[0297]

[0606] a) Synthesis of the pZiOZ-CRISPR plasmid.

[0607] b) The pZiOZ_CRISPR plasmids were strategically designed in silico to obtain DNA sequences for delivery to the CRISPR machinery. After obtaining the sequences in silico, they were submitted to companies in the field of DNA synthesis. After synthesis, the plasmids were sequenced by NGS and the obtained sequences were aligned with those designed in silico to observe the efficiency of gene synthesis. The correct synthesis of the pZiOZ_CRISPR plasmid was confirmed, obtaining 100% identity for each alignment group (Table 0).

[0608] c) Plasmid cloning. To obtain ideal quantities of plasmids for product development, the synthesized pZiOZ_CRISPR plasmid was cloned into chemocompetent E. coli bacteria by

[0609] REPLACEMENT SHEET (RULE 26) using the thermal shock technique and then seeded in LB-agar bacterial culture medium containing ampicillin for transformant selection. Two colonies from each plasmid were used for inoculation in liquid LB bacterial culture medium containing ampicillin for transformant selection and bacterial growth. These inocula were processed and plasmid extraction was performed using the MidiPrep technique.

[0610] d) For each colony, the generated plasmids were evaluated for their identity by enzymatic restriction of DNA, according to the cleavage site of each enzyme (Fig. 20) through individual digestion by the restriction enzymes BamHI, Xbal, and EcoRI (Table 11, Figs. 45-47). Correct identity was confirmed in all plasmids, as all predicted fragments were observed in agarose gel electrophoresis (Table 11, Figs. 45-47).

[0611] e) Plasmid sequencing.

[0612]

[0298]

[0613]

[0299] Evaluation of Luciferase Knockout in Luciferase-Expressing Cells: The viability of delivering CRISPR machinery via pZiOZ was evaluated through Luciferase knockout in Luciferase-expressing cells. To this end, pZiOZ_CRISPR was transfected into a cell with transient luciferase overexpression. Shortly after, a control plasmid containing Luciferase was transfected into HEK cells for luciferase reporter gene overexpression. 24 hours after reporter gene transfection, a second transfection with pZiOZ_CRISPR was performed.

[0614] 24 hours after delivery of the CRISPR machinery, luciferase levels were analyzed to confirm delivery and functionality of the CRISPR machinery followed by luciferase gene knockout.

[0615]

[0300] Evaluation of the Efficacy of the pZiOZ System for Gene Knockout by CRISPR / Cas12 - Luciferase Knockout Assay in Model Cells: The viability and functionality of the pZiOZ delivery vector for the machinery

[0616] CRISPR / Cas12 REPLACEMENT SHEET (RULE 26) was rigorously evaluated using a gene knockout assay targeting the luciferase enzyme gene in a model cell system. This study was crucial to confirm the ability of pZiOZ to efficiently transfect the gene editing complex into cells and subsequently perform target gene knockout. Details follow:

[0617]

[0301] Reporter System Preparation: Initially, to establish the detection system, HEK cells were transfected with a control plasmid containing the Luciferase gene (reporter gene). The transfection was performed using the lipofectamine 3000 reagent. This step ensured the transient and robust overexpression of the Luciferase protein in the cells, creating a measurable and stable baseline signal.

[0618]

[0302] Delivery of the CRISPR Machinery (pZiOZ_CRISPR): 24 hours after the initial transfection of the reporter gene (Luciferase), the same cells underwent a second round of transfection. At this crucial step, the pZiOZ_CRISPR delivery vector was introduced, carrying the essential components for gene editing: the nucleotidase Cas12 and the guide RNA (sgRNA) strand specifically designed to target the cleavage site in the Luciferase gene. The choice of the 24-hour interval was strategic to allow for maximum Luciferase expression before the introduction of the knockout machinery.

[0619]

[0303] Functional Assessment and Quantification of Knockout: The success of delivery and functionality of the CRISPR system were assessed 24 hours after the second transfection (delivery of pZiOZ_CRISPR). Quantification of Luciferase activity was performed using a standardized bioluminescence assay. The significant reduction in Luciferase activity levels in cells transfected with pZiOZ_CRISPR, compared to control cells (which express Luciferase but did not receive the functional knockout pZiOZ_CRISPR), served as direct and quantifiable evidence of efficient pZiOZ delivery and successful functionality of the CRISPR / Cas12 machinery, resulting in the knockout of the Luciferase gene.

[0620] SUBSTITUTION SHEET (RULE 26)

[0304] Result: A decrease in luciferase signal was observed 24 hours after transfection of pZiOZ containing the CRISPR machinery (Figure 49). The results demonstrated that the pZiOZ vector is a viable and efficient platform for intracellular delivery of the CRISPR / Cas12 machinery, culminating in specific knockout of the target gene (Luciferase). This assay validates the use of pZiOZ for future gene editing applications.

[0621]

[0305] 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.

[0622] REPLACEMENT SHEET (RULE 26)

Claims

Claims 1. Modified flavivirus nucleotide sequence characterized by comprising at least three functionally linked nucleotide sequences, these being: (i) the nucleotide sequence encoding the Zika virus NS4A protein defined by Seq ID No. 1; (ii) the complete sequence of the Zika virus 2K protein defined by Seq ID No. 2; and (iii) the nucleotide sequence encoding the Zika Virus NS4B proteins defined by Seq ID No.

3.

2. Modified flavivirus nucleotide sequence according to claim 1 characterized by comprising the nucleotide sequence encoding the Zika virus Capsid C protein defined by Seq ID No.

4.

3. Modified flavivirus nucleotide sequence according to claim 1, characterized by comprising the nucleotide sequence of the 5' UTR defined by Seq ID No.

14.

4. Modified flavivirus nucleotide sequence according to claim 1, characterized by comprising the nucleotide sequence encoding the 2K protein defined by Seq ID No.

62.

5. Modified flavivirus nucleotide sequence according to claim 1, characterized by further comprising: - the nucleotide sequence encoding the Zika virus NS5 protein defined by Seq ID No. 5 functionally linked to 3'; - the nucleotide sequence encoding the Zika virus NS3 protein defined by Seq ID No. 6 functionally linked to 5'; - the nucleotide sequences encoding the C, M, E, NS1, NS2A, NS2B proteins of ZIKV defined by Seq ID Nos. 29, 7, 8, 10, 11 and 9, respectively, functionally linked to each other from 5' to 3'; - optionally, it comprises the nucleotide sequences 5'UTR eMRE (5' CGUGCCUUA 3', as a target of miR-124-3p) functionally linked to each other and to the 5' defined by Seq ID No. 14; - optionally, it includes the MRE nucleotide sequences (5' CCGACCUGU 3', as a target of miR-219a-2-3p) in the 2K protein defined by Seq ID No.

62.

6. A nucleotide sequence according to any of the preceding claims, characterized by additionally comprising another nucleotide sequence(s) of up to 10 kb, encoding functional proteins.

7. Modified flavivirus nucleotide sequence according to claim 6, further characterized by comprising: - the nucleotide sequences encoding the GFP and Luciferase proteins defined by SEQ IDs NO 17 and 18, respectively, functionally linked to each other from 5' to 3'; or combinations thereof.

8. Modified flavivirus nucleotide sequence according to claim 6, further characterized by comprising: - the nucleotide sequences encoding the Shank3, IRES and NS5 proteins defined by SEQ ID No. 19, 20 and 5 respectively, functionally linked to each other from 5' to 3'; or combinations thereof.

9. Modified flavivirus nucleotide sequence according to claim 6, further characterized by comprising: - the nucleotide sequences encoding Cas 12 proteins defined by Seq ID Nos. 21 and 56 functionally linked to 5'; - the nucleotide sequences encoding the Pre crRNA proteins defined by Seq ID Nos. 22, 58 and 59 functionally linked to 3'; or combinations thereof.

10. Use of the nucleotide sequence as defined in any of claims 1-9 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).

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

12. Therapeutic agent according to claim 11 characterized in that said nucleotide sequence is encapsulated in a lysosome or nanoparticle, for use as a vaccine.

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

14. Therapeutic agent according to claim 11 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.

15. Use of oncolytic virus as defined in claim 14 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.

16. Process for obtaining the therapeutic agent defined in any one of claims 11-14, characterized by comprising the steps of: - obtain a modified nucleotide sequence as defined in one of claims 1-9; and - add an excipient, adjuvant, nanoparticles and / or liposome.

17. Process according to claim 16 characterized in that the step of obtaining said nucleotide sequence comprises the construction of two plasmid DNAs.

18. 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 one of claims 11-14.

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