WO2022186708A1 - Method for the development of stem cell lines - Google Patents

Method for the development of stem cell lines Download PDF

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WO2022186708A1
WO2022186708A1 PCT/PT2022/050010 PT2022050010W WO2022186708A1 WO 2022186708 A1 WO2022186708 A1 WO 2022186708A1 PT 2022050010 W PT2022050010 W PT 2022050010W WO 2022186708 A1 WO2022186708 A1 WO 2022186708A1
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seq
chromosome
cells
edited
cell
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Portuguese (pt)
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João Manuel Coelho CARREIRA
Domingos Manuel Pinto Henrique
Maria Margarida Fonseca Rodrigues DIOGO
Carlos André Vitorino RODRIGUES
Cláudia Daniela Canelas MIRANDA
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Instituto Superior Técnico
Instituto De Medicina Molecular
Faculdade de medicina da universidade de Lisboa
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Definitions

  • the present disclosure relates to a method for developing stem cell lines and derived products.
  • This method belongs to the fields of molecular biology and cell culture, and can be applied in medicine, preferably in the field of transplantation.
  • the method now disclosed allows obtaining off-the-shelf cell lines prepared for the loss of one or more specific native chromosomes, as well as their replacement by equivalent exogenous chromosomes.
  • the present method makes it possible to create human stem cell lines primed for the loss of the indigenous chromosome 6 pair (universal off-the-shelf lines) and simultaneously obtain their replacement by a patient's Chromosome 6 pair. In this way, all cells resulting from their subsequent differentiation will carry the exact and total major histocompatibility complex of the patient, solving the problem of major incompatibility in allotransplantation.
  • the rejections are the consequence of the recognition by the immune system of differences between the proteins expressed in the cell membrane of the cells of the donor and those of the host.
  • This recognition is made from human leukocyte antigens (Human Leukocyte Antigens - HLA), divided into HLA Class I receptors and HLA Class II receptors.
  • Class I HLA receptors are expressed on the cell membrane of almost every cell in the human body.
  • Class II receptors are expressed in cells belonging to the immune system, called Antigen Presenting Cells (APC), as is the case with some subtypes of dendritic cells, macrophages and B cells.
  • APC Antigen Presenting Cells
  • the result could be the death of the patient and/or the destruction of the allo-transplant, so currently the post-transplant treatment includes immunosuppressants and other drugs to prevent rejection.
  • immunosuppression has harmful side effects for the patient's quality of life and can even put their survival in jeopardy. Infectious complications from viruses, bacteria and infestations by fungi or parasites will ensue, putting the patient's life in danger.
  • the loss of normal surveillance of the immune system can lead to the appearance of malignant tumors.
  • the allo-transplanted cells would be modified so as not to express HLA Class I or/and HLA Class II receptors, or the cells would express a high amount of specific HLA Class I receptors (HLA G), or other recipients, in order to obtain what has been called "transparent allo-transplants".
  • HLA G HLA Class I receptors
  • this "transparency" of the patient's immune system is a huge problem, because any infection, infestation or cancerous transformation in the allo-transplanted cells will be very difficult to detect in a timely manner.
  • MHC Major Histocompatibility Complex
  • b-2-microglobulin which is translated from a non-polymorphic gene residing on human chromosome 15 and which is an essential component of Class I HLA receptors.
  • the MHC in humans is composed of between 4.2 and 7 megabases of acid sequences.
  • deoxyribonucleic acid, (DNA) in each of the short arms of the pair of chromosomes 6.
  • DNA deoxyribonucleic acid
  • the cells produced according to the protocol by Tada et.al. can be completely compatible with the patient because they express their exact and complete HLA set, but at the same time they can add the characteristic of being healthy, since the replacement of chromosome 6 is performed on hPSCs that are free of pathogenic mutations.
  • Document US 20090264312 describes a method for obtaining the induced loss of a specific chromosome using a technique that makes use of a Cre nuclease with inverted LoxP recombination sites. Activation of the system by the introduction of Cre nuclease induces transformation of the chromosome prepared with inverted LoxP sites into a dicentric chromosome, that is, with two centromeres, which the cell rejects because it is not compatible with the replication/segregation during cell mitosis.
  • this method never evolved into clinical use, as the induction of dicentric chromosomes, which allows the induced loss of said chromosomes by the cells, is also the cause of serious genomic mutations in the cells that could survive.
  • CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9
  • CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9
  • these strategies can leave chromosomal material without means of selection and identification, which can lead to mutations/translocations.
  • the authors cannot guarantee the safety of the final product for its use in Medicine, since it is not possible to guarantee that all the fragments in which the chromosome is cleaved will be eliminated from the cells, and that they will not give rise to mutations/translocations. .
  • the authors thought of their strategy to solve polyploidy, such as trisomies. They do not refer at any time to the replacement of equivalent chromosomes to solve a clinical problem. They only recognize its usefulness for the development of disease models.
  • microcells which are composed of: a membrane with similar composition with that of the cytoplasmic membrane; cytoplasmic components of the donor cell; and the isolated chromosome/s inside it. From its first description to the present, small modifications have been introduced improving the efficiency of its isolation, conservation and the fusion capacity of microcells with euploid or aneuploid cells. This fusion of cells with microcells allows the transfer of the isolated chromosome/s to its interior, becoming part of its genome.
  • the present disclosure describes a new, industrializable and inventive method capable of producing off-the-shelf universal stem cell lines, edited so that they can lose a chromosome or more, in eukaryotic cells, or the chromosome of prokaryotic cells, with the possibility of replacing it with another equivalent exogenous chromosome/s, having genes of different or corrected sequences.
  • the present method allows the triple control of the phases of cell selection, which represents a safety advantage for the subsequent use of cells in a clinical scenario, namely in the therapy or treatment of monogenic, multigenic diseases or in transplants of replacement by loss of cellular function of cells without pathogenic mutations (e.g. myocardial infarction from atherosclerosis).
  • two cassettes of DNA sequences are edited into closed genes - ie genes not transcribed at the stem stage of the cells in question - in proximity to and on either side of the centromere.
  • These novel cassettes comprise promoters, fluorescent protein genes, genes encoding proteins that confer antibiotic resistance and/or sensitivity to anti-virals (eg acyclovir) or other drugs (eg tamoxifen).
  • Each of these cassettes also comprises a DNA sequence absent from the genome of the species whose stem cells are to be modified and susceptible to cleavage of the DNA double strand by the editor nuclease systems (clustered and regularly interspaced short palindromic repeat system nucleases). (CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats), zinc fingers (ZNF), Transcription Activator-like Effector Nucleases (TALENs), or others).
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • ZNF zinc fingers
  • TALENs
  • the DNA sequence susceptible to double-stranded DNA cleavage by genomic editing systems is a numeromer.
  • nullomer is to be understood as a short sequence of DNA that is not present in the genome of the unedited cells of the organism in question.
  • all cells, microtissues, tissues, or organs obtained from the differentiation of stem cells edited in their genome by the method described in the present disclosure are considered derivative products. Being different from the stem cells that gave rise to them, they can only be derived or differentiated from them by the method described in the present disclosure, in order to create other stem cell lines with the same edition or cellular, tissue or organ products for transplantation.
  • the method for developing stem cell lines and derived products is to edit the stem cell lines so that they are available as off-the-shelf universal stem cell lines, with the ability from losing one or more chromosomes or as a patient's stem cell lines (Ex. Hematopoietic, CD34+, mesenchymal cells, etc.), for use in the treatment of monogenic or multigenic diseases.
  • the induced loss of said chromosomes is achieved using nucleases (CRISPR, ZNF, TALENs, or others), which cause double-stranded DNA cleavages in specific sequences, located near and on each side of the centromere of the edited chromosomes.
  • the specific sequences where the cleavages in the DNA double strand take place are part of two gene cassettes (Cassette 1 and Cassette 2), whose composition is important both in the identification and selection phase of correctly edited stem cell clones. , as well as later when the deleted chromosomes are replaced by equivalent chromosomes.
  • the fluorescence and drug resistance and/or sensitivity markers, included in the cassettes, are responsible for these functional and technical characteristics in the edited cells and, later, in the cells in which the deleted chromosome is replaced by an equivalent one.
  • multipotent cells eg hematopoietic progenitor cells
  • fully differentiated cells eg pancreatic beta cells
  • the fluorescence proteins, antibiotic resistance and drug susceptibility allow choosing only those cell clones that produce them, and eliminating all others.
  • the developmental stage of transplants allow you to select only cells that do not produce them, a sign that they have completely lost the edited pair of chromosomes and that they only have the replacement pair of chromosomes.
  • stem cells are harvested from the patients themselves, and native chromosomes with pathological mutations replaced with chromosomes from healthy donors, by the method described in the present disclosure.
  • the derived products are then used in the treatment of monogenic or multigenic diseases.
  • off-the-shelf universal stem cell lines are obtained, prepared from the method described in the present disclosure, wherein the loss of the chromosome containing the genes that translate HLA receptors occurs.
  • these genes are found on chromosome 6.
  • the donor's indigenous chromosome 6 is removed and replaced by an exogenous chromosome 6, isolated from the cells of the patient and future host. Consequently, cells differentiated from off-the-shelf human stem cells begin to express exactly the same HLA receptors as the patient, being completely compatible with the patient.
  • clusters of cells, micro-tissues, tissues or organs derived from the modified stem cells are also compatible, and can be transplanted into the patient without the usual risks of rejection.
  • the original stem cells are healthy, showing no disease-causing mutations. Therefore, products derived from off-the-shelf universal stem cell lines edited by the method described in the present disclosure are healthy and can be used in the treatment of diseases that depend on single or multigene mutations, unless the mutation in question is found on the patient's chromosome 6.
  • either the patient's human chromosome 6, which will replace the indigenous chromosome 6 of off-the-shelf universal stem cells, or the healthy donor chromosome that will replace the patient's mutated chromosome can be transferred by the microcell Mediated Chromosome Transfer (MMCT) technique.
  • MMCT microcell Mediated Chromosome Transfer
  • exogenous chromosomes can be transferred by microinjecting the contents of the microcells into the target cells. In both cases, the genetic material is transferred together with the nuclease system responsible for the subsequent cleavage of sequences normally absent from the genome, but present on edited native chromosomes (CRISPR, ZNF, TALENs, or others).
  • Methods for aligning sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA.
  • the BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10 ) calculates the percent sequence identity and performs a statistical analysis of the similarity between the two sequences.
  • the software to perform the BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • Overall percentages of similarity and identity can also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10; 4:29.
  • MatGAT an application that generates similarity/identity matrices using proteins or DNA sequences). Sequence identity values, which are reported in the present disclosure as a percentage, were determined on the entire base sequence using BLAST with default parameters.
  • the present disclosure relates to a method for editing the stem cell genome comprising the following steps: (i) preparing at least one gene cassette, wherein the gene cassette comprises a promoter, a marker, and a gene sequence absent from the genome of the stem cells to be edited, wherein the marker comprises at least one gene encoding a protein of: fluorescence, antibiotic resistance, drug sensitivity, or combinations thereof; and wherein the genetic sequence absent from the genome of the stem cells to be edited is cleavable by at least one genomic editing nuclease system, preferably the CRISPR, ZNF, TALENs system, or mixtures thereof; (ii) inserting at least one genetic cassette into a chromosome of the cell to be edited by means of cleavages in the double strand of DNA near and on either side of the centromere of the chromosome of the cell to be edited, these cleavages of the double strand of DNA being produced by nucleases preferably from the CRISPR, ZNF, TALENS, Trans
  • the genetic sequence absent from the stem cell genome to be edited comprises a nullomer.
  • the nullomer comprises at least one sequence having at least 90% identity of the following sequences: Seq. ID No. 24, Seq. ID No. 25, Seq. ID No. 26, Seq. ID No. 27, Seq. ID No. 28, Seq. ID No. 29, Seq. ID No. 30, Seq. ID No. 31, Seq. ID No. 32, Seq. ID No. 33, Seq. ID No. 34, Seq. ID No. 35, Seq. ID No. 36, Seq. ID No. 37, Seq. ID No. 38, Seq. ID No. 39, Seq. ID No. 40, Seq. ID No. 41, Seq. ID No. 42 or mixtures, multiples or fractions thereof.
  • the nucleomer sequence comprises 95% identity to Seq. ID No. 24, Seq. ID No. 25, Seq. ID No. 26, Seq. ID No. 27, Seq. ID No. 28, Seq. ID No. 29, Seq. ID No. 30, Seq. ID No. 31, Seq. ID No. 32, Seq. ID No. 33, Seq. ID No. 34, Seq. ID No. 35, Seq. ID No. 36, Seq. ID No. 37, Seq. ID No. 38, Seq. ID No. 39, Seq. ID No. 40, Seq. ID No. 41, Seq. ID No. 42, or mixtures, multiples or fractions thereof; preferably 96%, 97%, 98%, 99% or the like.
  • the method described in the present disclosure further comprises a step of replace the indigenous chromosome comprising the genetic cassette with an unedited exogenous equivalent chromosome.
  • replacement of the chromosome comprising the gene cassette with an equivalent chromosome is done by microcell-mediated chromosomal transfer.
  • the marker encodes a protein that confers sensitivity to drugs, in preference to tamoxifen, acyclovir or combinations thereof.
  • editing the genome of the cell to be edited aims at the total deletion of at least one chromosome, preferably a pair of chromosomes.
  • the chromosome of the cell to be edited is chromosome 6.
  • DNA double strand cleavages occur by the addition of nucleases, preferably CRISPR nucleases, ZNF, TALENs, Transposases, tyrosine site-specific recombinases, T-SSRs ), site-specific serine recombinases (S-SSRs), or others.
  • nucleases preferably CRISPR nucleases, ZNF, TALENs, Transposases, tyrosine site-specific recombinases, T-SSRs ), site-specific serine recombinases (S-SSRs), or others.
  • the present disclosure further comprises a genetic cassette comprising at least one promoter, a marker, and a genetic sequence absent from the genome of the stem cells to be edited, wherein the marker comprises at least one gene encoding a protein of fluorescence, antibiotic resistance, drug sensitivity, or combinations thereof; and wherein the genetic sequence absent from the genome of the stem cells to be edited is cleavable by at least one nuclease system of genome editing, preferably CRISPR, ZNF, TALENs, or mixtures thereof.
  • the genetic sequence absent from the stem cell genome to be edited comprises a nullomer.
  • the nullomer comprises at least one sequence with at least 90% sequence identity Seq. ID No. 24, Seq. ID No. 25, Seq. ID No. 26, Seq. ID No. 27, Seq. ID No. 28, Seq. ID No. 29, Seq. ID No. 30, Seq. ID No. 31, Seq. ID No. 32, Seq. ID No. 33, Seq. ID No. 34, Seq. ID No. 35, Seq. ID No. 36, Seq. ID No. 37, Seq. ID No. 38, Seq. ID No. 39, Seq. ID No. 40, Seq. ID No. 41, Seq. ID No. 42 or mixtures, multiples or fractions thereof.
  • the present disclosure further describes a genetic cassette as described herein for use in medicine or veterinary medicine.
  • the gene cassette is for use in the treatment of genetic diseases, namely monogenic or multigenic diseases, preferably hemophilia, sickle cell disease, thalassemia, Fanconi anemia, Alagille syndrome, congenital glycosylation disease, or retinitis pigmented.
  • genetic diseases namely monogenic or multigenic diseases, preferably hemophilia, sickle cell disease, thalassemia, Fanconi anemia, Alagille syndrome, congenital glycosylation disease, or retinitis pigmented.
  • the gene cassette comprises at least 90% identity with Seq. ID No. 43, or Seq. ID No. 44.
  • the gene cassette comprises at least 95% identity with Seq. ID No. 43, or Seq. ID No. 44. [0049] In another embodiment, the gene cassette comprises at least 99% identity with Seq. ID No. 43, or Seq. ID No. 44.
  • the present disclosure also describes a vector comprising at least one genetic cassette described in the present disclosure.
  • One aspect of the present disclosure comprises edited stem cells obtained by the method described in the present disclosure, which comprise the cassette described in the present disclosure.
  • the edited stem cells comprise at least the replacement of an indigenous chromosome with an exogenous chromosome, without comprising residues, i.e. and. chromosomal material, from the indigenous chromosome.
  • the present disclosure also comprises a cell, microtissue, tissue or organ obtained from the differentiation of the edited stem cells described in the present disclosure after replacement of the edited chromosome pair with its exogenous equivalent.
  • One aspect of the present disclosure comprises the cell, microtissue, tissue or organ described in the present disclosure for use in medicine.
  • the cell, microtissue, tissue or organ is for use in the treatment of genetic diseases, preferably monogenic or multigenic diseases, most preferably hemophilia, sickle cell disease, thalassemia, Fanconi anemia, Alagille, congenital disease of glycosylation, or retinitis pigmentosa.
  • genetic diseases preferably monogenic or multigenic diseases, most preferably hemophilia, sickle cell disease, thalassemia, Fanconi anemia, Alagille, congenital disease of glycosylation, or retinitis pigmentosa.
  • the cell, microtissue, tissue or organ is for use in decreasing rejection of the transplanted cell, microtissue, tissue or organ.
  • the cell, microtissue, tissue or organ is for use in eliminating or lessening autoimmune disease.
  • the cell, microtissue, tissue or organ is for use in eliminating or lessening an infectious disease in a patient, in particular Acquired Immunodeficiency Syndrome (AIDS).
  • AIDS Acquired Immunodeficiency Syndrome
  • the cell, microtissue, tissue, or organ is for use in transplantation, for integral material or functional replacement, or for complementation of a diminished function of a subject's cells, tissues, or organs .
  • the cell, microtissue, tissue or organ lessens rejection by the transplanted subject.
  • the immune response of the transplanted subject is eliminated or diminished.
  • the cell, microtissue, tissue or organ is for use in replacing a patient's hematopoietic progenitors, (known as a bone marrow transplant), following therapeutic or accidental exposure or in a warfare environment to ionizing radiation, chemotherapy, toxic substance or combinations thereof.
  • hematopoietic progenitors known as a bone marrow transplant
  • the present disclosure further comprises a cell, microtissue, tissue or organ described in the present disclosure for use in medicine or veterinary medicine.
  • the cell, microtissue, tissue or organ are for use in the treatment of genetic disorders, preferably monogenic or multigenic disorders, most preferably hemophilia, sickle cell disease, thalassemia, Fanconi anemia, Alagille syndrome, congenital glycosylation disease, or retinitis pigmentosa.
  • the cell, microtissue, tissue or organ is for use in the treatment of Hemophilia A, Glanzmann's Thrombasthenia, Equine Atypical Thrombasthenia, von Willebrand Disease, Prekallikrein Deficiency, Polysaccharide Storage Myopathy Type 1 and Type 2, Glycogen Branching Enzyme Deficiency, or Myosin 2X heavy chain myopathy, in horses; beta-mannosidase deficiency, Bovine Leukocyte Adhesion Deficiency, Hereditary Zinc Deficiency, or Citrulemia in cattle; Hemophilia A; or von Willebrand disease, in canines.
  • Figure 1 Schematic representation of human chromosome 6 and location of the BAG2 1 gene near centromere 2 in the long arm 4.
  • the LGSN 3 gene is located near the centromere 2, but in the short arm 5.
  • Figure 2 Representation of an embodiment of transfection of virgin stem cell line, with CRISPR/Cas9 + sgRNA/BAG2 6 and Cassette 17 for editing the Human Chromosome 6 BAG2 Gene.
  • CRISPR/Cas9 + sgRNA/BAG2 6 nuclease causes double-stranded DNA cleavage in the BAG2 gene. This cleavage is then repaired by the cell with the integration of the set of sequences comprised in Cassette 17, with the exception of the homology arms HA5' and HA3' (insert 8), using Homology Directed Repair. HDR).
  • [PGKpmt] means phosphoglycerate kinase promoter; [PuroR] means Puromycin resistance transferase; [GFP] means Green Fluorescence Protein; [2pA] represents the polyadenylation sequence.
  • FIG. 3 Representation of an embodiment of the transfection of the stem cell line previously edited in the BAG gene 2, with CRISPR/Cas9 + sgRNA/LGSN 11 and Cassette 29 for editing the Human Chromosome 6 LGSN Gene.
  • CRISPR/Cas9 + sgRNA/LGSN 11 nuclease causes double stranded DNA cleavage in the LGSN gene. This cleavage is then repaired by the cell with the integration of the set of sequences comprised in Cassette 29, with the exception of the homology arms HA5' and HA3' (insert 10), using Homology Directed Repair (HDR).
  • HDR Homology Directed Repair
  • [EOS pmt] corresponds to the SSR2 enhancer sequence downstream of the SOX2 gene sequence;
  • [TK] corresponds to the truncated sequence of the Herpes simplex virus Thymidine kinase gene;
  • [SV40pA] corresponds to the SV40 polyadenylation sequence;
  • [EFloc pmt] corresponds to the human elongation factor EFl promoter;
  • [NeoR] corresponds to the aminoglycoside transferase sequence;
  • [mCherry] corresponds to the sequence of the monomeric derivative of the red fluorescent protein DSRed;
  • [bGH pA] corresponds to the bovine growth hormone polyadenylation sequence.
  • Figure 4 Representation of an embodiment of the Human Chromosome 6 deletion.
  • the double-edited stem cell line near and on either side of the centromere (BAG2 gene and LGSN gene) is transfected with CRISPR/Cas9/gRNA Nulomer 12 and 13.
  • the nuclease produces two cleavages of the DNA double strand of the chromosome, separating it into 3 parts.
  • a chromosome in these circumstances is deleted in 40% of the cells because it cannot secrete during mitosis.
  • Figure 5 Polymerase chain reaction (PCR) test to evaluate the correct insertion of the insert in the BAG2 gene in hiPSCs.
  • the numerals 15;16;17;18;19;20;21 represent the primers that correspond to SEQ. ID NO: 15, SEQ. ID NO: 16, SEQ. ID NO: 17, SEQ. ID NO: 18, SEQ. ID NO: 19, SEQ. ID NO: 20 and SEQ. ID No. 21, respectively.
  • WT Naive cell genomic DNA (Negative control).
  • FIG. 6 Flow Cytometry, percentage of Green Fluorescent Protein (GFP) expression in 4 samples of human induced Pluripotent Stem Cell (hiPSCs). of CRISPR/Cas9-BAG2sgRNA + Cassetel according to the protocol described in this document.
  • Figure 7 Flow Cytometry, percent of Green Fluorescent Protein (GFP) expression in 2 samples (10 6 cells/sample) of hiPSCs. Day 6 of CRISPR/Cas9-BAG2sgRNA + Cassetel Co-transfection according to protocol described in this document.
  • the cells Prior to performing the flow cytometry, the cells were stained using the kit: LIVE/DEADTMFixable Dead Cell Stain Kit, ThermoFisher ScientificTM. This method allows for a more accurate assessment of the percentage of GFP+ cells that are viable in the culture.
  • hPSC human pluripotent stem cells
  • hiPSC human induced pluripotent stem cells
  • the hiPSCs are obtained from adult human cells, namely fibroblasts.
  • the induction of pluripotency occurs through the over-expression of the Yamanaka Factors, i.e. Oct4, Sox2, C-mic and KLF4, which can be made from the transfection of the recombinant factors themselves, from retroviral vectors, from small chemical molecules, from microRNAs, from Piggy-Bac transposons, which promote their over-expression in adult cells, leading to their reprogramming to the condition of pluripotent cells.
  • the Yamanaka Factors i.e. Oct4, Sox2, C-mic and KLF4
  • retroviral vectors from small chemical molecules
  • microRNAs from Piggy-Bac transposons
  • the genetic alterations introduced into inactive genes of the cells to be altered ensure that there is no change to the normal cell functioning in the culture phase, in which the edited cells are kept until their further use.
  • the edited indigenous chromosome has already been lost by the cells, and replaced by its equivalent that does not contain the aforementioned edit.
  • the presence of fluorescence and antibiotic selection markers and drug sensitization genes make it possible to obtain correctly edited clones, making it possible to select clones, free from possible contamination with unedited cells.
  • the presence of a nullomeric sequence is yet another inventive device, which allows for simultaneous double-stranded DNA cleavages on both sides of the centromere, leading to the deletion/loss of the chromosome carrying this edition. Furthermore, the presence of the nullomeric sequence prevents double-stranded DNA cleavages from the replacement equivalent chromosomes from occurring. As normal cells require a normal set of chromosomes (euploidy), whenever the induced loss/deletion of a specific chromosome pair takes place in that cell, another equivalent pair of chromosomes must be provided. This chromosomal replacement must be simultaneous with the loss, so that there are no hypoploid times.
  • the replacement chromosomes do not have the said nulleric sequence. Therefore, the nuclease editor system (ZNF, TALENs, CRISPR, or other system) cannot promote its loss by the cell, as this system only cleaves the double strand of DNA that contains the nullomeric sequence. The presence of the nullomeric sequence thus ensures that cleavages in the DNA double strand, and consequent loss/deletion of the edited pair of chromosomes, do not occur in the replacement pair of exogenous chromosomes. As the replacement chromosome pair does not have this sequence, it is free of double strand cleavages of its DNA, maintaining all its sequential and functional integrity, and the cell will return to its normal euploidy.
  • correctly edited clones can give off-the-shelf universal or personal stem cell lines from which a specific chromosomal pair deletion can be obtained.
  • This feature opens the door to industrialization of universal cell lines as well as personal stem cell lines, as they will be available for immediate use (off-the-shelf) for use in the preparation of derived products.
  • An off-the-shelf product acquires advantages of scale and immediate availability with great savings in laboratory time, and facilitating its availability in time for patients.
  • the question of the speed of preparation of the final derivative product to be transplanted to the patient has very important implications in increasing the probability of patient survival, as well as in reducing the total (economic and social) costs of the treatment and in increasing the quality of life. of the patient and family.
  • the replacement pair of chromosomes can be delivered to cells using the Microcell Mediated Chromosome Transfer (MMCT) technique (Fournier and Ruddle 1977), in conjunction with components of the nuclease editor system (CRISPR, ZNF, TALENs, or others) that cleave the numeromer, or another suitable sequence absent from the normal stem cell genome of the species to be edited, on either side of the centromere.
  • MMCT Microcell Mediated Chromosome Transfer
  • the replacement chromosome pair can be delivered to the target cells by microinjection techniques along with the nuclease editor system (CRISPR, ZNF, TALENs, or others).
  • CRISPR nuclease editor system
  • cells that after the chromosomal replacement protocol may contain some portion of the deleted chromosome pair are positively and/or negatively selected by markers of fluorescence and antibiotic resistance and drug sensitivity (e.g. , acyclovir, tamoxifen, or mixtures thereof), present in the inserts.
  • markers of fluorescence and antibiotic resistance and drug sensitivity e.g. , acyclovir, tamoxifen, or mixtures thereof
  • the correctly replaced cells show no fluorescence, no antibiotic resistance, or programmed drug sensitivity.
  • the cells obtained from the method described in the present disclosure can be used in the treatment of various monogenic pathologies, such as hemophilia, sickle cell disease, thalassemia, among others.
  • the chromosome carrying the mutated gene is replaced in stem cells taken from the patient (with the exception of chromosome 6), the chromosome being replaced by an equivalent healthy chromosome taken from a healthy donor.
  • the resulting cells are subsequently differentiated and transplanted into the patient.
  • off-the-shelf universal stem cells are used with replacement of the indigenous chromosome 6 pair with the patient's chromosome 6 pair.
  • the resulting cells are thus compatible with the patient and can be transplanted without host rejection.
  • the patient's mutated chromosome is absent from the cells to be transplanted.
  • Hemophilia A Factor VIII
  • Hemophilia B Hemophilia B
  • Factor IX are two human congenital bleeding disorders that depend on mutations in the Human X chromosome. Production of 6% of the normal value of Factor VIII or Factor IX is sufficient to allow a normal level of blood clotting.
  • Universal male off-the-shelf stem cells contain a normal X chromosome, which produces normal factors VIII and IX.
  • transplantation of a minimum amount of differentiated cells capable of producing 6% or more of the normal level of factors VIII and IX, can provide adequate levels of the mentioned factors, curing the sick, despite in all other cells of the body of the patient mutations responsible for the previously low levels of Factor VIII or Factor IX still exist.
  • sickle cell disease a congenital human hematological disease
  • this disease can be cured by replacing the patient's chromosome 11 pair with a normal chromosome 11 pair in the patient's stem cells and further differentiating them into hematopoietic progenitor cells; or by transplantation of differentiated hematopoietic progenitor cells from off-the-shelf universal stem cells whose indigenous chromosome 11 pair is normal and where the indigenous chromosome 6 pair has been replaced by the patient's chromosome 6 pair to nullify rejection of the transplanted cells.
  • Molecular Biology has techniques capable of identifying and reproducing the processes involved in the replication of cellular DNA, as well as in its transcription into ribonucleic acid (RNA) and the translation of RNA into proteins.
  • RNA ribonucleic acid
  • Genomic Editing In the present disclosure it is necessary to proceed with the integration (from English - knock-in) of new DNA at precise locations within the genome of the stem cells to be modified. For this, it is necessary to have access to that genetic material with the appropriate nucleotide sequence or sequences. Said genetic material is obtained by industrial synthesis, either in the entire sequence or in portions that are later artificially linked, or by subcloning its various promoters and genes. In the latter case, the construct is obtained using PCR techniques for the production of each of the homology and subcloning arms of plasmids containing the sequences of the promoters and genes of the insert. The sequence thus obtained is called the "construct". - construct).
  • the construct is composed of two homology arms, (5' or Left homology arm, and 3' or Right homology arm), interspersed by the insert.
  • the insert is the new sequence that will be part of the stem cell genome that is to be modified/prepared/edited. It is the homology arms that, by complementarity with the DNA of the place where we want to integrate the insert, allow it to be led and copied to the specific location and not to any other random location in the genome.
  • the present disclosure describes the construction of the entire Cassette 1 (SEQ ID NO:43) construct in the laboratory.
  • the subcloning comprised the usual steps of digestion with suitable restriction enzymes (nucleases), of plasmids prepared artificially or acquired from the industry, subsequent ligation of the nucleotide sequences in said plasmids and transfection of competent cells acquired to the industry, (E. Coli specially prepared), following the protocol recommended by the manufacturers. Subsequently, clones of these competent cells were isolated correctly edited by PCR and/or sequencing technique. One or more desired clones were kept frozen at
  • DNA Double Strand Cleavages - Insert integration is facilitated when, concomitantly with the presence of the construct, the DNA at the site where the insert is integrated is cleaved/opened.
  • the CRISPR/Cas9 system was used in the present disclosure as a tool to cleave the DNA double strand (DSB) at the exact location of the genome to be edited.
  • HDR Homology Directed Repair
  • cleavage of the two DNA strands if the construct is simultaneously co-transfected within the cell, by HDR the cell can copy the insert to the cut site, passing thus the cell genome to express the new genetic information.
  • This technique has a very low efficiency, especially in human stem cells. But it is currently the most efficient to carry out the intended genomic editing.
  • Transfection - Transfection is the technique that allows the introduction into the cells to be edited of the genetic material prepared for that edition, as well as other elements capable of promoting it, for example CRISPR/Cas9 nuclease and sgRNA.
  • the main transfection methods are Lipofection, electroporation, or microinjection.
  • FACS Fluorescence Associated Cell Sorting
  • Stem Cell - Stem cells are considered to be all cells that have not yet undergone a final differentiation process and that have the ability to divide into cells equal to themselves (symmetrical division), or that have the ability to divide giving rise to a cell equal to itself and another that goes on to a differentiation step (asymmetric division).
  • Pluripotent cells were used in the present disclosure. These cells can give rise. ie, differentiate into any of the three types of embryonic tissues: ectoderm, mesoderm and endoderm, and can be kept in culture for very long periods maintaining all their original properties. Appropriate media are currently available for their culture, called serum and feeder-free, as they do not contain animal components in their composition, and do not need to be cultivated on a layer of cells, normally a layer of mouse embryonic fibroblasts. Cell culture in serum and feeder-free media makes it possible to overcome some questions of safety issues raised by regulatory authorities, when these cells or products derived from them are intended to be used in the clinical area.
  • hiPSCs were used.
  • transplantation is understood to mean the displacement of cells, tissues or organs, from their place of origin to another place within the same individual (self-transplantation), or between individuals of the same species (allo -transplantation), or between individuals of different species (xeno-transplantation).
  • the method of the present disclosure may be employed in editing non-human embryonic pluripotent stem cells, which may be used in treatments in the field of medicine or veterinary medicine.
  • Non-human embryonic pluripotent stem cells can differentiate into all types of cells derived from embryonic ectoderm, mesoderm and endoderm, that is, they maintain the ability to form any type of cell, micro-tissue, tissue or organ present in the organism. Therefore, they become important for the development of off-the-shelf universal stem cell lines, prepared to lose their pair of chromosomes responsible for encoding the major histocompatibility complex (MHC) of the species in question.
  • MHC major histocompatibility complex
  • the method of developing stem cell lines comprises editing the stem cell lines on one or more pairs of chromosomes, in proximity to and either side of the centromere, with gene cassettes comprising: Cassette 1 (Seq. ID No. 43) - comprises a 5' or Left Homology Arm, a promoter, a fluorescent protein gene, an antibiotic resistance gene, a numeromer suitable to be cleaved by the various nuclease systems (ZNF , TALEN, CRISPR, or others), 2pA sequence, 3' or Right Homology Arm and respective linker sequences.
  • This Cassette 1 should preferably edit a non-active stem cell gene, located near and on one side (3' or 5') of the centromere;
  • Cassette 2 (Seq. ID No. 44) - comprises a 5' or Left Homology Arm, a promoter, a drug sensitivity gene (tamoxifen, acyclovir), an SV40 virus polyadenylation sequence, (SV40pA), a nullomer with the same sequence as the nullomer present in the Cassette 1 sequence, a promoter, an antibiotic resistance gene different from the gene present in Cassette 1, a fluorescent protein gene different from the gene present in Cassette 1, a growth hormone polyadenylation sequence bovine, (bGHpA), and a 3' or Right Homology Arm.
  • a promoter a drug sensitivity gene (tamoxifen, acyclovir), an SV40 virus polyadenylation sequence, (SV40pA), a nullomer with the same sequence as the nullomer present in the Cassette 1 sequence, a promoter, an antibiotic resistance gene different from the gene present in Cassette 1,
  • This Cassette 2 should preferably edit an inactive stem cell gene, located close to and on the other side of the centromere (relative to Cassette 1).
  • the editing of Cassette 1 and Cassette 2 should preferably be obtained at different editing times, since it is necessary to cleave the DNA double strand by any of the editing systems (CRISPR, ZNF, TALEN, or others) simultaneously with the introduction of the constructs, more than 40% of the cells to be edited would suffer the deletion of the chromosomes in question, and consequent cell death, greatly reducing the efficiency of the present method.
  • the method for developing stem cell lines comprises the inclusion of nullomeric sequences complemented by a sequence called the Protospacer Adjacent Motif (PAM) in the gene cassettes to be expressed in stem cell lines.
  • PAM Protospacer Adjacent Motif
  • the present method for developing stem cell lines but in which the nucleomer sequences have been replaced in Cassette 1 and Cassette 2 by sequences normally non-existent in the genome of the species to which the stem cells to be edited belong, and which are suitable for cleavage by genomic editing nuclease systems (ZNF, TALENs, CRISPR, or others).
  • ZNF genomic editing nuclease systems
  • Nulomeric sequences are also suitable for the use of other nuclease systems in addition to the CRISPR system, namely ZNF, TALEN, and others that may be invented or discovered and that are based on the principle of driving the nuclease to specific sequences of the genome.
  • the disclosure comprises introducing nullomeric or other sequences normally absent from the genome of the organism to be modified, making use of genomic editing systems, (CRISPR, ZNF, TALEN or others).
  • CRISPR genomic editing systems
  • the CRISPR nuclease system comprises a large diversity of nucleases of bacterial origin that have the particularity of being able to cause cleavages of the double strand of DNA with great specificity, because the nuclease is carried to the cleavage site by a sequence of no more than 20 bases.
  • RNA called single-lead RNA (sgRNA).
  • PAM Protospacer Adjacent Motif
  • the optimal CRISPR-Cas9 (crRNA) cleavage site was chosen in silic as well as the Cassette 1 and Cassette-bearing vector plasmids.
  • Cassette 2 for editing the inactive centromere flanking genes.
  • inactive genes were selected in the hiPSCs of the line to be genetically modified.
  • genes BAG2 inactive gene in hiPSCs, centromere flanking on the long arm of human chromosome 6 were chosen; and LGSN, a gene that is also inactive in hiPSCs and flanks the centromere on the short arm of human chromosome 6 ( Figure 1).
  • LGSN a gene that is also inactive in hiPSCs and flanks the centromere on the short arm of human chromosome 6 ( Figure 1).
  • the importance that they are inactive genes is related to the fact that after editing, these genes become inoperative (knockout). Thus, its inactivation by editing would make it impossible for the modified cells to survive or correctly expand, if their activity were essential for these functions.
  • the annotated sequence of the chosen genes (e.g.
  • BAG2 - CRCh38:6:57171726:57190433:1 and LGSN - CRCh38:6:63275351:63320583:1) can be obtained from Emsembl.org, (e.g. GenBank format or MicrosoftTM WordTM compatible).
  • the sequences obtained were then imported into one of the various CRISPR-Cas9 bioinformatics tools (eg CRISPR Finder; CT-Finder; CCTop-CRISPR-Cas9 or any other available).
  • the CRISPR bioinformatics tool presents several sites in selected sequences where the CRISPR-Cas9 nuclease can produce DNA double strand cleavages (DSBs). Two or three with the best scores were selected among the presented sequences.
  • CRISPR-Cas9 nuclease is guided to the cleavage site by an RNA sequence called guide RNA, gRNA (guide-RNA).
  • guide-RNA guide-RNA
  • these sequences are complementary and immediately upstream of an NGG-like sequence in the DNA of the chosen gene, known as PAM.
  • the sequences obtained are tested, in order to select the most effective ones after cell transfection. After transfections, the best gRNAs were selected using a T7 endonuclease protocol gene 3 DNA endonuclease 1 (T7E1), (e.g. EnGenTMMutation Detection Kit from New England BioLabsTM (NEB), following the instructions recommended by the manufacturer.
  • T7E1 T7 endonuclease protocol gene 3 DNA endonuclease 1
  • Seq. ID No. 1 for the BAG2 gene cleavage site by the CRISPR/Cas9 system and Seq. ID No. 2 for the cleavage site of the LGSN gene by the CRISPR/Cas9 system.
  • Cassette 1/Gene BAG2 (Seq. ID No. 43) comprises a 5' or Left Homology Arm (5'HA or Left Homology Arm or LeftHA, 1783 base pairs, bp); a PGK promoter (Phosphoglycerate Kinase promoter - PGK pmt); a puromycin resistance gene; the Enhanced Green Fluorescent Protein (EGFP) gene, a polyadenylation sequence (Poly-A or pA); a numeromer; and a 3' or Right homology arm (3'HA or R ⁇ ght Homology Arm or R ⁇ ght HA, 1590 bp).
  • PGK promoter Phosphoglycerate Kinase promoter - PGK pmt
  • EGFP Enhanced Green Fluorescent Protein
  • Poly-A or pA polyadenylation sequence
  • numeromer a 3' or Right homology arm
  • plasmid p925 is a plasmid expressing ampicillin resistance for the selection of clones perfectly edited, as well as a cassette comprising the PGK promoter, the Puromycin Resistance gene and the EGFP protein gene and a Poly A sequence.
  • Seq. ID No. 7 and Seq. ID No. 8 were used as primers for the synthesis of the Sequence Arm of
  • Seq. ID No. 9 and Seq. ID No. 10 were used as primers for the synthesis of the Sequence Arm of
  • Seq primers. ID No. 11 and Seq. ID No. 12 were used for the synthesis of the polyadenylation and nullomer sequences prepared for subcloning (or insertion) into recognition sites for the restriction endonucleases XbaI and NotI in the plasmid (p925).
  • the BAG2 gene editing vector can be obtained in two different ways.
  • the entire vector can be purchased from industry, delivered as a plasmid in frozen competent cells.
  • the culture of these competent cells makes it possible to obtain the vector after its isolation using purification systems such as the WizardTM Plus SV MINIPREPS DNA Purification System (PromegaTM) or other equivalent system.
  • the vector can be prepared by subcloning its various promoters and genes, either into a virgin plasmid (empty backbone plasmid), or another more complete and suitable plasmid.
  • subcloning was performed in three steps.
  • the first step comprises subcloning the Poly A + nullomer sequence into XbaI and NotI sites in plasmid p925.
  • the Poly A and nucleomer sequences were subcloned downstream of the EGFP gene sequence, to allow both the selection of correctly edited off-the-shelf cell clones and, at a stage after the chromosomal replacement, the elimination of the cell clones. with maintenance of the expression of the PuroR gene and the EGFP gene. ( Figure 4).
  • Seq primers. ID No. 11 and Seq. ID No. 12 were used in polymerase chain reaction (PCR) for the synthesis of the Poly A + nucleomer sequences prepared for their subcloning (ligation) into the appropriate XbaI and NotI sites on plasmid p925.
  • a solution was prepared by mixing the various reagents (MIX), according to Table 1. After preparation, 47.5uL of the obtained MIX was mixed at 2.5uL/40ng p925DNA [20ng/uL]. The PCR reaction proceeded in a thermocycler (Applied Systems ThermocyclerTM), under the conditions indicated in Table 2.
  • MIX various reagents
  • Table 2 PCR conditions in a thermal cycler.
  • PCR 0.8% agarose gel electrophoresis was performed on a 5uL sample of the PCR product, to confirm the presence of a 470bp band, which contains the PCR product composed of the Polyadenylation sequence and numeromer. After verifying the presence of the band, the electrophoresis of all remaining PCR product on a 0.8% agarose gel.
  • the 470bp band was extracted from the gel, with an appropriate kit, (eg WizardTM SV Gel and PCR clean-up System - PROMEGATM), following the manufacturer's instructions.
  • the product obtained (PCR DNA) was resuspended in 20uL of PCRH2O, that is, water free of RNase and DNase enzymes.
  • Table 3 Conditions used in the digestion of DNA obtained in the PCR reaction by restriction enzymes XbaI and NotIHF.
  • the product obtained was purified with a mixture of phenol/chloroform/isoamyl alcohol, precipitated in ethanol and resuspended in 20uL of PCR H2O. After purification and precipitation, the obtained insert contains the Poly A + Nunomer sequence, ready to be ligated into the p925 vector.
  • the p925 vector is prepared for ligation to the obtained insert.
  • a double digestion with restriction endonucleases XbaI and NotI, and a dephosphorylation was performed.
  • XbaI and NotI double digestion was performed with a 5ug sample of p925 plasmid DNA, using the conditions described in Table 4. After the solution was prepared, the sample was incubated for 2 hours at 37°C.
  • Table 4 Experimental conditions for XbaI and NotIHF double digestion of plasmid p925.
  • a MIDI or MAXI-prep (eg QIAGENTM Midi or Maxi Kit) was extracted to obtain a sufficient amount of plasmid to be used in the following steps.
  • the method described in the present disclosure comprises a second subcloning step, for ligating the 5' or Left Homology Arm, upstream of the PGK promoter on plasmid p925/PolyA+nulomer).
  • the 5' or Left Homology Arm (1873bp) was ligated (subcloned) into an EcoRI site immediately upstream of the PGK promoter on plasmid p925/PolyA+nulomer.
  • the presence of a BamHI site at the 5' end of the sequence was provided to allow isolation of the complete vector for editing the BAG2 gene.
  • the PCR product was obtained from genomic DNA (gDNA) from HEK293T cells, using the NZY Tissue gDNA Isolation Kit (NZYtech®), following the instructions of the manufacturer.
  • gDNA genomic DNA
  • NZYtech® NZY Tissue gDNA Isolation Kit
  • Seq primers. ID No. 7 and Seq. ID No. 8 a solution was prepared with the mixture of the various reagents (MIX), according to Table 7, and then 45uL of the MIX obtained was mixed with 5uL (200ng) of gDNA [41ng/uL] isolated from the HEK293T.
  • the PCR reaction proceeded in a thermocycler (Applied Systems ThermocyclerTM), under the conditions indicated in Table 8.
  • Table 8 PCR conditions in the thermal cycler.
  • 0.8% agarose gel electrophoresis was performed on a 2uL sample of the PCR product, to confirm the presence of a band of 1783bp corresponding to the DNA fragment that contains the sequence of the Arm of Cassette Left Homology 1.
  • Checking for the presence of the band the remaining DNA obtained in the PCR reaction was purified by phenol/chloroform/isoamyl alcohol, and digested with the restriction enzyme EcoRI, according to Table 9, for 2 hours at 37°C.
  • Table 9 Experimental conditions for digestion of PCR product (Left Homology Arm BAG2) by EcoRI enzyme.
  • the p925/PolyA+nulomer vector was ligated to the digested BAG2 Left Homology Arm insert.
  • the p925/PolyA+nunomer plasmid was digested with the restriction enzyme EcoRI (Table 10), to prepare the vector for ligation (subcloning) with the PCR product Left Homology Arm also digested with EcoRI, previously isolated. Digestion took place for 2 hours at 37°C.
  • Table 10 Experimental conditions for digestion of p925/PolyA+nulomer plasmid vector by EcoRI enzyme.
  • the digested and dephosphorylated p925/PolyA vector was ligated to the BAG2 Left Homology Arm insert, also already digested by EcoRI, using the conditions described in Table 12.
  • Table 12 Preparation of reagent solution (MIX) for binding the digested and dephosphorylated p925/PolyA vector to the BAG2 Left Homology Arm insert.
  • a third subcloning step was initiated, to effect binding of the Right Homology Arm downstream of the nullomer to the p925 vector /PolyA+ Nunomer/Left Homology Arm.
  • the 3' or Right Homology Arm (1590bp), was ligated (subcloned) at a NotI site immediately downstream of the nullomer on plasmid p925/PolyA+Nulomer/Left Homology Arm. The presence of a BamHI site at the 3' end of the sequence was provided to allow future isolation of the complete vector for editing the BAG2 gene.
  • the PCR product was obtained from genomic DNA from HEK293T cells using the NZY Tissue gDNA Isolation Kit (NZYtech®) following the manufacturer's instructions. Using Seq primers. ID No. 9 and Seq. ID No.
  • MIX various reagents
  • Table 13 a solution was prepared with the mixture of the various reagents (MIX), according to Table 13, and then 45uL of the obtained MIX was mixed with 5uL (200ng) of gDNA [41ng/uL] isolated from the HEK293T.
  • the PCR reaction proceeded in a thermocycler (Applied Systems ThermocyclerTM), under the conditions indicated in Table 14.
  • Table 14 PCR conditions in the thermal cycler.
  • Table 16 Treatment of p925/PolyA+nulomer/Left Homology Arm BAG2 vector with Plasmid-SafeTM ATP-Dependent DNase (EPICENTRE®).
  • reaction product was purified by phenol/chloroform/isoamyl alcohol, ethanol precipitated and resuspended in 20ul PCR H2O.
  • Plasmid-SafeTM After treatment with Plasmid-SafeTM, plasmid p925/PolyA+nunomer/Left Homology Arm was digested with restriction enzyme NotIHF for 2 hours at 37°C in a water bath, according to the protocol indicated in the Table 17.
  • Table 17 Conditions used in the digestion of plasmid p925/PolyA+nulomer/Left Homology Arm by the restriction enzyme NotIHF [00149] After complete digestion, confirmed by 0.8% agarose gel electrophoresis, the resulting plasmid was purified by phenol/chloroform/isoamyl alcohol, ethanol precipitated, and resuspended in 20ul PCR H2O. Finally, the product was dephosphorylated, following the protocol mentioned in Table 18. After incubation for 10 minutes at 37°C in a water bath, the reaction was terminated by incubating 5 minutes at 75°C. The product obtained was purified by phenol/chloroform/isoamyl alcohol, precipitated in ethanol, and resuspended in 50uL PCR H2O.
  • Table 18 Dephosphorylation protocol of treated and digested p925/PolyA+nulomer/Left Homology Arm vector.
  • the digested and dephosphorylated p925/PolyA+nulomer/Left Homology Arm vector was ligated to the BAG2 Right Homology Arm insert, also already digested by Notl, using the conditions described in Table 19.
  • Table 19 Preparation of MIX solution for binding the digested and dephosphorylated PolyA+nulomer/Left Homology Arm vector to the BAG2 Right Homology Arm insert.
  • SV Minipreps DNA Purification System 250 preps, PROMEGATM, and the DNA obtained quantified using the NanodropTM.
  • the correct insertion of the plasmids was confirmed by restriction enzymes and/or sequencing, using methods widely known in the state of the art.
  • the R52 construct, prepared for editing the BAG2 gene is flanked by two recognition sites for the restriction enzyme BamHI. Taking advantage of this intentional feature, the vector for editing the BAG2 gene was prepared by excision by digestion with BamHI, through the reaction described in Table 20.
  • Table 20 Mixture of reagents prepared for the digestion of the construct R52 by the restriction enzyme BamHI
  • 94uL of the MIX and 6uL of R52 plasmid DNA were pipetted into a microtube, which was then incubated for 2 hours at 37°C in a water bath. After digestion with BamHI, the DNA was purified by phenol/chloroform/isoamyl alcohol, followed by precipitation in ethanol. After precipitation, the R52 construct was ready for cell transfection, where transfection designates the procedure in which DNA is made to enter cells in order to achieve changes in their genome.
  • human pluripotent stem cells (hiPSCs) were transfected into the BAG2 gene, with the R52 vector that contains the Cassette
  • the culture of hiPSC cells immediately after thawing was done in 6-well adherent cell culture plates, previously coated with MatrigelTM CorningTM, following the manufacturer's instructions.
  • the hiPSCs were seeded in each well, in the presence of mTeSRTMl STEMCELL TechnologiesTM culture medium supplemented at 1% with a solution of Penicillin 10,000IU/mL/10.000ug/mL Streptomycin (Sigma®), and then incubated at 37°C in a humidified atmosphere of 5% CC> 2 .
  • the culture medium was changed daily.
  • the culture medium was supplemented with Y-27632 ROCK inhibitor STEMCELL TechnologiesTM (Rocki), followed by an incubation for 2 hours prior to the introduction of the transfection materials.
  • CRISPR-Cas9/BAG2gRNA ( ⁇ 250ng DNA/well) was co-transfected with the R52 construct ( ⁇ 250ng DNA/well) in 24-well plates, using the cells previously prepared in the referred wells when reaching 10% confluence. Co-transfection was achieved by lipofection using LipofectamineTM 3000 Transfection Reagent, Thermo Fisher Scientific, following the manufacturer's instructions.
  • Figure 5 shows the results of PCR reactions obtained on samples of genomic DNA from VIRGIN hiPSCs (WT) as a negative control, and genomic DNA from hiPSCs of the same line as WT but co-transfected, using SEQ primers. ID NO: 15, SEQ. ID NO:16, SEQ.
  • A- 30uL of WT Virgin genomic DNA was added to microtube 1;
  • E-46uL of the reagent mixture from microtube 1 was pipetted into each of 5 PCR reaction microtubes (C-) (WT genomic) added 2uL of each of labeled FWD and REV primers (SEQ. ID No. 15 to 21), and
  • F-46uL of reagent mixture from microtube 2 was pipetted into each of 5 PCR reaction microtubes (D-) (co-transfected) with 2uL of each of labeled FWD and REV primers added (SEQ ID No. 15 to 21).
  • Table 22 PCR conditions in the thermocycler. [00164] After PCR, a 3uL sample from each of the microtubes was electrophoresed on a 0.8% agarose gel ( Figure 5).
  • FIG. 6 shows the results of the evaluation by Flow Cytometry (Becton Dickinson FACS Calibur® Cytometer) of the percentages of cells expressing Green Fluorescent Protein (GFP) in four different samples of an induced Human Pluripotent Stem Cell line. (hiPSCs), (TCLab line, Passage 37), co-transfected in accordance with the present disclosure.
  • the analysis was performed using the Flowing Software software.
  • Figure 7 shows the results of the evaluation by Flow Cytometry (Becton Dickinson FACS Calibur® Cytometer) of the percentages of cells expressing Green Fluorescent Protein (GFP) in two different samples from another line of Human Pluripotent Stem Cells (hiPSCs), (GEpi/GIBCO Line, Passage 50).
  • This evaluation was performed after labeling non-viable cells using the Kit: LIVE/DEADTMFixable Dead Cell Stain Kit, ThermoFisher ScientificTM. This labeling of non-viable cells allows for increased certainty regarding transfected and GFP-expressing cells that can be expanded in culture (viable).
  • the analysis was performed using the Flowing software® software.
  • the first vector is prepared from commercially obtained CRISPR-Cas9 nuclease, (e.g. TrueCut Cas9 Protein v2® ThermoFisher®), and synthetic LGSNgARN (TrueGuide Synthetic gRNA® ThermoFisher®), following the manufacturer's instructions.
  • Seq.ID No. 2 was the guide chosen after testing with T7E1, (EnGen® Mutation Detection Kit, NewEngland Biolabs, Inc.), for inserting cassette 2 insert into Gene LGSN.
  • the second vector the Cassette 2 vector for the LGSN gene ( Figure 3, Seq. ID No. 44) is purchased from external suppliers.
  • the culture of hiPSCs/BAG2-Cassette 1 is done in 6-well adherent cell culture plates, previously coated with MatrigelTM CorningTM, following the manufacturer's instructions.
  • the hiPSC cells are seeded in each of the wells, in the presence of mTeSRTM culture medium! STEMCELL TechnologiesTM supplemented at 1% with a solution of Penicillin 10,000IU/mL/Streptomycin 10,000ug/mL (Sigma®), then incubated at 37°C in a humidified atmosphere of 5% CC> 2 .
  • the culture medium was changed daily.
  • the culture medium was supplemented with Y-27632 ROCK inhibitor STEMCELL TechnologiesTM (Rocki), followed by an incubation for 2 hours prior to co-transfection.
  • selection of the edited cells resistant to puromycin and neomycin is initiated.
  • selection of mCherry+/neomycin-resistant clones is done by increasing the concentration of the neomycin in the culture medium in 10ug/mL increments in the culture medium starting with a concentration of 40ug/mL up to 200ug/mL depending on the hiPSC line used.
  • FACS Fluorescence Associated Cell Sorting
  • GFP Green Fluorescent Protein
  • mCherry Red Fluorescent Protein
  • the correctly edited clones are selected.
  • PCR reactions are performed, followed by sequencing the Gene BAG2 and Gene LGSN of the cultivated clones using primers amplifying sequences from outside to inside the constructs of Gene BAG2 (Cassette 1) and Gene LGSN (Cassette 2), in both the 5' and 3' ends. Confirmation is also made that the inserted sequences are correct, from the PCR amplification of sequences within the inserts.
  • primers with the following sequences can be used: Seq. ID No. 13, Seq. ID No. 14, Seq. ID No. 15, Seq. ID No. 16, Seq.
  • the corresponding primers can be used: Seq. ID No. 45, Seq. ID No. 46, Seq. ID No. 47, Seq. ID No. 48, Seq ID No. 49, seq ID No. 50, Seq. ID No. 51, Seq. ID No. 52, Seq ID No. 53 and Seq. ID No. 54.
  • these cells allow the replacement of their indigenous pair of chromosomes 6, by the pair of chromosomes 6 of a patient and the obtaining of healthy cells, (not carrying any mutations present in the patient's genome) and with the patient's exact and complete set of HLA receptors. Therefore, these off-the-shelf cells are of great relevance in the field of transplantation, as the problem of rejection due to HLA incompatibility is overcome.
  • a CRISPR-Cas9/gRNA-nulomer vector was prepared.
  • This vector comprises a CRISPR-Cas9 nuclease and gRNA nullomer, which cleaves chromosome 6 pair of edited hiPSCs/BAG2-LSGN, flanking the centromeres.
  • the Seq. ID No. 42 was used as the nullomer for vector construction, and Seq. ID No. 22 Seq. ID No. 23 as primers for the modification of the ADDGENETM plasmid #71814 eCas9, capable of produce CRISPR/Cas9 nuclease and the sgRNA corresponding to the nullomer.
  • CRISPR-Cas9 nuclease [00182] In another embodiment, CRISPR-Cas9 nuclease
  • ThermoFisher® Cas9 nuclease (TrueCut Cas9 Protein v2® ThermoFisher®) and the synthetic gRNA nullomer (TrueGuide Synthetic gRNA® ThermoFisher®).
  • the following sequences of nucleomers, mixtures, multiples or fractions thereof, complemented by a PAM-like NGG or NGA 5' sequence for CRISPR/Cas9 can be used: Seq. ID No. 24, Seq. ID No. 25, Seq. ID No. 26, Seq. ID No. 27, Seq. ID No. 28, Seq. ID No. 29, Seq. ID No. 30, Seq. ID No. 31, Seq. ID No. 32, Seq. ID No. 33, Seq. ID No. 34, Seq. ID No. 35, Seq. ID No. 36, Seq. ID No. 37, Seq. ID No. 38, Seq. ID No. 39, Seq. ID No. 40, Seq. ID No. 41, Seq. ID No. 42.
  • the PAM sequence and its respective position are variable, which should be taken into account if they are being used instead of the CRISPR/Cas9 nuclease.
  • cassette 1 with the PGK promoter, plasmid R52 comprises Seq. ID No. 43, a construct originating from synthetic DNA.
  • cassette 2 comprises Seq.ID No. 44, which is a construct originating from synthetic DNA.
  • Table 23 Sequences related to gRNA (complementary DNA)
  • Table 24 Sequences of Primers and Numomers.
  • ABSENT SEQUENCES NULLOMERS AND PRIMES, - GREG HAMPIKIAN and TIM ANDERSEN - Pacific Symposium on Biocomputing 12:355-366(2007)

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Abstract

The present disclosure describes a method for stem cell genome editing, as well as a gene cassette comprising at least one promoter, a marker, and a nullomer, wherein the marker comprises at least one gene encoding a fluorescence protein, antibiotic resistance, drug sensitivity, or combinations thereof; and wherein the nullomer is cleavable by at least one genome editing nuclease system, preferably the clustered and regularly interspaced short palindromic repeat system, zinc-finger, transcription activator-like effector nucleases (TALENs ), or mixtures thereof.

Description

D E S C R I Ç Ã O DESCRIPTION
MÉTODO PARA O DESENVOLVIMENTO DE LINHAS DE CÉLULASMETHOD FOR THE DEVELOPMENT OF CELL LINES
ESTAMINAIS STAMINALS
Domínio Técnico Technical Domain
[0001] A presente divulgação refere-se a um método para o desenvolvimento de linhas celulares estaminais e produtos derivados. Este método pertence aos campos da biologia molecular e cultura de células, e pode ser aplicado em medicina, de preferência na área de transplantação. [0001] The present disclosure relates to a method for developing stem cell lines and derived products. This method belongs to the fields of molecular biology and cell culture, and can be applied in medicine, preferably in the field of transplantation.
[0002] O método agora divulgado permite a obtenção de linhas celulares para uso imediato (do inglês off-the-shelf) preparadas para a perda de um ou mais cromossomas nativos específicos, bem como a sua substituição por cromossomas exógenos equivalentes. Numa realização, o presente método permite criar linhas celulares estaminais humanas preparadas para a perda do par de cromossomas 6 indígena (linhas universais off-the-shelf) e simultaneamente obter a sua substituição pelo par de Cromossomas 6 de um paciente. Deste modo, todas as células resultantes da sua posterior diferenciação, serão portadoras do exato e total complexo principal de histocompatibilidade do paciente, solucionando o problema da incompatibilidade principal em alo- transplantação . [0002] The method now disclosed allows obtaining off-the-shelf cell lines prepared for the loss of one or more specific native chromosomes, as well as their replacement by equivalent exogenous chromosomes. In one embodiment, the present method makes it possible to create human stem cell lines primed for the loss of the indigenous chromosome 6 pair (universal off-the-shelf lines) and simultaneously obtain their replacement by a patient's Chromosome 6 pair. In this way, all cells resulting from their subsequent differentiation will carry the exact and total major histocompatibility complex of the patient, solving the problem of major incompatibility in allotransplantation.
[0003] A presente divulgação descreve um novo tipo de transplantação : alo-auto-transplantação. Antecedentes [0003] The present disclosure describes a new type of transplantation: allo-autotransplantation. background
[0004] Apesar de ser uma prática comum na atualidade, existem ainda constrangimentos na transplantação de células, tecidos ou órgãos entre seres humanos. Para além da enorme falta de órgãos para transplantar, várias síndromas de rejeição podem acontecer após transplante, levando à destruição do órgão transplantado pelo sistema imunológico do hospedeiro ou, como no caso dos transplantes de Progenitores Hematopoiéticos, à lesão de múltiplos órgãos do hospedeiro por células transplantadas, conhecida como doença do enxerto contra o hospedeiro, (do inglês Graft Versus Host Dísease - GVHD). [0004] Despite being a common practice today, there are still constraints in the transplantation of cells, tissues or organs between human beings. In addition to the huge lack of organs to be transplanted, several rejection syndromes can occur after transplantation, leading to the destruction of the transplanted organ by the host's immune system or, as in the case of Hematopoietic Progenitor transplants, to the damage of multiple organs of the host by cells. transplanted, known as graft-versus-host disease (Graft Versus Host Disease - GVHD).
[0005] As rejeições são a consequência do reconhecimento pelo sistema imunitário de diferenças entre as proteínas expressas na membrana celular das células do dador e as do hospedeiro. Este reconhecimento é feito a partir dos antigénios de leucócitos humanos (do inglês Human Leukocyte Antígens - HLA), dividindo-se em recetores HLA Classe I e recetores HLA Classe II. Os recetores HLA Classe I são expressos na membrana celular de quase todas as células do corpo humano. Os recetores da Classe II são expressos em células pertencentes ao sistema imunitário, designadas Células Apresentadoras de Antigénios (do inglês: Antígen Presentíng Cells - APC), como é o caso de alguns subtipos de células dendríticas, macrófagos e células B. [0005] The rejections are the consequence of the recognition by the immune system of differences between the proteins expressed in the cell membrane of the cells of the donor and those of the host. This recognition is made from human leukocyte antigens (Human Leukocyte Antigens - HLA), divided into HLA Class I receptors and HLA Class II receptors. Class I HLA receptors are expressed on the cell membrane of almost every cell in the human body. Class II receptors are expressed in cells belonging to the immune system, called Antigen Presenting Cells (APC), as is the case with some subtypes of dendritic cells, macrophages and B cells.
[0006] No entanto, este rigoroso e eficiente mecanismo, transforma-se num enorme problema sempre que um paciente necessita de um alo-transplante. Cada diferença, ainda que mínima, entre os recetores HLA das células do dador e o HLA das células do paciente/hospedeiro, desencadeará o seu tipo de Síndroma de Rejeição. A diferença é interpretada pelo sistema imunológico do hospedeiro como se uma ameaça estivesse a acontecer e por isso, uma rápida eliminação dessa ameaça é desencadeada. [0006] However, this rigorous and efficient mechanism becomes a huge problem whenever a patient needs an allo-transplant. Every difference, however small, between the HLA receptors of the donor's cells and the HLA of the patient/host cells, will trigger your type of Rejection Syndrome. The difference is interpreted by the host's immune system as if a threat were happening and therefore a rapid elimination of that threat is triggered.
[0007] Se no caso de órgãos duplos, como é o dos rins, é possível a realização de um transplante entre seres humanos potencialmente "compatíveis", no caso de órgãos únicos, como é o caso do coração, essa doação só pode ocorrer post mortem. Isto reflete-se numa carência imensa de órgãos para transplantação, agravado pelo fato de que o grau de compatibilidade é sempre parcial. Ou seja, podendo existir igualdade na composição de alguns dos recetores HLA à superfície das células, sempre existirão outros recetores diferentes, os quais condicionarão o aparecimento de graus variáveis de fenómenos de rejeição. [0007] If in the case of double organs, such as the kidneys, it is possible to carry out a transplant between potentially "compatible" human beings, in the case of single organs, such as the heart, this donation can only occur after death. This is reflected in an immense shortage of organs for transplantation, exacerbated by the fact that the degree of compatibility is always partial. In other words, there may be equality in the composition of some of the HLA receptors on the surface of the cells, there will always be other different receptors, which will condition the appearance of varying degrees of rejection phenomena.
[0008] Face a um episódio de rejeição, o resultado poderá ser a morte do paciente e/ou a destruição do alo-transplante, pelo que atualmente o tratamento pós-transplante inclui imunossupressores e outros medicamentos para evitar a rejeição. No entanto, a imunossupressão tem efeitos secundários nefastos para a qualidade de vida do paciente e pode mesmo colocar a sua sobrevivência em perigo. Complicações infeciosas por vírus, bactérias e infestações por fungos ou parasitas acontecerão, colocando a vida do paciente em perigo. Do mesmo modo, a baixa da vigilância normal do sistema imunológico pode levar ao aparecimento de tumores malignos. [0008] Faced with an episode of rejection, the result could be the death of the patient and/or the destruction of the allo-transplant, so currently the post-transplant treatment includes immunosuppressants and other drugs to prevent rejection. However, immunosuppression has harmful side effects for the patient's quality of life and can even put their survival in jeopardy. Infectious complications from viruses, bacteria and infestations by fungi or parasites will ensue, putting the patient's life in danger. Likewise, the loss of normal surveillance of the immune system can lead to the appearance of malignant tumors.
[0009] Apesar destas bem conhecidas complicações, ainda não existem outras alternativas para doentes alo- transplantados . Por isso, protocolos de imunossupressão são prescritos em todos os serviços de aio-transplantação em todo o mundo, a fim de tentar maximizar a sobrevivência, quer dos pacientes quer dos alo-transplantes. [0009] Despite these well-known complications, there are still no other alternatives for allotransplanted patients. Therefore, immunosuppression protocols are prescribed in all ayo-transplantation services in around the world, in order to try to maximize the survival of both patients and allotransplants.
[0010] Recentemente, foram descritas algumas propostas com o objetivo de possibilitar a aio-transplantação sem imunossupressão . Resumidamente, com recurso a diferentes mecanismos, as células alo-transplantadas seriam modificadas de forma a não expressar recetores HLA Classe I ou/e HLA Classe II, ou as células expressariam uma elevada quantidade de recetores HLA Class I específicos (HLA G), ou outros recetores, no sentido de se obterem o que se designou como "alo-transplantes transparentes". Estes alo-transplantes que o sistema imunológico do paciente não consegue reconhecer como não-do-próprio (em inglês: non-self) conferem uma espécie de tolerância imune ao alo-transplante. Contudo, esta "transparência" do sistema imunológico do paciente constitui um enorme problema, porque qualquer infeção, infestação ou transformação cancerosa nas células alo- transplantadas será muito dificilmente detetada em tempo útil. [0010] Recently, some proposals have been described with the aim of enabling aio-transplantation without immunosuppression. Briefly, using different mechanisms, the allo-transplanted cells would be modified so as not to express HLA Class I or/and HLA Class II receptors, or the cells would express a high amount of specific HLA Class I receptors (HLA G), or other recipients, in order to obtain what has been called "transparent allo-transplants". These allo-transplants that the patient's immune system cannot recognize as non-self confer a kind of immune tolerance to the allo-transplant. However, this "transparency" of the patient's immune system is a huge problem, because any infection, infestation or cancerous transformation in the allo-transplanted cells will be very difficult to detect in a timely manner.
[0011] Um facto extremamente vantajoso, presente nos humanos, é que a totalidade dos genes muito polimórficos que são traduzidos em recetores HLA, estão reunidos num grande, mas único, complexo de genes conhecido como Complexo de Histocompatibilidade Major (MHC, do inglês Major Histocompatibility Complex), localizado no braço curto do cromossoma 6. Uma exceção é a b-2-microglobulina que é traduzida de um gene não-polimórfico residente no cromossoma 15 humano e que é um componente essencial dos recetores HLA da Classe I. [0011] An extremely advantageous fact, present in humans, is that all the highly polymorphic genes that are translated into HLA receptors are gathered in a large, but unique, gene complex known as the Major Histocompatibility Complex (MHC). Histocompatibility Complex), located on the short arm of chromosome 6. An exception is b-2-microglobulin which is translated from a non-polymorphic gene residing on human chromosome 15 and which is an essential component of Class I HLA receptors.
[0012] Dependendo dos autores o MHC em humanos é composto por entre 4,2 e 7 megabases de sequências de ácido desoxirribonucleico, (ADN) em cada um dos braços curtos do par de cromossomas 6. Com as técnicas atuais de Biologia Molecular, torna-se impossível realizar a substituição de duas sequências tão longas de ADN. [0012] Depending on the authors, the MHC in humans is composed of between 4.2 and 7 megabases of acid sequences. deoxyribonucleic acid, (DNA) in each of the short arms of the pair of chromosomes 6. With the current techniques of Molecular Biology, it becomes impossible to carry out the substitution of two such long sequences of DNA.
[0013] Outras duas soluções aparentemente muito promissoras, foram também preparadas com o objetivo de poderem resolver o problema das rejeições na aio- transplantação. A proposta mais recente foi da autoria do grupo do Professor Yamanaka, com a criação de linhas de células estaminais a partir de células adultas usando um protocolo de reprogramação. A partir da diferenciação destas células, designadas por Células Estaminais Pluripotentes Induzidas Humanas (hiPSCs, do inglês human induced Pluripotent Stem Cells), seria possível obter as células, tecidos ou órgãos que um paciente necessite, sendo que estes contêm o mesmo, exato e completo conjunto de recetores HLA que todas as restantes células do seu corpo. Assim, a compatibilidade é total e por isso nenhuma síndroma de rejeição será desencadeada. No entanto, todas as células, tecidos ou órgãos produzidos por reprogramação continuarão a expressar as doenças resultantes das mutações de que o paciente é portador, caso estas mutações não sejam corrigidas previamente ou após a reprogramação. Para além disso, as hiPSCs são muito dispendiosas, levam bastante tempo a serem preparadas e levantam algumas questões de segurança, nomeadamente a possibilidade de transformação cancerígena, entre outros. [0013] Two other apparently very promising solutions were also prepared with the objective of being able to solve the problem of rejections in ayo-transplantation. The most recent proposal was authored by Professor Yamanaka's group, with the creation of stem cell lines from adult cells using a reprogramming protocol. From the differentiation of these cells, called Human Induced Pluripotent Stem Cells (hiPSCs), it would be possible to obtain the cells, tissues or organs that a patient needs, and they contain the same, exact and complete set of HLA receptors than all other cells in your body. Thus, the compatibility is total and therefore no rejection syndrome will be triggered. However, all cells, tissues or organs produced by reprogramming will continue to express the diseases resulting from mutations carried by the patient, if these mutations are not corrected before or after reprogramming. Furthermore, hiPSCs are very expensive, take a long time to prepare and raise some safety issues, namely the possibility of cancerous transformation, among others.
[0014] Uma outra proposta foi apresentada em 2006 por Tada et.al. O protocolo apresentado baseia-se no facto de que o conjunto de recetores HLA de cada indivíduo é traduzido a partir do MHC, e que o MHC reside no braço curto do cromossoma 6. Sendo possível a substituição do par de cromossomas 6 indígena em Células Estaminais Pluripotentes Humanas (hPSCs, do inglês Human Plurípotent Stem Cells), pelo par de cromossomas 6 de um paciente, poderia tornar-se possível obter qualquer célula, tecido ou órgão que um paciente necessite, conseguindo-se a completa coincidência HLA ou compatibilidade entre as células do transplante e as células do paciente. Esta solução é muito parecida nos seus objetivos e resultados com as hiPSCs, mas contém também uma enorme vantagem sobre as hiPSCs. Do mesmo modo que as hiPSCs, as células produzidas segundo o protocolo de Tada et.al., conseguem ser completamente compatíveis com o paciente porque expressam o seu exato e completo conjunto HLA, mas conseguem simultaneamente adicionar a característica de serem saudáveis, pois a substituição do cromossoma 6 é realizada em hPSCs que estão isentas de mutações patogénicas. [0014] Another proposal was presented in 2006 by Tada et.al. The protocol presented is based on the fact that the set of HLA receptors of each individual is translated from the MHC, and that the MHC resides on the short arm of chromosome 6. It is possible to replace the pair of chromosomes 6 in Human Pluripotent Stem Cells (hPSCs), by the pair of chromosomes 6 from a patient, it could become possible to obtain any cell, tissue or organ that a patient needs, achieving complete HLA matching or compatibility between transplant cells and patient cells. This solution is very similar in its goals and results to hiPSCs, but it also has a huge advantage over hiPSCs. In the same way as hiPSCs, the cells produced according to the protocol by Tada et.al., can be completely compatible with the patient because they express their exact and complete HLA set, but at the same time they can add the characteristic of being healthy, since the replacement of chromosome 6 is performed on hPSCs that are free of pathogenic mutations.
[0015] Esta estratégia, permitiria a ausência de todas as doenças mono- ou multigénicas de que o doente possa padecer, ao contrário do que sucede com as hiPSCs, e simultaneamente permite uma compatibilidade HLA total com o paciente. Apenas as doenças genéticas devidas a mutações patogénicas do cromossoma 6 do paciente não poderão ser corrigidas, a menos que essas mutações sejam corrigidas previamente ou posteriormente à substituição cromossómica nas hPSCs off- the-shelf. [0015] This strategy would allow the absence of all mono- or multigenic diseases that the patient may suffer, contrary to what happens with hiPSCs, and simultaneously allows full HLA compatibility with the patient. Only genetic diseases due to pathogenic mutations on the patient's chromosome 6 cannot be corrected unless those mutations are corrected before or after chromosomal replacement in off-the-shelf hPSCs.
[0016] O documento US 20090264312 descreve um método para a obtenção da perda induzida de um cromossoma específico com recurso a uma técnica que faz uso de uma nuclease Cre com sítios de recombinação LoxP invertidos.A ativação do sistema pela introdução da nuclease Cre induz a transformação do cromossoma preparado com sítios LoxP invertidos, num cromossoma dicêntrico isto é, com dois centrómeros, que a célula rejeita por não ser compatível com a replicação/segregação durante a mitose celular. Contudo, este método nunca evoluiu para o uso clinico, pois a indução de cromossomas dicêntricos, que permite a perda induzida dos referidos cromossomas pelas células, é também causa de graves mutações genómicas nas células que pudessem sobreviver. [0016] Document US 20090264312 describes a method for obtaining the induced loss of a specific chromosome using a technique that makes use of a Cre nuclease with inverted LoxP recombination sites. Activation of the system by the introduction of Cre nuclease induces transformation of the chromosome prepared with inverted LoxP sites into a dicentric chromosome, that is, with two centromeres, which the cell rejects because it is not compatible with the replication/segregation during cell mitosis. However, this method never evolved into clinical use, as the induction of dicentric chromosomes, which allows the induced loss of said chromosomes by the cells, is also the cause of serious genomic mutations in the cells that could survive.
[0017] Através do sistema de nuclease CRISPR/Cas9 (do inglês Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated proteín 9) é também possível induzir a perda de um cromossoma específico. Foi já demonstrado que é possível eliminar o cromossoma humano Y usando diferentes estratégias. Por exemplo, provocando múltiplas clivagens na cadeia dupla no ADN em ambos os braços do cromossoma, induzindo múltiplas clivagens no ADN centromérico específico do cromossoma humano, e clivando o ADN em dois locais próximos e de cada lado do centrómero. Contudo, estas estratégias podem deixar material cromossómico sem meios de seleção e identificação, o que pode levar ao aparecimento de mutações/translocações. Por razões de segurança este método, tal como descrito, não tem aplicação em Medicina. Por outro lado, ainda não foi descrito como é possível manter vivas as células após a perda do cromossoma Y, sem o desenvolvimento de uma metodologia para prover à sua substituição. Esta é outra razão pela qual a descrição de um método de perda específica de um cromossoma só por si não tem aplicação em Medicina, com a eventual exceção de poder vir a ser útil, resolvidas outras dificuldades técnicas, no caso das trissomias. [0017] Through the nuclease system CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) it is also possible to induce the loss of a specific chromosome. It has already been shown that it is possible to eliminate the human Y chromosome using different strategies. For example, causing multiple double-stranded cleavages in the DNA on both arms of the chromosome, inducing multiple cleavages in the centromeric DNA specific to the human chromosome, and cleaving the DNA at two sites near and on either side of the centromere. However, these strategies can leave chromosomal material without means of selection and identification, which can lead to mutations/translocations. For safety reasons this method, as described, has no application in Medicine. On the other hand, it has not yet been described how it is possible to keep cells alive after the loss of the Y chromosome, without the development of a methodology to provide for its replacement. This is another reason why the description of a specific method for the loss of a chromosome by itself has no application in Medicine, with the possible exception of being useful, once other technical difficulties are resolved, in the case of trisomies.
[0018] Em Zuo et al., os autores descrevem a perda de um cromossoma específico em células estaminais humanas, recorrendo ao sistema de nuclease CRISPR/Cas9, nomeadamente a perda de um cromossoma 21 humano em células portadoras de trissomia 21. No mesmo documento é também descrita a perda especifica do cromossoma Y humano. Estas perdas foram obtidas através do uso de múltiplos ácido ribonucleicos de guia único (sgARN, do inglês single guide ribonucleic acid) específicos para o cromossoma em causa, ou um sgARN específico para sequências repetidas no cromossoma em causa, presentes quer em agrupamentos (do inglês clusters), quer dispersas ao longo do cromossoma. Também neste caso os autores não conseguem garantir a segurança do produto final para o seu uso em Medicina, posto que não é possível garantir que todos os fragmentos em que o cromossoma é clivado serão eliminados das células, e que não darão origem a mutações/translocações. Por outro lado, os autores pensaram a sua estratégia para resolver poliploidias, como podem ser os casos de trissomias. Não se referem em momento algum à substituição de cromossomas equivalentes para resolverem algum problema clínico. Apenas reconhecem a sua utilidade para o desenvolvimento de modelos de doenças. [0018] In Zuo et al., the authors describe the loss of a specific chromosome in human stem cells, using the CRISPR/Cas9 nuclease system, namely the loss of a human chromosome 21 in cells carrying trisomy 21. In the same document loss is also described. specific to the human Y chromosome. These losses were achieved through the use of multiple single-guide ribonucleic acid (sgRNA) specific for the chromosome in question, or a sgRNA specific for repeated sequences on the chromosome in question, present either in clusters. clusters), or dispersed along the chromosome. Also in this case, the authors cannot guarantee the safety of the final product for its use in Medicine, since it is not possible to guarantee that all the fragments in which the chromosome is cleaved will be eliminated from the cells, and that they will not give rise to mutations/translocations. . On the other hand, the authors thought of their strategy to solve polyploidy, such as trisomies. They do not refer at any time to the replacement of equivalent chromosomes to solve a clinical problem. They only recognize its usefulness for the development of disease models.
[0019] Fournier e Ruddle em 1977 descrevem um método que consiste na possibilidade de isolamento de cromossomas inteiros únicos ou em grupos pequenos (2 ou 3), no interior de estruturas designadas por microcélulas e que são compostas por: uma membrana com semelhanças de composição com a da membrana citoplasmática; componentes de citoplasma da célula dadora; e o/s cromossoma/s isolado/s no seu interior. Desde a sua primeira descrição até à atualidade, pequenas modificações foram introduzidas melhorando a eficiência do seu isolamento, conservação e a capacidade de fusão das microcélulas com células euploides ou aneuploides. Esta fusão das células com as microcélulas permite a transferência do/s cromossoma/s isolado/s para o seu interior, passando a fazer parte do seu genoma. Descrição Geral da Divulgação [0019] Fournier and Ruddle in 1977 describe a method that consists in the possibility of isolating single entire chromosomes or in small groups (2 or 3), within structures called microcells and which are composed of: a membrane with similar composition with that of the cytoplasmic membrane; cytoplasmic components of the donor cell; and the isolated chromosome/s inside it. From its first description to the present, small modifications have been introduced improving the efficiency of its isolation, conservation and the fusion capacity of microcells with euploid or aneuploid cells. This fusion of cells with microcells allows the transfer of the isolated chromosome/s to its interior, becoming part of its genome. General Description of Disclosure
[0020] A presente divulgação descreve um novo método, industrializável e inventivo capaz de produzir linhas de células estaminais universais off-the-shelf, editadas de forma a poderem perder um cromossoma ou mais, em células eucarióticas, ou o cromossoma das células procarióticas, com a possibilidade de se proceder à sua substituição por outro/s cromossoma/s exógeno/s equivalente/s, possuindo genes de sequências diferentes ou corrigidas. Ao contrário dos demais métodos descritos, o presente método permite o controlo triplo das fases de seleção celular, o que representa uma vantagem de segurança para posterior utilização das células em cenário clinico, nomeadamente na terapia ou tratamento de doenças monogénicas, multigénicas ou em transplantes de substituição por perda da função celular, de células sem mutações patogénicas (p.ex. enfarte do miocárdio por aterosclerose) . [0020] The present disclosure describes a new, industrializable and inventive method capable of producing off-the-shelf universal stem cell lines, edited so that they can lose a chromosome or more, in eukaryotic cells, or the chromosome of prokaryotic cells, with the possibility of replacing it with another equivalent exogenous chromosome/s, having genes of different or corrected sequences. Unlike the other methods described, the present method allows the triple control of the phases of cell selection, which represents a safety advantage for the subsequent use of cells in a clinical scenario, namely in the therapy or treatment of monogenic, multigenic diseases or in transplants of replacement by loss of cellular function of cells without pathogenic mutations (e.g. myocardial infarction from atherosclerosis).
[0021] Numa forma de realização, duas cassetes de sequências de ADN são editadas em genes fechados - i.e. genes não transcritos na fase estaminal das células em causa - na proximidade e de cada lado do centrómero. Estas novas cassetes compreendem promotores, genes de proteínas fluorescentes, genes codificadores de proteínas que conferem resistência a antibióticos e/ou sensibilidade a anti-virais (por exemplo aciclovir) ou a outras drogas (por exemplo tamoxifeno) . Cada uma destas cassetes compreende, ainda, uma sequência de ADN ausente do genoma da espécie cujas células estaminais se pretendem modificar e suscetível de sofrer clivagem da cadeia dupla do ADN pelos sistemas de nucleases editoras (nucleases do sistema de repetições palindrómicas curtas agrupadas e regularmente interespaçadas (CRISPR, do inglês Clustered Regularly Interspaced Short Palindromic Repeats), dedo de zinco (ZNF, do inglês zinc fingers), nucleases efetoras semelhantes a ativadores de transcrição (TALENs, do inglês Transcription Activator-like Effector Nucleases), ou outras). [0021] In one embodiment, two cassettes of DNA sequences are edited into closed genes - ie genes not transcribed at the stem stage of the cells in question - in proximity to and on either side of the centromere. These novel cassettes comprise promoters, fluorescent protein genes, genes encoding proteins that confer antibiotic resistance and/or sensitivity to anti-virals (eg acyclovir) or other drugs (eg tamoxifen). Each of these cassettes also comprises a DNA sequence absent from the genome of the species whose stem cells are to be modified and susceptible to cleavage of the DNA double strand by the editor nuclease systems (clustered and regularly interspaced short palindromic repeat system nucleases). (CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats), zinc fingers (ZNF), Transcription Activator-like Effector Nucleases (TALENs), or others).
[0022] Numa forma de realização, a sequência de ADN suscetível de sofrer clivagens da cadeia dupla do ADN pelos sistemas de edição genómica (CRISPR/Cas9, ZNF, TALENs, ou outras), e que não está presente no genoma das células não editadas do organismo em causa é um nulómero. [0022] In one embodiment, the DNA sequence susceptible to double-stranded DNA cleavage by genomic editing systems (CRISPR/Cas9, ZNF, TALENs, or others), and which is not present in the genome of the unedited cells of the organism concerned is a numeromer.
[0023] Para os efeitos da presente divulgação, um nulómero deve ser entendido como uma sequência curta de ADN que não está presente no genoma das células não editadas do organismo em causa. [0023] For the purposes of the present disclosure, a nullomer is to be understood as a short sequence of DNA that is not present in the genome of the unedited cells of the organism in question.
[0024] Na presente realização, são considerados produtos derivados todas as células, micro-tecidos (do inglês microtissues), tecidos, ou órgãos obtidos a partir da diferenciação de células estaminais editadas no seu genoma pelo método descrito na presente divulgação. Sendo diferentes das células estaminais que lhe deram origem, só podem delas ser derivadas ou diferenciadas pelo método descrito na presente divulgação, de forma a criar outras linhas celulares estaminais com a mesma edição ou produtos celulares, teciduais ou órgãos para transplantação. [0024] In the present embodiment, all cells, microtissues, tissues, or organs obtained from the differentiation of stem cells edited in their genome by the method described in the present disclosure are considered derivative products. Being different from the stem cells that gave rise to them, they can only be derived or differentiated from them by the method described in the present disclosure, in order to create other stem cell lines with the same edition or cellular, tissue or organ products for transplantation.
[0025] Numa forma de realização, o método para o desenvolvimento de linhas de células estaminais e produtos derivados consiste na edição de linhas de células estaminais de modo a que fiquem disponíveis como linhas de células estaminais universais off-the-shelf, com a capacidade de perderem um ou mais cromossomas ou como linhas de células estaminais do paciente (Ex. Células Progenitoras Hematopoiétcas, CD34+, células mesenquimatosas, etc), para uso no tratamento de doenças monogénicas ou multigénicas. A perda induzida dos referidos cromossomas é conseguida com recurso a nucleases (CRISPR, ZNF, TALENs, ou outras), que provocam clivagens da cadeia dupla do ADN em sequências especificas, situadas perto e de cada lado do centrómero dos cromossomas editados. Por outro lado, as sequências especificas onde têm lugar as clivagens na cadeia dupla do ADN fazem parte de duas cassetes de genes (Cassete 1 e Cassete 2), cuja composição é importante tanto na fase de identificação e seleção dos clones de células estaminais corretamente editados, como também mais tarde aquando da substituição dos cromossomas deletados por cromossomas equivalentes. Os marcadores de fluorescência e de resistência e/ou de sensibilidade a drogas, incluídos nas cassetes, são os responsáveis por estas características funcionais e técnicas nas células editadas e, mais tarde, nas células em que o cromossoma deletado é substituído por um equivalente. [0025] In one embodiment, the method for developing stem cell lines and derived products is to edit the stem cell lines so that they are available as off-the-shelf universal stem cell lines, with the ability from losing one or more chromosomes or as a patient's stem cell lines (Ex. Hematopoietic, CD34+, mesenchymal cells, etc.), for use in the treatment of monogenic or multigenic diseases. The induced loss of said chromosomes is achieved using nucleases (CRISPR, ZNF, TALENs, or others), which cause double-stranded DNA cleavages in specific sequences, located near and on each side of the centromere of the edited chromosomes. On the other hand, the specific sequences where the cleavages in the DNA double strand take place are part of two gene cassettes (Cassette 1 and Cassette 2), whose composition is important both in the identification and selection phase of correctly edited stem cell clones. , as well as later when the deleted chromosomes are replaced by equivalent chromosomes. The fluorescence and drug resistance and/or sensitivity markers, included in the cassettes, are responsible for these functional and technical characteristics in the edited cells and, later, in the cells in which the deleted chromosome is replaced by an equivalent one.
[0026] Estes artifícios técnicos inovadores permitem resolver problemas relacionados com a segurança da utilização dos produtos derivados, em particular células multipotentes (p. ex. células progenitoras hematopoiéticas) ou células completamente diferenciadas (p. ex. células Beta- pancreáticas), garantindo que provêm de origem clonal e que não se mantém material genómico do cromossoma deletado no interior das células a transplantar. Numa forma de realização, na fase de desenvolvimento das linhas celulares, as proteínas de fluorescência, a resistência a antibióticos e suscetibilidade a drogas, permitem escolher apenas os clones de células que as produzam, e eliminar todas as outras. Na fase de desenvolvimento dos transplantes, permitem selecionar apenas células que não as produzam, sinal de que perderam completamente o par de cromossomas editado e que têm apenas o par de cromossomas de substituição. [0026] These innovative technical devices make it possible to solve problems related to the safety of the use of derived products, in particular multipotent cells (eg hematopoietic progenitor cells) or fully differentiated cells (eg pancreatic beta cells), ensuring that come from clonal origin and that genomic material from the deleted chromosome does not remain inside the cells to be transplanted. In one embodiment, at the stage of cell line development, the fluorescence proteins, antibiotic resistance and drug susceptibility allow choosing only those cell clones that produce them, and eliminating all others. In the developmental stage of transplants, allow you to select only cells that do not produce them, a sign that they have completely lost the edited pair of chromosomes and that they only have the replacement pair of chromosomes.
[0027] Numa forma de realização, as células estaminais são colhidas dos próprios pacientes, e os cromossomas nativos com mutações patológicas, substituídos por cromossomas de dadores saudáveis, através do método descrito na presente divulgação. Os produtos derivados são então utilizados no tratamento de doenças monogénicas ou multigénicas. [0027] In one embodiment, stem cells are harvested from the patients themselves, and native chromosomes with pathological mutations replaced with chromosomes from healthy donors, by the method described in the present disclosure. The derived products are then used in the treatment of monogenic or multigenic diseases.
[0028] Numa outra forma de realização, são obtidas linhas de células estaminais universais off-the-shelf, preparadas a partir do método descrito na presente divulgação, em que ocorre a perda do cromossoma que contém os genes que traduzem os recetores HLA. No caso da espécie humana, estes genes encontram-se no cromossoma 6. Assim, o cromossoma 6 indígena do dador é removido, e substituído por um cromossoma 6 exógeno, isolado a partir de células do paciente e futuro hospedeiro. Consequentemente, as células diferenciadas a partir de células estaminais humanas off-the-shelf passam a expressar exatamente os mesmos recetores HLA do paciente, sendo completamente compatíveis com o paciente. Da mesma forma, aglomerados de células, micro-tecidos, tecidos ou órgãos derivados das células estaminais modificadas são também compatíveis, e podem ser transplantados no paciente sem os habituais riscos de rejeição. [0028] In another embodiment, off-the-shelf universal stem cell lines are obtained, prepared from the method described in the present disclosure, wherein the loss of the chromosome containing the genes that translate HLA receptors occurs. In the case of the human species, these genes are found on chromosome 6. Thus, the donor's indigenous chromosome 6 is removed and replaced by an exogenous chromosome 6, isolated from the cells of the patient and future host. Consequently, cells differentiated from off-the-shelf human stem cells begin to express exactly the same HLA receptors as the patient, being completely compatible with the patient. Likewise, clusters of cells, micro-tissues, tissues or organs derived from the modified stem cells are also compatible, and can be transplanted into the patient without the usual risks of rejection.
[0029] No caso da substituição do par de cromossomas 6 indígena nas células estaminais humanas pelo par de cromossomas 6 de um paciente, não só se consegue evitar ou reduzir a necessidade de recurso à terapêutica imunossupressora, mas igualmente importante, mantem-se a integridade do funcionamento normal de todo o sistema imunológico do paciente. Isto representa uma mudança radical em relação ao paradigma atual que assenta exclusivamente no combate/redução da atividade do sistema imunológico para tentar reduzir o mais possível os fenómenos de rejeição. Por outro lado, os custos da terapêutica imunossupressora, anti- infeciosa e tempos de internamento hospitalar são substancialmente reduzidos. Também os custos sociais (familiares e institucionais), relacionados com a terapêutica crónica imunossupressora e anti-infeciosa das complicações, são substancialmente reduzidos. [0029] In the case of replacement of the pair of chromosomes 6 indigenous in human stem cells by the pair of chromosomes 6 of a patient, not only can the need to resort to immunosuppressive therapy be avoided or reduced, but equally important, the integrity is maintained. the normal operation of the entire system patient's immune system. This represents a radical change from the current paradigm that is based exclusively on combating/reducing the activity of the immune system to try to reduce rejection phenomena as much as possible. On the other hand, the costs of immunosuppressive and anti-infectious therapy and hospital stay are substantially reduced. The social costs (family and institutional) related to the chronic immunosuppressive and anti-infectious therapy of complications are also substantially reduced.
[0030] Numa forma de realização, as células estaminais originais são saudáveis, não apresentando mutações causadoras de doenças. Logo, os produtos derivados das linhas de células estaminais universais off-the-shelf editadas pelo método descrito na presente divulgação, são saudáveis, podendo ser utilizadas no tratamento de doenças que dependem de mutações mono ou multigénicas, exceto se a mutação em causa se encontrar no cromossoma 6 do doente. [0030] In one embodiment, the original stem cells are healthy, showing no disease-causing mutations. Therefore, products derived from off-the-shelf universal stem cell lines edited by the method described in the present disclosure are healthy and can be used in the treatment of diseases that depend on single or multigene mutations, unless the mutation in question is found on the patient's chromosome 6.
[0031] Numa forma de realização, quer o cromossoma 6 humano do paciente, que irá substituir o cromossoma 6 indígena das células estaminais universais off-the-shelf, quer o cromossoma de dador saudável que irá substituir o cromossoma mutado do paciente, podem ser transferidos pela técnica de transferência cromossómica mediada por microcélulas (do inglês: Microcell Mediated Cromossome Transfer (MMCT)). Noutra outra forma de realização, os cromossomas exógenos podem ser transferidos através da microinjeção do conteúdo das microcélulas nas células de destino. Em ambos os casos, o material genético é transferido juntamente com o sistema de nuclease responsável pela posterior clivagem das sequências normalmente ausentes do genoma, mas presentes nos cromossomas nativos editados, (CRISPR, ZNF, TALENs, ou outras). [0031] In one embodiment, either the patient's human chromosome 6, which will replace the indigenous chromosome 6 of off-the-shelf universal stem cells, or the healthy donor chromosome that will replace the patient's mutated chromosome, can be transferred by the microcell Mediated Chromosome Transfer (MMCT) technique. In another embodiment, exogenous chromosomes can be transferred by microinjecting the contents of the microcells into the target cells. In both cases, the genetic material is transferred together with the nuclease system responsible for the subsequent cleavage of sequences normally absent from the genome, but present on edited native chromosomes (CRISPR, ZNF, TALENs, or others).
[0032] Métodos para o alinhamento de sequências para comparação são bem conhecidos na técnica, tais métodos incluem GAP, BESTFIT, BLAST, FASTA e TFASTA. O algoritmo BLAST (Altschul et al. (1990) J Mol Biol 215: 403-10) calcula a identidade percentual da sequência e realiza uma análise estatística da semelhança entre as duas sequências. O software para executar a análise BLAST está disponível ao público no National Center for Biotechnology Information (NCBI). As percentagens globais de similaridade e identidade também podem ser determinadas usando um dos métodos disponíveis no pacote de software MatGAT (Campanella et al., BMC Bioinformatics. 2003 Jul 10; 4:29. MatGAT: um aplicativo que gera matrizes de similaridade/identidade usando proteínas ou sequências de ADN). Os valores de identidade de sequência, que são indicados na presente divulgação como uma percentagem, foram determinados em toda a sequência de bases, utilizando BLAST com os parâmetros padrão. [0032] Methods for aligning sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10 ) calculates the percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software to perform the BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI). Overall percentages of similarity and identity can also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10; 4:29. MatGAT: an application that generates similarity/identity matrices using proteins or DNA sequences). Sequence identity values, which are reported in the present disclosure as a percentage, were determined on the entire base sequence using BLAST with default parameters.
[0033] A presente divulgação diz respeito a um método para a edição do genoma de células estaminais compreendendo os seguintes passos: (i) preparar pelo menos uma cassete genética, em que a cassete genética compreende um promotor, um marcador, e uma sequência genética ausente do genoma das células estaminais a editar, em que o marcador compreende pelo menos um gene codificador de uma proteína de: fluorescência, resistência a antibiótico, sensibilidade a drogas, ou suas combinações; e em que a sequência genética ausente do genoma das células estaminais a editar é clivável por pelo menos um sistema de nucleases de edição genómica, de preferência o sistema de CRISPR, ZNF, TALENs, ou as suas misturas; (ii) inserir pelo menos uma cassete genética num cromossoma da célula a editar por meio de clivagens na cadeia dupla do ADN perto e de cada lado do centrómero do cromossoma da célula a editar, sendo estas clivagens da cadeia dupla do ADN produzidas por nucleases de preferência do sistema CRISPR, ZNF, TALENS, Transposases , recombinases sítio- específicas de tirosina (do inglês tyrosine síte-specífíc recombinases, T-SSRs), recombinases sítio-específicas da serina (S-SSRs); e (iii) selecionar as células editadas com o marcador introduzido na referida cassete genética. [0033] The present disclosure relates to a method for editing the stem cell genome comprising the following steps: (i) preparing at least one gene cassette, wherein the gene cassette comprises a promoter, a marker, and a gene sequence absent from the genome of the stem cells to be edited, wherein the marker comprises at least one gene encoding a protein of: fluorescence, antibiotic resistance, drug sensitivity, or combinations thereof; and wherein the genetic sequence absent from the genome of the stem cells to be edited is cleavable by at least one genomic editing nuclease system, preferably the CRISPR, ZNF, TALENs system, or mixtures thereof; (ii) inserting at least one genetic cassette into a chromosome of the cell to be edited by means of cleavages in the double strand of DNA near and on either side of the centromere of the chromosome of the cell to be edited, these cleavages of the double strand of DNA being produced by nucleases preferably from the CRISPR, ZNF, TALENS, Transposases system , site-specific tyrosine recombinases (T-SSRs), site-specific serine recombinases (S-SSRs); and (iii) selecting edited cells with the marker introduced into said genetic cassette.
[0034] Numa forma de realização, a sequência genética ausente do genoma das células estaminais a editar compreende um nulómero. [0034] In one embodiment, the genetic sequence absent from the stem cell genome to be edited comprises a nullomer.
[0035] Numa forma de realização, o nulómero compreende pelo menos uma sequência com pelo menos 90% de identidade das seguintes sequências: Seq. ID N° 24, Seq. ID N° 25, Seq. ID N° 26, Seq. ID N° 27, Seq. ID N° 28, Seq. ID N° 29, Seq. ID N° 30, Seq. ID N° 31, Seq. ID N° 32, Seq. ID N° 33, Seq. ID N° 34, Seq. ID N° 35, Seq. ID N° 36, Seq. ID N° 37, Seq. ID N° 38, Seq. ID N° 39, Seq. ID N° 40, Seq. ID N° 41, Seq. ID N° 42 ou suas misturas, múltiplos ou frações. [0035] In one embodiment, the nullomer comprises at least one sequence having at least 90% identity of the following sequences: Seq. ID No. 24, Seq. ID No. 25, Seq. ID No. 26, Seq. ID No. 27, Seq. ID No. 28, Seq. ID No. 29, Seq. ID No. 30, Seq. ID No. 31, Seq. ID No. 32, Seq. ID No. 33, Seq. ID No. 34, Seq. ID No. 35, Seq. ID No. 36, Seq. ID No. 37, Seq. ID No. 38, Seq. ID No. 39, Seq. ID No. 40, Seq. ID No. 41, Seq. ID No. 42 or mixtures, multiples or fractions thereof.
[0036] Numa outra forma de realização, a sequência de nulómero compreende 95% de identidade com a Seq. ID N° 24, Seq. ID N° 25, Seq. ID N° 26, Seq. ID N° 27, Seq. ID N° 28, Seq. ID N° 29, Seq. ID N° 30, Seq. ID N° 31, Seq. ID N° 32, Seq. ID N° 33, Seq. ID N° 34, Seq. ID N° 35, Seq. ID N° 36, Seq. ID N° 37, Seq. ID N° 38, Seq. ID N° 39, Seq. ID N° 40, Seq. ID N° 41, Seq. ID N° 42, ou suas misturas,seus múltiplos ou frações; de preferência 96%, 97%, 98%, 99% ou idênticas. [0036] In another embodiment, the nucleomer sequence comprises 95% identity to Seq. ID No. 24, Seq. ID No. 25, Seq. ID No. 26, Seq. ID No. 27, Seq. ID No. 28, Seq. ID No. 29, Seq. ID No. 30, Seq. ID No. 31, Seq. ID No. 32, Seq. ID No. 33, Seq. ID No. 34, Seq. ID No. 35, Seq. ID No. 36, Seq. ID No. 37, Seq. ID No. 38, Seq. ID No. 39, Seq. ID No. 40, Seq. ID No. 41, Seq. ID No. 42, or mixtures, multiples or fractions thereof; preferably 96%, 97%, 98%, 99% or the like.
[0037] Numa forma de realização, o método descrito na presente divulgação compreende adicionalmente um passo de substituir o cromossoma indígena que compreende a cassete genética por um cromossoma equivalente exógeno não editado. [0037] In one embodiment, the method described in the present disclosure further comprises a step of replace the indigenous chromosome comprising the genetic cassette with an unedited exogenous equivalent chromosome.
[0038] Numa forma de realização, a substituição do cromossoma que compreende a cassete genética por um cromossoma equivalente é feita por transferência cromossómica mediada por microcélulas. [0038] In one embodiment, replacement of the chromosome comprising the gene cassette with an equivalent chromosome is done by microcell-mediated chromosomal transfer.
[0039] Numa forma de realização, o marcador codifica para uma proteína que confere sensibilidade a drogas, de preferência ao tamoxifeno, aciclovir ou suas combinações. [0039] In one embodiment, the marker encodes a protein that confers sensitivity to drugs, in preference to tamoxifen, acyclovir or combinations thereof.
[0040] Numa forma de realização, a edição do genoma da célula a editar tem por objeto a deleção total de pelo menos um cromossoma, de preferência um par de cromossomas. [0040] In one embodiment, editing the genome of the cell to be edited aims at the total deletion of at least one chromosome, preferably a pair of chromosomes.
[0041] Numa forma de realização, o cromossoma da célula a editar é o cromossoma 6. [0041] In one embodiment, the chromosome of the cell to be edited is chromosome 6.
[0042] Numa forma de realização, as clivagens na cadeia dupla do ADN ocorrem pela adição de nucleases, de preferência nucleases CRISPR, ZNF, TALENs, Transposases, recombinases sítio-específicas de tirosina (do inglês tyrosine site- specific recombinases, T-SSRs), recombinases sítio- específicas da serina (S-SSRs) ou outras. [0042] In one embodiment, DNA double strand cleavages occur by the addition of nucleases, preferably CRISPR nucleases, ZNF, TALENs, Transposases, tyrosine site-specific recombinases, T-SSRs ), site-specific serine recombinases (S-SSRs), or others.
[0043] A presente divulgação compreende, ainda, uma cassete genética que compreende pelo menos um promotor, um marcador, e uma sequência genética ausente do genoma das células estaminais a editar, em que o marcador compreende pelo menos um gene codificador de uma proteína de fluorescência, de resistência a antibiótico, de sensibilidade a drogas, ou suas combinações; e em que a sequência genética ausente do genoma das células estaminais a editar é clivável por pelo menos um sistema de nucleases de edição genómica, de preferência CRISPR, ZNF, TALENs, ou as suas misturas. Numa forma de realização, a sequência genética ausente do genoma das células estaminais a editar compreende um nulómero. [0043] The present disclosure further comprises a genetic cassette comprising at least one promoter, a marker, and a genetic sequence absent from the genome of the stem cells to be edited, wherein the marker comprises at least one gene encoding a protein of fluorescence, antibiotic resistance, drug sensitivity, or combinations thereof; and wherein the genetic sequence absent from the genome of the stem cells to be edited is cleavable by at least one nuclease system of genome editing, preferably CRISPR, ZNF, TALENs, or mixtures thereof. In one embodiment, the genetic sequence absent from the stem cell genome to be edited comprises a nullomer.
[0044] Numa forma de realização, o nulómero compreende pelo menos uma sequência com pelo menos 90% de identidade das sequências Seq. ID N° 24, Seq. ID N° 25, Seq. ID N° 26, Seq. ID N° 27, Seq. ID N° 28, Seq. ID N° 29, Seq. ID N° 30, Seq. ID N° 31, Seq. ID N° 32, Seq. ID N° 33, Seq. ID N° 34, Seq. ID N° 35, Seq. ID N° 36, Seq. ID N° 37, Seq. ID N° 38, Seq. ID N° 39, Seq. ID N° 40, Seq. ID N° 41, Seq. ID N° 42 ou suas misturas, múltiplos ou frações. [0044] In one embodiment, the nullomer comprises at least one sequence with at least 90% sequence identity Seq. ID No. 24, Seq. ID No. 25, Seq. ID No. 26, Seq. ID No. 27, Seq. ID No. 28, Seq. ID No. 29, Seq. ID No. 30, Seq. ID No. 31, Seq. ID No. 32, Seq. ID No. 33, Seq. ID No. 34, Seq. ID No. 35, Seq. ID No. 36, Seq. ID No. 37, Seq. ID No. 38, Seq. ID No. 39, Seq. ID No. 40, Seq. ID No. 41, Seq. ID No. 42 or mixtures, multiples or fractions thereof.
[0045] A presente divulgação descreve ainda uma cassete genética tal como descrita neste documento para o uso em medicina ou em medicina veterinária. [0045] The present disclosure further describes a genetic cassette as described herein for use in medicine or veterinary medicine.
[0046] Numa forma de realização, a cassete genética é para o uso no tratamento de doenças genéticas, nomeadamente doenças monogénicas ou multigénicas, de preferência hemofilia, drepanocitose, talassémia, anemia de Fanconi, síndroma de Alagille, doença congénita da glicosilação, ou retinite pigmentosa. [0046] In one embodiment, the gene cassette is for use in the treatment of genetic diseases, namely monogenic or multigenic diseases, preferably hemophilia, sickle cell disease, thalassemia, Fanconi anemia, Alagille syndrome, congenital glycosylation disease, or retinitis pigmented.
[0047] Numa forma de realização, a cassete genética compreendende pelo menos 90% de identidade com a Seq. ID N°43, ou Seq. ID N° 44. [0047] In one embodiment, the gene cassette comprises at least 90% identity with Seq. ID No. 43, or Seq. ID No. 44.
[0048] Noutra forma de realização, a cassete genética compreende pelo menos 95% de identidade com Seq. ID N° 43, ou Seq. ID N° 44. [0049] Numa outra forma de realização, a cassete genética compreende pelo menos 99% de identidade com Seq. ID N° 43, ou Seq. ID N° 44. [0048] In another embodiment, the gene cassette comprises at least 95% identity with Seq. ID No. 43, or Seq. ID No. 44. [0049] In another embodiment, the gene cassette comprises at least 99% identity with Seq. ID No. 43, or Seq. ID No. 44.
[0050] A presente divulgação descreve também um vetor que compreende pelo menos uma cassete genética descrita na presente divulgação. [0050] The present disclosure also describes a vector comprising at least one genetic cassette described in the present disclosure.
[0051] Um aspeto da presente divulgação compreende as células estaminais editadas, obtidas pelo método descrito na presente divulgação, que compreendem a cassete descrita na presente divulgação. [0051] One aspect of the present disclosure comprises edited stem cells obtained by the method described in the present disclosure, which comprise the cassette described in the present disclosure.
[0052] Numa forma de realização, as células estaminais editadas compreendem pelo menos a substituição de um cromossoma indígena por um cromossoma exógeno, sem compreender resíduos, i. e. material cromossómico, do cromossoma indígena. [0052] In one embodiment, the edited stem cells comprise at least the replacement of an indigenous chromosome with an exogenous chromosome, without comprising residues, i.e. and. chromosomal material, from the indigenous chromosome.
[0053] A presente divulgação compreende também uma célula, micro-tecido, tecido ou órgão obtido a partir da diferenciação das células estaminais editadas, descritas na presente divulgação, após substituição do par de cromossomas editado pelo seu equivalente exógeno. [0053] The present disclosure also comprises a cell, microtissue, tissue or organ obtained from the differentiation of the edited stem cells described in the present disclosure after replacement of the edited chromosome pair with its exogenous equivalent.
[0054] Um aspeto da presente divulgação compreende a célula, micro-tecido, tecido ou órgão, descritas na presente divulgação, para o uso em medicina. [0054] One aspect of the present disclosure comprises the cell, microtissue, tissue or organ described in the present disclosure for use in medicine.
[0055] Numa forma de realização, a célula, micro-tecido, tecido ou órgão são para o uso no tratamento de doenças genéticas, de preferência doenças monogénicas ou multigénicas, de maior preferência hemofilia, drepanocitose, talassémia, anemia de Fanconi, síndroma de Alagille, doença congénita da glicosilação, ou retinite pigmentosa. [0056] Numa outra forma de realização, a célula, micro- tecido, tecido ou órgão são para o uso na diminuição da rejeição da célula, micro-tecido, tecido ou órgão transplantados . [0055] In one embodiment, the cell, microtissue, tissue or organ is for use in the treatment of genetic diseases, preferably monogenic or multigenic diseases, most preferably hemophilia, sickle cell disease, thalassemia, Fanconi anemia, Alagille, congenital disease of glycosylation, or retinitis pigmentosa. [0056] In another embodiment, the cell, microtissue, tissue or organ is for use in decreasing rejection of the transplanted cell, microtissue, tissue or organ.
[0057] Ainda noutra forma de realização, a célula, micro- tecido, tecido ou órgão são para o uso na eliminação ou diminuição de doença autoimune. [0057] In yet another embodiment, the cell, microtissue, tissue or organ is for use in eliminating or lessening autoimmune disease.
[0058] Numa forma de realização, a célula, micro-tecido, tecido ou órgão são para o uso na eliminação ou diminuição de uma doença infeciosa num paciente, em particular da Síndroma da imunodeficiência humana adquirida (SIDA). [0058] In one embodiment, the cell, microtissue, tissue or organ is for use in eliminating or lessening an infectious disease in a patient, in particular Acquired Immunodeficiency Syndrome (AIDS).
[0059] Numa outra forma de realização, a célula, micro- tecido, tecido ou órgão são para o uso em transplantes, para a substituição material ou funcional integral, ou para complementação de uma função diminuída de células, tecidos ou órgãos de um sujeito. Numa forma de realização, a célula, micro-tecido, tecido ou órgão diminuem a rejeição por parte do sujeito transplantado. Numa outra forma de realização, a resposta imune do sujeito transplantado é eliminada ou diminuída. [0059] In another embodiment, the cell, microtissue, tissue, or organ is for use in transplantation, for integral material or functional replacement, or for complementation of a diminished function of a subject's cells, tissues, or organs . In one embodiment, the cell, microtissue, tissue or organ lessens rejection by the transplanted subject. In another embodiment, the immune response of the transplanted subject is eliminated or diminished.
[0060] Numa forma de realização, a célula, micro-tecido, tecido ou órgão são para o uso na substituição de progenitores hematopoiéticos de um paciente, (conhecido como transplante de medula óssea), após exposição terapêutica ou acidental ou em ambiente de guerra a radiações ionizantes, quimioterapia, substância tóxica ou suas combinações. [0060] In one embodiment, the cell, microtissue, tissue or organ is for use in replacing a patient's hematopoietic progenitors, (known as a bone marrow transplant), following therapeutic or accidental exposure or in a warfare environment to ionizing radiation, chemotherapy, toxic substance or combinations thereof.
[0061] A presente divulgação compreende, ainda, uma célula, micro-tecido, tecido ou órgão, descritas na presente divulgação, para uso em medicina ou medicina veterinária. Numa forma de realização, a célula, micro-tecido, tecido ou órgão são para o uso no tratamento de doenças genéticas, de preferência doenças monogénicas ou multigénicas, de maior preferência hemofilia, drepanocitose, talassémia, anemia de Fanconi, síndroma de Alagille, doença congénita da glicosilação, ou retinite pigmentosa. Numa outra forma de realização, a célula, micro-tecido, tecido ou órgão são para o uso no tratamento de Hemofilia A, Trombastenia de Glanzmann, Trombastenia Atípica Equina, Doença de von Willebrand, Deficiência de pre-calicreína, Miopatia do armazenamento de polissacaridos Tipo 1 e Tipo 2, Deficiência da Enzima de Ramificação do Glicogénio, ou Miopatia das cadeias pesadas 2X da miosina, em equinos; deficiência da beta-manosidase, Deficiência da adesão leucocitária bovina, Deficiência hereditária do Zinco, ou Citrulémia, em bovinos; Hemofilia A; ou Doença de von Willebrand, em caninos. [0061] The present disclosure further comprises a cell, microtissue, tissue or organ described in the present disclosure for use in medicine or veterinary medicine. In one embodiment, the cell, microtissue, tissue or organ are for use in the treatment of genetic disorders, preferably monogenic or multigenic disorders, most preferably hemophilia, sickle cell disease, thalassemia, Fanconi anemia, Alagille syndrome, congenital glycosylation disease, or retinitis pigmentosa. In another embodiment, the cell, microtissue, tissue or organ is for use in the treatment of Hemophilia A, Glanzmann's Thrombasthenia, Equine Atypical Thrombasthenia, von Willebrand Disease, Prekallikrein Deficiency, Polysaccharide Storage Myopathy Type 1 and Type 2, Glycogen Branching Enzyme Deficiency, or Myosin 2X heavy chain myopathy, in horses; beta-mannosidase deficiency, Bovine Leukocyte Adhesion Deficiency, Hereditary Zinc Deficiency, or Citrulemia in cattle; Hemophilia A; or von Willebrand disease, in canines.
Breve Descrição das Figuras Brief Description of Figures
[0062] Para uma mais fácil compreensão, juntam-se em anexo as figuras, as quais representam realizações preferenciais que não pretendem limitar o objeto da presente descrição. [0062] For an easier understanding, the figures are attached, which represent preferred embodiments that do not intend to limit the object of the present description.
[0063] Figura 1: Representação esquemática do cromossoma 6 humano e localização do gene BAG2 1 junto ao centrómero 2 no braço longo 4. O gene LGSN 3 está localizado junto ao centrómero 2, mas no braço curto 5. [0063] Figure 1: Schematic representation of human chromosome 6 and location of the BAG2 1 gene near centromere 2 in the long arm 4. The LGSN 3 gene is located near the centromere 2, but in the short arm 5.
[0064] Figura 2: Representação de uma forma de realização da transfeção da linha virgem de células estaminais, com CRISPR/Cas9 + sgARN/BAG2 6 e Cassete 17 para a edição do Gene BAG2 do Cromossoma 6 Humano. A nuclease CRISPR/Cas9 + sgARN/BAG2 6 provoca uma clivagem da cadeia dupla do ADN no gene BAG2. Essa clivagem é depois reparada pela célula com a integração do conjunto das sequências compreendidas na Cassete 17 com exceção dos braços de homologia HA5' e HA3' (inserto 8), com recurso a Reparação Dirigida por Homologia, (do inglês - Homology Directed Repair - HDR). No inserto 8 o [PGKpmt] significa promotor da fosfoglicerato quinase; [PuroR] significa transferase da resistência à Puromicina; [GFP] significa Proteína de Fluorescência Verde; [2pA] representa a sequência de poliadenilação. [0064] Figure 2: Representation of an embodiment of transfection of virgin stem cell line, with CRISPR/Cas9 + sgRNA/BAG2 6 and Cassette 17 for editing the Human Chromosome 6 BAG2 Gene. CRISPR/Cas9 + sgRNA/BAG2 6 nuclease causes double-stranded DNA cleavage in the BAG2 gene. This cleavage is then repaired by the cell with the integration of the set of sequences comprised in Cassette 17, with the exception of the homology arms HA5' and HA3' (insert 8), using Homology Directed Repair. HDR). In insert 8 [PGKpmt] means phosphoglycerate kinase promoter; [PuroR] means Puromycin resistance transferase; [GFP] means Green Fluorescence Protein; [2pA] represents the polyadenylation sequence.
[0065] Figura 3: Representação de uma forma de realização da transfeção da linha de células estaminais previamente editada no gene BAG 2, com CRISPR/Cas9 + sgARN/LGSN 11 e Cassete 2 9 para a edição do Gene LGSN do Cromossoma 6 Humano. A nuclease CRISPR/Cas9 + sgARN/LGSN 11 provoca uma clivagem da cadeia dupla do ADN no gene LGSN. Essa clivagem é depois reparada pela célula com a integração do conjunto das sequências compreendidos na Cassete 29 com exceção dos braços de homologia HA5' e HA3' (inserto 10), com recurso a Reparação Dirigida por Homologia (HDR). No inserto 10, [EOS pmt] corresponde à sequência do aumentador (do inglês: enhancer) SSR2 a jusante da sequência do gene SOX2; [TK] corresponde à sequência truncada do gene da Timidina quinase do vírus Herpes simples; [SV40pA] corresponde à sequência de poliadenilação do SV40; [EFloc pmt] corresponde ao promotor do fator de elongamento humano EFl ; [NeoR] corresponde à sequência da aminoglicosídeo transferase; [mCherry] corresponde à sequência do derivado monomérico da proteína fluorescente vermelha DSRed; [bGH pA] corresponde à sequência de poliadenilação da hormona de crescimento bovina. [0065] Figure 3: Representation of an embodiment of the transfection of the stem cell line previously edited in the BAG gene 2, with CRISPR/Cas9 + sgRNA/LGSN 11 and Cassette 29 for editing the Human Chromosome 6 LGSN Gene. CRISPR/Cas9 + sgRNA/LGSN 11 nuclease causes double stranded DNA cleavage in the LGSN gene. This cleavage is then repaired by the cell with the integration of the set of sequences comprised in Cassette 29, with the exception of the homology arms HA5' and HA3' (insert 10), using Homology Directed Repair (HDR). In insert 10, [EOS pmt] corresponds to the SSR2 enhancer sequence downstream of the SOX2 gene sequence; [TK] corresponds to the truncated sequence of the Herpes simplex virus Thymidine kinase gene; [SV40pA] corresponds to the SV40 polyadenylation sequence; [EFloc pmt] corresponds to the human elongation factor EFl promoter; [NeoR] corresponds to the aminoglycoside transferase sequence; [mCherry] corresponds to the sequence of the monomeric derivative of the red fluorescent protein DSRed; [bGH pA] corresponds to the bovine growth hormone polyadenylation sequence.
[0066] Figura 4: Representação de uma forma de realização da deleção do Cromossoma 6 Humano. Para ser produzida a deleção do Cromossoma 6 humano, a linha de células estaminais duplamente editada perto e de cada lado do centrómero (gene BAG2 e gene LGSN) é transfectada com CRISPR/Cas9/Nulómero gARN 12 e 13. A nuclease produz duas clivagens da dupla cadeia do ADN do cromossoma, separando-o em 3 partes. Um cromossoma nestas circunstâncias sofre deleção em 40% das células por não poder segregar durante a mitose. [0066] Figure 4: Representation of an embodiment of the Human Chromosome 6 deletion. to be produced at Deletion of human Chromosome 6, the double-edited stem cell line near and on either side of the centromere (BAG2 gene and LGSN gene) is transfected with CRISPR/Cas9/gRNA Nulomer 12 and 13. The nuclease produces two cleavages of the DNA double strand of the chromosome, separating it into 3 parts. A chromosome in these circumstances is deleted in 40% of the cells because it cannot secrete during mitosis.
[0067] Figura 5: Teste da reação em cadeia da polimerase (PCR, do inglês polymerase chain reactíon) para avaliação da inserção correta do inserto no gene BAG2 em hiPSCs. Imagem da electroforese em gel de agarose a 0,8% dos produtos de PCR/ADN genómico de células co-transfetadas com CRISPR/Cas9 + sgARN/BAG2 + Cassetel. Os algarismos 15;16;17;18;19;20;21 representam os iniciadores que corrrespondem à SEQ. ID N° 15, SEQ. ID N° 16, SEQ. ID N° 17, SEQ. ID N° 18, SEQ. ID N° 19, SEQ. ID N° 20 e SEQ. ID N° 21, respetivamente. 15+16 = 5'fora do construto até ao promotor PGK (PGKpmt) (~1900bp); 15+17 = 5'fora do construto até ao Braço de Homologia 5'(5'BH) (~500bp); 20+21= 3'fora do construto até ao Braço de Homologia 3'(3'BH)(~900bp); 16+18 = Braço de Homologia 5'(5'BH) até ao promotor PGK (PGKpmt)(~600bp); 19+20= 3' fora do construto até ao Nulómero (~1800bp). WT= ADN genómico de células Virgens (Controlo negativo). [0067] Figure 5: Polymerase chain reaction (PCR) test to evaluate the correct insertion of the insert in the BAG2 gene in hiPSCs. Image of 0.8% agarose gel electrophoresis of PCR/genomic DNA products from cells co-transfected with CRISPR/Cas9 + sgRNA/BAG2 + Cassetel. The numerals 15;16;17;18;19;20;21 represent the primers that correspond to SEQ. ID NO: 15, SEQ. ID NO: 16, SEQ. ID NO: 17, SEQ. ID NO: 18, SEQ. ID NO: 19, SEQ. ID NO: 20 and SEQ. ID No. 21, respectively. 15+16 = 5' outside the construct to the PGK promoter (PGKpmt) (~1900bp); 15+17 = 5'out of construct to Homology Arm 5'(5'BH) (~500bp); 20+21= 3'out of the construct to the Homology Arm 3'(3'BH)(~900bp); 16+18 = Homology Arm 5'(5'BH) to PGK promoter (PGKpmt)(~600bp); 19+20= 3' outside the construct to the Nunomer (~1800bp). WT= Naive cell genomic DNA (Negative control).
[0068] Figura 6: Citometria de Fluxo, percentagem da expressão de Proteína Fluorescente Verde (do inglês green fluorescent protein, GFP) em 4 amostras de Células Estaminais Pluripotentes Humanas Induzidas (do inglês human induced Pluripotent Stem Cell (hiPSCs). Co-transfeção de CRISPR/Cas9-BAG2sgRNA + Cassetel de acordo com o protocolo descrito neste documento. [0069] Figura 7: Citometria de Fluxo, percentagem da expressão de Proteína Fluorescente Verde (GFP) em 2 amostras (106 células/amostra) de hiPSCs. Dia 6 da Co-transfeção de CRISPR/Cas9-BAG2sgRNA + Cassetel de acordo com protocolo descrito neste documento. Previamente à realização da citometria de fluxo as células foram coradas com recurso ao kit: LIVE/DEAD™Fixable Dead Cell Stain Kit, ThermoFisher Scientific™. Este método permite ter uma avaliação mais rigorosa da percentagem das células GFP+ que estão viáveis na cultura. [0068] Figure 6: Flow Cytometry, percentage of Green Fluorescent Protein (GFP) expression in 4 samples of human induced Pluripotent Stem Cell (hiPSCs). of CRISPR/Cas9-BAG2sgRNA + Cassetel according to the protocol described in this document. [0069] Figure 7: Flow Cytometry, percent of Green Fluorescent Protein (GFP) expression in 2 samples (10 6 cells/sample) of hiPSCs. Day 6 of CRISPR/Cas9-BAG2sgRNA + Cassetel Co-transfection according to protocol described in this document. Prior to performing the flow cytometry, the cells were stained using the kit: LIVE/DEAD™Fixable Dead Cell Stain Kit, ThermoFisher Scientific™. This method allows for a more accurate assessment of the percentage of GFP+ cells that are viable in the culture.
Descrição Detalhada da Divulgação Detailed Description of the Disclosure
[0070] Na presente divulgação não foram utilizadas linhas de células estaminais que tenham implicado a destruição de embriões humanos. Na presente divulgação foram utilizadas células estaminais pluripotentes humanas (hPSC, do inglês human plurípotent stem cells), nomeadamente: células estaminais pluripotentes induzidas humanas (hiPSC, do inglês human induced plurípotent stem cells). [0070] In the present disclosure, no stem cell lines that have involved the destruction of human embryos have been used. In the present disclosure, human pluripotent stem cells (hPSC) were used, namely: human induced pluripotent stem cells (hiPSC).
[0071] Numa forma de realização, as hiPSCs são obtidas a partir de células adultas humanas, nomeadamente fibroblastos . A indução de pluripotência ocorre através da sobre-expressão dos Fatores de Yamanaka, i.e. Oct4, Sox2, C- mic e KLF4, que pode ser feita a partir da transfecção dos próprios fatores recombinantes, a partir de vetores retrovirais, a partir de pequenas moléculas químicas, a partir de microRNAs, a partir de Transposões Piggy-Bac, que promovem a sua sobre-expressão nas células adultas, levando à sua reprogramação para a condição de células pluripotentes. [0071] In one embodiment, the hiPSCs are obtained from adult human cells, namely fibroblasts. The induction of pluripotency occurs through the over-expression of the Yamanaka Factors, i.e. Oct4, Sox2, C-mic and KLF4, which can be made from the transfection of the recombinant factors themselves, from retroviral vectors, from small chemical molecules, from microRNAs, from Piggy-Bac transposons, which promote their over-expression in adult cells, leading to their reprogramming to the condition of pluripotent cells.
[0072] Numa forma de realização, as alterações genéticas introduzidas em genes inativos das células a alterar garantem que não existe nenhuma alteração ao normal funcionamento celular na fase de cultura, em que as células editadas são mantidas até à sua posterior utilização. Numa fase posterior de diferenciação celular já não existem genes editados, porque o cromossoma indígena editado já foi perdido pelas células, e substituído pelo seu equivalente que não contém a referida edição. Assim, não existirão genes passíveis de ficarem inoperativos (do inglês knockout) por edição, nas células, micro-tecidos, tecidos, ou órgãos preparados por este método, para uso em transplantação. [0072] In one embodiment, the genetic alterations introduced into inactive genes of the cells to be altered ensure that there is no change to the normal cell functioning in the culture phase, in which the edited cells are kept until their further use. At a later stage of cell differentiation, there are no longer edited genes, because the edited indigenous chromosome has already been lost by the cells, and replaced by its equivalent that does not contain the aforementioned edit. Thus, there will be no genes capable of being knocked out by editing, in cells, micro-tissues, tissues, or organs prepared by this method, for use in transplantation.
[0073] Numa forma de realização, a presença de marcadores de fluorescência e de seleção antibiótica e de genes de sensibilização a drogas (p.ex. aciclovir, tamoxifeno ou outras), permitem obter clones corretamente editados, tornando possível a seleção de clones, livres de eventuais contaminações com células não editadas. [0073] In one embodiment, the presence of fluorescence and antibiotic selection markers and drug sensitization genes (e.g. acyclovir, tamoxifen or others) make it possible to obtain correctly edited clones, making it possible to select clones, free from possible contamination with unedited cells.
[0074] Numa forma de realização, a presença de uma sequência nulomérica constitui mais um artifício inventivo, que permite que possam existir clivagens da cadeia dupla do ADN simultâneas, junto a ambos os lados do centrómero, levando à deleção/perda do cromossoma portador dessa edição. Além disso, a presença da sequência nulomérica evita que se produzam clivagens da cadeia dupla do ADN dos cromossomas equivalentes de substituição. Como as células normais necessitam de um conjunto de cromossomas normais (euploidia), sempre que a perda/deleção induzida de um par de cromossomas específicos tenha lugar nessa célula, outro par de cromossomas equivalente tem de lhe ser fornecido. Essa substituição cromossómica deve ser simultânea com a perda, para que não existam tempos de hipoploidia. Mesmo coabitando por momentos dentro da célula (tetraploidia temporária), os cromossomas de substituição não possuem a referida sequência nulomérica. Logo, o sistema de nuclease editora (ZNF, TALENs, CRISPR, ou outro sistema) não pode promover a sua perda pela célula, pois este sistema apenas cliva a cadeia dupla do ADN que contém a sequência nulomérica. A presença da sequência nulomérica garante assim que as clivagens na cadeia dupla do ADN, e consequente perda/deleção do par de cromossomas editados, não acontece no par de cromossomas exógenos substituto/s. Como o par de cromossomas substituto não possui essa sequência, fica livre de clivagens da cadeia dupla do seu ADN, mantendo toda a sua integridade sequencial e funcional, e a célula retornará à sua euploidia normal. [0074] In one embodiment, the presence of a nullomeric sequence is yet another inventive device, which allows for simultaneous double-stranded DNA cleavages on both sides of the centromere, leading to the deletion/loss of the chromosome carrying this edition. Furthermore, the presence of the nullomeric sequence prevents double-stranded DNA cleavages from the replacement equivalent chromosomes from occurring. As normal cells require a normal set of chromosomes (euploidy), whenever the induced loss/deletion of a specific chromosome pair takes place in that cell, another equivalent pair of chromosomes must be provided. This chromosomal replacement must be simultaneous with the loss, so that there are no hypoploid times. Even though they cohabit for moments inside the cell (temporary tetraploidy), the replacement chromosomes do not have the said nulleric sequence. Therefore, the nuclease editor system (ZNF, TALENs, CRISPR, or other system) cannot promote its loss by the cell, as this system only cleaves the double strand of DNA that contains the nullomeric sequence. The presence of the nullomeric sequence thus ensures that cleavages in the DNA double strand, and consequent loss/deletion of the edited pair of chromosomes, do not occur in the replacement pair of exogenous chromosomes. As the replacement chromosome pair does not have this sequence, it is free of double strand cleavages of its DNA, maintaining all its sequential and functional integrity, and the cell will return to its normal euploidy.
[0075] Numa forma de realização, os clones corretamente editados podem originar linhas de células estaminais universais ou pessoais off-the-shelf, a partir das quais é possível obter a deleção de um par cromossómico específico. Esta característica abre uma porta à industrialização de linhas de células universais bem como de linhas de células estaminais pessoais, uma vez que ficarão disponíveis para uso imediato (off-the-shelf) para a utilização na preparação de produtos derivados. Um produto off-the-shelf adquire vantagens de escala e de disponibilidade imediata com grande poupança de tempo de laboratório, e facilitando a sua disponibilização a tempo para os pacientes. A questão da rapidez de preparação do produto derivado final a transplantar ao paciente, tem implicações muito importantes no aumento da probabilidade de sobrevivência do paciente, bem como na redução dos custos totais, (económicos e sociais) do tratamento e no aumento da qualidade de vida do paciente e família. [0075] In one embodiment, correctly edited clones can give off-the-shelf universal or personal stem cell lines from which a specific chromosomal pair deletion can be obtained. This feature opens the door to industrialization of universal cell lines as well as personal stem cell lines, as they will be available for immediate use (off-the-shelf) for use in the preparation of derived products. An off-the-shelf product acquires advantages of scale and immediate availability with great savings in laboratory time, and facilitating its availability in time for patients. The question of the speed of preparation of the final derivative product to be transplanted to the patient has very important implications in increasing the probability of patient survival, as well as in reducing the total (economic and social) costs of the treatment and in increasing the quality of life. of the patient and family.
[0076] Numa forma de realização, o par de cromossomas de substituição pode ser fornecido às células utilizando a técnica de transferência cromossómica mediada por microcélulas (MMCT, do inglês Microcell Mediated Chromosome Transfer) (Fournier e Ruddle 1977), em simultâneo com os componentes do sistema de nuclease editora, (CRISPR, ZNF, TALENs, ou outros) que clivam o nulómero, ou uma outra sequência adequada, ausente do genoma normal das células estaminais da espécie a editar, de cada lado do centrómero. [0076] In one embodiment, the replacement pair of chromosomes can be delivered to cells using the Microcell Mediated Chromosome Transfer (MMCT) technique (Fournier and Ruddle 1977), in conjunction with components of the nuclease editor system (CRISPR, ZNF, TALENs, or others) that cleave the numeromer, or another suitable sequence absent from the normal stem cell genome of the species to be edited, on either side of the centromere.
[0077] Numa outra forma de realização, o par de cromossomas de substituição pode ser fornecido às células destinatárias por técnicas de microinjeção juntamente com o sistema de nuclease editora (CRISPR, ZNF, TALENs, ou outros). [0077] In another embodiment, the replacement chromosome pair can be delivered to the target cells by microinjection techniques along with the nuclease editor system (CRISPR, ZNF, TALENs, or others).
[0078] Numa forma de realização, as células que após o protocolo de substituição cromossómica possam conter alguma porção do par de cromossomas deletado, são positivamente e/ou negativamente selecionadas pelos marcadores de fluorescência e de resistência antibiótica e de sensibilidade a drogas (por exemplo, aciclovir, tamoxifeno, ou suas misturas), presentes nos insertos (do inglês insert). [0078] In one embodiment, cells that after the chromosomal replacement protocol may contain some portion of the deleted chromosome pair are positively and/or negatively selected by markers of fluorescence and antibiotic resistance and drug sensitivity (e.g. , acyclovir, tamoxifen, or mixtures thereof), present in the inserts.
[0079] Numa forma de realização, as células corretamente substituídas não apresentam fluorescência, nem resistência antibiótica nem sensibilidade às drogas programadas. As células obtidas a partir do método descrito na presente divulgação podem ser utilizadas no tratamento de diversas patologias monogénicas, tais como hemofilias, drepanocitose, talassémia, entre outras. [0079] In one embodiment, the correctly replaced cells show no fluorescence, no antibiotic resistance, or programmed drug sensitivity. The cells obtained from the method described in the present disclosure can be used in the treatment of various monogenic pathologies, such as hemophilia, sickle cell disease, thalassemia, among others.
[0080] Numa forma de realização, é feita a substituição do cromossoma portador do gene mutado em células estaminais colhidas do doente (com exceção do cromossoma 6), sendo o cromossoma substituído por um cromossoma equivalente saudável, colhido de dador saudável. As células resultantes são posteriormente diferenciadas e transplantadas no paciente. [0080] In one embodiment, the chromosome carrying the mutated gene is replaced in stem cells taken from the patient (with the exception of chromosome 6), the chromosome being replaced by an equivalent healthy chromosome taken from a healthy donor. The resulting cells are subsequently differentiated and transplanted into the patient.
[0081] Numa outra forma de realização, são utilizadas células estaminais universais off-the-shelf com substituição do par de cromossomas 6 indígena pelo par de cromossomas 6 do paciente. As células resultantes são, assim, compatíveis com o paciente, podendo ser transplantadas sem ocorrer rejeição por parte do hospedeiro. Mais ainda, visto que as células são preparadas a partir de células estaminais off- the-shelf saudáveis, o cromossoma mutado do paciente está ausente das células a transplantar. [0081] In another embodiment, off-the-shelf universal stem cells are used with replacement of the indigenous chromosome 6 pair with the patient's chromosome 6 pair. The resulting cells are thus compatible with the patient and can be transplanted without host rejection. Furthermore, since the cells are prepared from healthy off-the-shelf stem cells, the patient's mutated chromosome is absent from the cells to be transplanted.
[0082] De seguida, são apresentados dois exemplos de forma a expor as implicações práticas, industriais, e clínicas do método da presente divulgação. A Hemofilia A (Fator VIII) e a Hemofilia B (Fator IX) são duas doenças hemorrágicas congénitas humanas que dependem de mutações no cromossoma X Humano. A produção de 6% do valor normal de Fator VIII ou do Fator IX é suficiente para permitir um nível normal da coagulação sanguínea. As células estaminais universais off-the-shelf masculinas contêm um cromossoma X normal, produtor dos fatores VIII e IX normais. Por isso o transplante de uma quantidade mínima de células diferenciadas, capaz de produzir 6% ou mais do nível normal dos fatores VIII e IX pode providenciar os níveis adequados dos fatores referidos, curando os doentes, apesar de em todas as outras células do corpo do paciente continuarem a existir mutações responsáveis pelos anteriores baixos níveis de Fator VIII ou Fator IX. [0082] Next, two examples are presented in order to expose the practical, industrial, and clinical implications of the method of the present disclosure. Hemophilia A (Factor VIII) and Hemophilia B (Factor IX) are two human congenital bleeding disorders that depend on mutations in the Human X chromosome. Production of 6% of the normal value of Factor VIII or Factor IX is sufficient to allow a normal level of blood clotting. Universal male off-the-shelf stem cells contain a normal X chromosome, which produces normal factors VIII and IX. Therefore, the transplantation of a minimum amount of differentiated cells, capable of producing 6% or more of the normal level of factors VIII and IX, can provide adequate levels of the mentioned factors, curing the sick, despite in all other cells of the body of the patient mutations responsible for the previously low levels of Factor VIII or Factor IX still exist.
[0083] Como segundo exemplo, a drepanocitose, doença hematológica humana congénita, é dependente de uma única mutação na posição 6 do gene da cadeia b da hemoglobina. Numa forma de realização, esta doença pode ser curada a partir da substituição do par de cromossomas 11 do doente por um par de cromossomas 11 normais em células estaminais do paciente e sua posterior diferenciação em células progenitoras hematopoiéticas; ou pela transplantação de células progenitoras hematopoiéticas diferenciadas a partir de células estaminais universais off-the-shelf, cujo par de cromossomas 11 indígena é normal e onde o par de cromossomas 6 indígena foi substituído pelo par de cromossomas 6 do paciente, para anular a rejeição das células transplantadas. [0083] As a second example, sickle cell disease, a congenital human hematological disease, is dependent on a single mutation at position 6 of the hemoglobin b-chain gene. In one embodiment, this disease can be cured by replacing the patient's chromosome 11 pair with a normal chromosome 11 pair in the patient's stem cells and further differentiating them into hematopoietic progenitor cells; or by transplantation of differentiated hematopoietic progenitor cells from off-the-shelf universal stem cells whose indigenous chromosome 11 pair is normal and where the indigenous chromosome 6 pair has been replaced by the patient's chromosome 6 pair to nullify rejection of the transplanted cells.
[0084] A Biologia Molecular possui técnicas capazes de identificar e reproduzir os processos envolvidos na replicação do ADN celular, bem como na sua transcrição em ácido ribonucleico (ARN) e a tradução do ARN em proteínas. [0084] Molecular Biology has techniques capable of identifying and reproducing the processes involved in the replication of cellular DNA, as well as in its transcription into ribonucleic acid (RNA) and the translation of RNA into proteins.
[0085] São técnicas comummente utilizadas em Biologia Molecular e previstas no âmbito da presente divulgação as seguintes : [0085] The following are techniques commonly used in Molecular Biology and foreseen within the scope of this disclosure:
[0086] Edição Genómica - Na presente divulgação é necessário proceder à integração (do ingês - knock-in) de ADN novo em localizações precisas dentro do genoma das células estaminais que se pretende modificar. Para isso, é necessário ter acesso a esse material genético com a sequência ou sequências de nucleótidos adequada. O referido material genético é obtido por síntese industrial, quer na totalidade da sequência quer em parcelas que posteriormente se ligam artificialmente, ou por subclonagem dos seus diversos promotores e genes. No último caso, o construto é obtido utilizando técnicas de PCR para a produção de cada um dos braços de homologia e de subclonagem de plasmídeos contendo as sequências dos promotores e genes do inserto. A sequência assim obtida é designada o "construto" (do inglês - construct). O construto é composto por dois braços de homologia, (braço de homologia 5' ou Esquerdo, e braço de homologia 3' ou Direito), intercalados pelo inserto. O inserto é a nova sequência que irá fazer parte do genoma da célula estaminal que se pretende modificar/preparar/editar. São os braços de homologia que permitem, por complementaridade com ADN do local onde pretendemos integrar o inserto, que este seja conduzido e copiado para o local especifico e não noutro qualquer local aleatório no genoma. [0086] Genomic Editing - In the present disclosure it is necessary to proceed with the integration (from English - knock-in) of new DNA at precise locations within the genome of the stem cells to be modified. For this, it is necessary to have access to that genetic material with the appropriate nucleotide sequence or sequences. Said genetic material is obtained by industrial synthesis, either in the entire sequence or in portions that are later artificially linked, or by subcloning its various promoters and genes. In the latter case, the construct is obtained using PCR techniques for the production of each of the homology and subcloning arms of plasmids containing the sequences of the promoters and genes of the insert. The sequence thus obtained is called the "construct". - construct). The construct is composed of two homology arms, (5' or Left homology arm, and 3' or Right homology arm), interspersed by the insert. The insert is the new sequence that will be part of the stem cell genome that is to be modified/prepared/edited. It is the homology arms that, by complementarity with the DNA of the place where we want to integrate the insert, allow it to be led and copied to the specific location and not to any other random location in the genome.
[0087] Numa forma de realização, a presente divulgação descreve a construção da totalidade do construto Cassete 1 (SEQ. ID N° 43) no laboratório. [0087] In one embodiment, the present disclosure describes the construction of the entire Cassette 1 (SEQ ID NO:43) construct in the laboratory.
[0088] Numa forma de realização, a subclonagem compreendeu os habituais passos de digestão com enzimas de restrição adequadas (nucleases), de plasmideos preparados artificialmente ou adquiridos à indústria, posterior ligação das sequências de nucleótidos nos referidos plasmideos e transfecção de células competentes adquiridas à indústria, (E. Coli especialmente preparadas), seguindo o protocolo recomendado pelos fabricantes. Posteriormente procedeu-se ao isolamento dos clones destas células competentes corretamente editadas por técnica de PCR e/ou sequenciação. Um ou mais clones desejados foram conservados congelados a[0088] In one embodiment, the subcloning comprised the usual steps of digestion with suitable restriction enzymes (nucleases), of plasmids prepared artificially or acquired from the industry, subsequent ligation of the nucleotide sequences in said plasmids and transfection of competent cells acquired to the industry, (E. Coli specially prepared), following the protocol recommended by the manufacturers. Subsequently, clones of these competent cells were isolated correctly edited by PCR and/or sequencing technique. One or more desired clones were kept frozen at
-80°C. -80°C.
[0089] Clivagens da Cadeia Dupla do ADN - A integração do inserto é facilitada quando concomitantemente à presença do construto, o ADN do local onde o inserto é integrado estiver clivado/aberto. Existem vários sistemas de nucleases para proceder a essa clivagem da dupla hélice do ADN: ZFN, TALENs, transposases, CRISPR, T-SSRs, S-SSRs e outras. [0090] Numa forma de realização, na presente divulgação foi utilizado o sistema CRISPR/Cas9 como ferramenta para proceder à clivagem da dupla cadeia do ADN (DSB, do inglês: Double Strand Break) no local exato do genoma a editar. [0089] DNA Double Strand Cleavages - Insert integration is facilitated when, concomitantly with the presence of the construct, the DNA at the site where the insert is integrated is cleaved/opened. There are several nuclease systems to carry out this cleavage of the DNA double helix: ZFN, TALENs, transposases, CRISPR, T-SSRs, S-SSRs and others. [0090] In one embodiment, the CRISPR/Cas9 system was used in the present disclosure as a tool to cleave the DNA double strand (DSB) at the exact location of the genome to be edited.
[0091] Reparação dirigida por homologia (HDR, do inglês Homology Directed Repaír) - Este é um dos processos que uma célula que sofreu uma lesão do seu ADN pode utilizar, para fazer a sua reparação. Esta estratégia é frequentemente utilizada em técnicas de Biologia Molecular para proceder à edição genómica por inserção de ADN novo no genoma. [0091] Homology Directed Repair (HDR) - This is one of the processes that a cell that has suffered a DNA damage can use to make its repair. This strategy is frequently used in Molecular Biology techniques to carry out genomic editing by inserting new DNA into the genome.
[0092] Numa forma de realização, uma vez obtida a clivagem das duas cadeias do ADN, (DSB), se dentro da célula estiver simultaneamente co-transfectado o construto, por HDR a célula poderá copiar o inserto para o local do corte, passando assim o genoma da célula a expressar a nova informação genética. Esta técnica tem uma eficiência muito baixa, sobretudo em células estaminais humanas. Mas é atualmente a mais eficiente para realizar a edição genómica pretendida . [0092] In one embodiment, once the cleavage of the two DNA strands (DSB) is obtained, if the construct is simultaneously co-transfected within the cell, by HDR the cell can copy the insert to the cut site, passing thus the cell genome to express the new genetic information. This technique has a very low efficiency, especially in human stem cells. But it is currently the most efficient to carry out the intended genomic editing.
[0093] Transfecção - Designa-se por transfecção a técnica que permite a introdução dentro das células a editar do material genético preparado para essa edição, bem como de outros elementos capazes de o promoverem, por exemplo a nuclease CRISPR/Cas9 e o sgARN. Os principais métodos de transfecção são a Lipofecção, a electroporação, ou microinjecção . [0093] Transfection - Transfection is the technique that allows the introduction into the cells to be edited of the genetic material prepared for that edition, as well as other elements capable of promoting it, for example CRISPR/Cas9 nuclease and sgRNA. The main transfection methods are Lipofection, electroporation, or microinjection.
[0094] Numa forma de realização, na presente divulgação foi usada a lipofecção com Lipofectamine™ 3000 (Thermo Fisher Scientific) . [0095] Separação de células ativadas por fluorescência (FACS, do inglês Fluorescence Associated Cell Sortíng) Trata-se de uma técnica que permite a seleção e enriquecimento de uma população celular tendo por base a expressão de uma ou mais proteínas fluorescentes. É uma técnica que pode ser utilizada como única ferramenta de seleção ou, previamente ou posteriormente à seleção por expressão de uma molécula de resistência antibiótica. [0094] In one embodiment, lipofection with Lipofectamine™ 3000 (Thermo Fisher Scientific) was used in the present disclosure. [0095] Fluorescence Associated Cell Sorting (FACS) This is a technique that allows the selection and enrichment of a cell population based on the expression of one or more fluorescent proteins. It is a technique that can be used as the only selection tool or, before or after the selection by expression of an antibiotic resistance molecule.
[0096] Célula Estaminal - Consideram-se células estaminais todas as células que ainda não sofreram um processo de diferenciação final e que apresentam a capacidade de se dividirem em células iguais a si mesmas (divisão simétrica), ou que apresentam a capacidade de se dividirem dando origem a uma célula igual a si mesma e uma outra que segue para um passo de diferenciação (divisão assimétrica). [0096] Stem Cell - Stem cells are considered to be all cells that have not yet undergone a final differentiation process and that have the ability to divide into cells equal to themselves (symmetrical division), or that have the ability to divide giving rise to a cell equal to itself and another that goes on to a differentiation step (asymmetric division).
[0097] Durante o período de vida dos mamíferos existem pelo menos 5 tipos de Células Estaminais: Células[0097] During the life span of mammals there are at least 5 types of Stem Cells:
Totipotentes, Células Pluripotentes, Células Multipotentes, Células Oligopotentes e Células Unipotentes. Totipotent, Pluripotent Cells, Multipotent Cells, Oligopotent Cells and Unipotent Cells.
[0098] Na presente divulgação foram utilizadas células pluripotentes. Estas células podem dar origem. i.e., diferenciar-se, em qualquer dos três tipos de tecidos embrionários: ectoderme, mesoderme e endoderme, e podem ser mantidas em cultura por períodos muito longos mantendo todas as suas propriedades originais. Atualmente estão disponíveis meios apropriados para a sua cultura designados serum e feeder-free por não possuírem na sua composição componentes de origem animal, e não necessitarem de ser cultivadas sobre uma camada de células, normalmente uma camada de fibroblastos embrionários de murganho. A cultura das células em meios serum e feeder-free permitem ultrapassar algumas questões de segurança levantadas pelas autoridades reguladoras, quando estas células ou os produtos delas derivados se destinam a ser utilizados na área clinica. [0098] Pluripotent cells were used in the present disclosure. These cells can give rise. ie, differentiate into any of the three types of embryonic tissues: ectoderm, mesoderm and endoderm, and can be kept in culture for very long periods maintaining all their original properties. Appropriate media are currently available for their culture, called serum and feeder-free, as they do not contain animal components in their composition, and do not need to be cultivated on a layer of cells, normally a layer of mouse embryonic fibroblasts. Cell culture in serum and feeder-free media makes it possible to overcome some questions of safety issues raised by regulatory authorities, when these cells or products derived from them are intended to be used in the clinical area.
[0099] Numa forma de realização, foram utilizadas hiPSCs. [0099] In one embodiment, hiPSCs were used.
[00100] No âmbito da presente divulgação entende-se por transplantação a deslocação de células, tecidos ou órgãos, desde o seu local de origem para outro local dentro de um mesmo indivíduo (auto-transplantação), ou entre indivíduos da mesma espécie (alo-transplantação), ou ainda entre indivíduos de espécies diferentes (xeno-transplantação). [00100] Within the scope of the present disclosure, transplantation is understood to mean the displacement of cells, tissues or organs, from their place of origin to another place within the same individual (self-transplantation), or between individuals of the same species (allo -transplantation), or between individuals of different species (xeno-transplantation).
[00101] Numa forma de realização, o método da presente divulgação poderá ser empregue na edição de células estaminais pluripotentes embrionárias não humanas, que poderão ser utilizadas em tratamentos no âmbito da medicina ou medicina veterinária. [00101] In one embodiment, the method of the present disclosure may be employed in editing non-human embryonic pluripotent stem cells, which may be used in treatments in the field of medicine or veterinary medicine.
[00102] As células estaminais pluripotentes embrionárias não humanas podem diferenciar-se em todos os tipos de células derivadas da ectoderme, mesoderme e endoderme embrionárias, ou seja, mantêm a capacidade de formarem qualquer tipo de célula, micro-tecido, tecido ou órgão presente no organismo. Tornam-se, por isso, importantes para o desenvolvimento de linhas de células estaminais universais off-the-shelf, preparadas para perderem o seu par de cromossomas responsável pela codificação do complexo de histocompatibilidade major (MHC) da espécie em causa. [00102] Non-human embryonic pluripotent stem cells can differentiate into all types of cells derived from embryonic ectoderm, mesoderm and endoderm, that is, they maintain the ability to form any type of cell, micro-tissue, tissue or organ present in the organism. Therefore, they become important for the development of off-the-shelf universal stem cell lines, prepared to lose their pair of chromosomes responsible for encoding the major histocompatibility complex (MHC) of the species in question.
[00103] Numa forma de realização o método de desenvolvimento de linhas de células estaminais compreende a edição de linhas de células estaminais num ou mais pares de cromossomas, na proximidade e de cada lado do centrómero, com cassetes de genes compreendendo: Cassete 1 (Seq. ID N° 43) - compreende um Braço de Homologia 5' ou Esquerdo, um promotor, um gene de proteína fluorescente, um gene de resistência antibiótica, um nulómero adequado a poder ser clivado pelos diversos sistemas de nucleases (ZNF, TALEN, CRISPR, ou outros), sequência 2pA, Braço de Homologia 3'ou Direito e respetivas sequências de ligação. Esta Cassete 1 deve editar de preferência um gene não ativo das células estaminais, situado nas proximidades e num dos lados (3' ou 5'), do centrómero; [00103] In one embodiment the method of developing stem cell lines comprises editing the stem cell lines on one or more pairs of chromosomes, in proximity to and either side of the centromere, with gene cassettes comprising: Cassette 1 (Seq. ID No. 43) - comprises a 5' or Left Homology Arm, a promoter, a fluorescent protein gene, an antibiotic resistance gene, a numeromer suitable to be cleaved by the various nuclease systems (ZNF , TALEN, CRISPR, or others), 2pA sequence, 3' or Right Homology Arm and respective linker sequences. This Cassette 1 should preferably edit a non-active stem cell gene, located near and on one side (3' or 5') of the centromere;
Cassete 2 (Seq. ID N° 44) - compreende um Braço de Homologia 5' ou Esquerdo, um promotor, um gene de sensibilidade a drogas (tamoxifeno, aciclovir), uma sequência de poliadenilação do vírus SV40, (SV40pA), um nulómero com a mesma sequência do nulómero presente na sequência da Cassete 1, um promotor, um gene de resistência antibiótica diferente do gene presente na Cassete 1, um gene de proteína fluorescente diferente do gene presente na Cassete 1, uma sequência de poliadenilação da hormona de crescimento bovina, (bGHpA), e um Braço de Homologia 3' ou Direito. Esta Cassete 2 deve editar de preferência um gene inativo das células estaminais, situado nas proximidades e do outro lado do centrómero (em relação à Cassete 1). A edição da Cassete 1 e da Cassete 2 deve ser preferencialmente obtida em tempos de edição diferentes, uma vez que sendo necessário proceder a clivagens da cadeia dupla do ADN por qualquer dos sistemas de edição (CRISPR, ZNF, TALEN, ou outros) simultâneos à introdução dos construtos, mais de 40% das células a editar sofreriam a deleção dos cromossomas em causa, e consequente morte celular, diminuindo muito a eficiência do presente método. Cassette 2 (Seq. ID No. 44) - comprises a 5' or Left Homology Arm, a promoter, a drug sensitivity gene (tamoxifen, acyclovir), an SV40 virus polyadenylation sequence, (SV40pA), a nullomer with the same sequence as the nullomer present in the Cassette 1 sequence, a promoter, an antibiotic resistance gene different from the gene present in Cassette 1, a fluorescent protein gene different from the gene present in Cassette 1, a growth hormone polyadenylation sequence bovine, (bGHpA), and a 3' or Right Homology Arm. This Cassette 2 should preferably edit an inactive stem cell gene, located close to and on the other side of the centromere (relative to Cassette 1). The editing of Cassette 1 and Cassette 2 should preferably be obtained at different editing times, since it is necessary to cleave the DNA double strand by any of the editing systems (CRISPR, ZNF, TALEN, or others) simultaneously with the introduction of the constructs, more than 40% of the cells to be edited would suffer the deletion of the chromosomes in question, and consequent cell death, greatly reducing the efficiency of the present method.
[00104] Numa forma de realização, o método para o desenvolvimento de linhas de células estaminais compreende a inclusão de sequências nuloméricas complementadas por uma sequência designada por motivo adjacente ao protoespaçador (PAM, do inglês Protospacer Adjacent Motíf), nas cassetes de genes a expressar em linhas de células estaminais. A introdução de uma sequência nulomérica no genoma complementada pela sequência PAM, sequência necessária para o reconhecimento do local de clivagem na dupla cadeia de ADN pelas nucleases do sistema CRISPR (CRISPR/Cas9 ou outra), permite garantir que a utilização do sistema CRISPR irá encontrar um único local de clivagem no genoma editado, ultrapassando o problema de clivagem fora do local adequado (do inglês off-targeting). Também dá garantias de que os outros sistemas de edição genómica, (ZNF, TALENs ou outros) irão encontrar uma única sequência no genoma para realizarem clivagens da cadeia dupla do ADN. [00104] In one embodiment, the method for developing stem cell lines comprises the inclusion of nullomeric sequences complemented by a sequence called the Protospacer Adjacent Motif (PAM) in the gene cassettes to be expressed in stem cell lines. The introduction of a nullomeric sequence in the genome complemented by the PAM sequence, a sequence necessary for the recognition of the cleavage site in the DNA double strand by the nucleases of the CRISPR system (CRISPR/Cas9 or other), makes it possible to guarantee that the use of the CRISPR system will find a single cleavage site in the edited genome, bypassing the off-targeting problem. It also guarantees that other genomic editing systems (ZNF, TALENs or others) will find a unique sequence in the genome to perform double strand DNA cleavages.
[00105] É este artificio importante que permite, a deleção dos cromossomas editados e por isso possuidores da sequência nulomérica e simultaneamente evita a deleção dos cromossomas de substituição, por não conterem sequências nuloméricas. No exemplo relativo à deleção do par de cromossomas 6 editado, é construído um sistema CRISPR que perante dois pares de cromossomas 6 (o par indígena e o par transferido/ substituto), encontre o par portador do nulómero e aí promova clivagens da cadeia dupla do ADN e respetiva deleção, poupando o par de cromossomas substituto. A partir desse momento as células possuirão apenas o exato MHC do paciente, ou seja, expressarão exatamente o mesmo conjunto de recetores HLA do paciente. [00105] It is this important artifice that allows the deletion of edited chromosomes and therefore possess the nullomeric sequence and simultaneously prevents the deletion of replacement chromosomes, as they do not contain nullomeric sequences. In the example concerning the deletion of the edited pair of chromosomes 6, a CRISPR system is constructed that, when faced with two pairs of chromosomes 6 (the indigenous pair and the transferred/substitute pair), finds the pair carrying the nullomere and there promotes cleavages of the double chain of the DNA and its deletion, sparing the replacement chromosome pair. From that moment on, the cells will only have the exact MHC of the patient, that is, they will express exactly the same set of HLA receptors as the patient.
[00106] Numa outra forma de realização o presente método para o desenvolvimento de linhas de células estaminais, mas em que as sequências de nulómeros foram substituídos na Cassete 1 e na Cassete 2 por sequências normalmente inexistentes no genoma da espécie a que as células estaminais a editar pertencem, e que sejam adequadas à clivagem pelos sistemas de nucleases de edição genómica (ZNF, TALENs, CRISPR, ou outros). [00106] In another embodiment the present method for developing stem cell lines, but in which the nucleomer sequences have been replaced in Cassette 1 and Cassette 2 by sequences normally non-existent in the genome of the species to which the stem cells to be edited belong, and which are suitable for cleavage by genomic editing nuclease systems (ZNF, TALENs, CRISPR, or others).
[00107] As sequêncas nuloméricas são também adequadas para a utilização de outros sistemas de nucleases além do sistema CRISPR, nomeadamente ZNF, TALEN, e outras que venham a ser inventadas ou descobertas e que se baseiem no principio de condução da nuclease a sequências especificas do genoma. [00107] Nulomeric sequences are also suitable for the use of other nuclease systems in addition to the CRISPR system, namely ZNF, TALEN, and others that may be invented or discovered and that are based on the principle of driving the nuclease to specific sequences of the genome.
[00108] Numa forma de realização, a divulgação compreende a introdução de sequências nuloméricas ou outras normalmente ausentes do genoma do organismo a modificar, fazendo uso dos sistemas de edição genómica, (CRISPR, ZNF, TALEN ou outros). O sistema de nucleases CRISPR compreende uma grande diversidade de nucleases de origem bacteriana que têm a particularidade de poderem provocar clivagens da cadeia dupla do ADN com grande especificidade, porque a nuclease é conduzida para o local da clivagem por uma sequência de não mais de 20 bases de ARN, designada ARN de guia único, (sgARN). No entanto, a clivagem só acontecerá se a sequência de ADN genómico complementar do sgARN for precedida ou seguida de uma sequência designada por motivo adjacente ao protoespaçador (PAM, do inglês Protospacer Adjacent Motíf). A sequência PAM e a sua posição relativa, depende da nuclease CRISPR que se utilizar. [00108] In one embodiment, the disclosure comprises introducing nullomeric or other sequences normally absent from the genome of the organism to be modified, making use of genomic editing systems, (CRISPR, ZNF, TALEN or others). The CRISPR nuclease system comprises a large diversity of nucleases of bacterial origin that have the particularity of being able to cause cleavages of the double strand of DNA with great specificity, because the nuclease is carried to the cleavage site by a sequence of no more than 20 bases. of RNA, called single-lead RNA (sgRNA). However, cleavage will only occur if the sgRNA complementary genomic DNA sequence is preceded or followed by a sequence called a Protospacer Adjacent Motif (PAM). The PAM sequence and its relative position depend on the CRISPR nuclease used.
EXEMPLO EXAMPLE
[00109] Numa forma de realização, foi feita a escolha ín sílíco do local ideal de clivagem da CRISPR-Cas9 (crARN) bem como dos plasmideos vetores portadores da Cassete 1 e da Cassete 2 para a edição (do inglês: gene editing) dos genes inativos flanqueadores do centrómero. [00109] In one embodiment, the optimal CRISPR-Cas9 (crRNA) cleavage site (crRNA) was chosen in silic as well as the Cassette 1 and Cassette-bearing vector plasmids. Cassette 2 for editing the inactive centromere flanking genes.
[00110] Numa primeira fase, foram selecionados dois genes inativos nas hiPSCs da linha que se pretende modificar geneticamente. Numa forma de realização, e de forma a editar o cromossoma 6 humano, foram escolhidos os genes BAG2, gene inativo em hiPSCs, flanqueador do centrómero no braço longo do cromossoma 6 humano; e LGSN, gene também inativo em hiPSCs e flanqueador do centrómero no braço curto do cromossoma 6 humano (Figura 1). A importância de que sejam genes inativos, prende-se com o facto de que após a edição, estes genes ficam inoperativos (do inglês, knockout). Assim, a sua inativação pela edição, impossibilitaria a sobrevivência ou a correta expansão das células modificadas, caso a sua atividade fosse essencial para estas funções. A sequência anotada dos genes escolhidos, (p. ex. BAG2 - CRCh38:6:57171726:57190433:1 e LGSN - CRCh38:6:63275351:63320583:1), pode ser obtida em Emsembl.org, (p. ex. GenBank format ou Word™ Microsoft™ compatível). [00110] In a first phase, two inactive genes were selected in the hiPSCs of the line to be genetically modified. In one embodiment, and in order to edit human chromosome 6, genes BAG2, inactive gene in hiPSCs, centromere flanking on the long arm of human chromosome 6 were chosen; and LGSN, a gene that is also inactive in hiPSCs and flanks the centromere on the short arm of human chromosome 6 (Figure 1). The importance that they are inactive genes is related to the fact that after editing, these genes become inoperative (knockout). Thus, its inactivation by editing would make it impossible for the modified cells to survive or correctly expand, if their activity were essential for these functions. The annotated sequence of the chosen genes, (e.g. BAG2 - CRCh38:6:57171726:57190433:1 and LGSN - CRCh38:6:63275351:63320583:1), can be obtained from Emsembl.org, (e.g. GenBank format or Microsoft™ Word™ compatible).
[00111] De seguida, procedeu-se à importação das sequências obtidas para uma das várias ferramentas bioinformáticas CRISPR-Cas9 (p.ex. CRISPR Finder; CT-Finder; CCTop-CRISPR-Cas9 ou qualquer outra disponível). A ferramenta bioinformática CRISPR apresenta diversos locais nas sequências selecionadas onde a nuclease CRISPR-Cas9 pode produzir clivagens na dupla cadeia do ADN (DSBs). Foram selecionadas entre as sequências apresentadas, duas ou três com as melhores pontuações (do inglês scores). A nuclease CRISPR-Cas9 é guiada para o local de clivagem por uma sequência de ARN designada ARN guia, gARN (do inglês: guide- RNA). Para a utilização do sistema CRISPR/Cas9, estas sequências são complementares e imediatamente a montante de uma sequência do tipo NGG, no ADN do gene escolhido, conhecida como PAM. [00111] The sequences obtained were then imported into one of the various CRISPR-Cas9 bioinformatics tools (eg CRISPR Finder; CT-Finder; CCTop-CRISPR-Cas9 or any other available). The CRISPR bioinformatics tool presents several sites in selected sequences where the CRISPR-Cas9 nuclease can produce DNA double strand cleavages (DSBs). Two or three with the best scores were selected among the presented sequences. CRISPR-Cas9 nuclease is guided to the cleavage site by an RNA sequence called guide RNA, gRNA (guide-RNA). For the use of the CRISPR/Cas9 system, these sequences are complementary and immediately upstream of an NGG-like sequence in the DNA of the chosen gene, known as PAM.
[00112] Numa forma de realização, as sequências obtidas são testadas, de forma a selecionar as mais eficazes após transfeção celular. Após as transfeções, os melhores gARNs foram eleitos com recurso a um Protocolo de endonuclease T7 gene 3 ADN endonuclease 1 (T7E1), (p.ex. EnGen™Mutation Detection Kit da New England BioLabs™ (NEB), seguindo as instruções recomendadas pelo fabricante. [00112] In one embodiment, the sequences obtained are tested, in order to select the most effective ones after cell transfection. After transfections, the best gRNAs were selected using a T7 endonuclease protocol gene 3 DNA endonuclease 1 (T7E1), (e.g. EnGen™Mutation Detection Kit from New England BioLabs™ (NEB), following the instructions recommended by the manufacturer.
[00113] Numa forma de realização, foi escolhida a Seq. ID N° 1 para o local de clivagem do gene BAG2 pelo sistema CRISPR/Cas9 e Seq. ID N° 2 para o local de clivagem do gene LGSN pelo sistema CRISPR/Cas9. Foram também utilizados os iniciadores (do inglês prímers) com Seq. ID N° 3 e Seq. ID N° 4 para o ensaio T7E1 no gene BAG2; e os iniciadores Seq. ID N° 5 e Seq. ID N° 6 para o ensaio T7E1 no gene LGSN. [00113] In one embodiment, Seq. ID No. 1 for the BAG2 gene cleavage site by the CRISPR/Cas9 system and Seq. ID No. 2 for the cleavage site of the LGSN gene by the CRISPR/Cas9 system. Primers with Seq. ID No. 3 and Seq. ID No. 4 for the T7E1 assay on the BAG2 gene; and Seq primers. ID No. 5 and Seq. ID No. 6 for the T7E1 assay on the LGSN gene.
[00114] Numa forma de realização, a Cassete 1/Gene BAG2 (Seq. ID N° 43) compreende um Braço de Homologia 5' ou Esquerdo (do inglês: 5'HA ou Left Homology Arm ou LeftHA, 1783 pares de bases, pb); um promotor PGK (do inglês: Phosphoglycerate Kínase promoter - PGK pmt); um gene de resistência à puromicina; o gene da Proteína de Fluorescência Verde Melhorado (do inglês: Enhanced Green Fluorescent Protein - EGFP), uma sequência de poliadenilação (do inglês: poliadenilation sequence - Poli-A ou pA); um nulómero; e um braço de homologia 3'ou Direito (do inglês: 3'HA ou Ríght Homology Arm ou Ríght HA, 1590 pb). [00114] In one embodiment, Cassette 1/Gene BAG2 (Seq. ID No. 43) comprises a 5' or Left Homology Arm (5'HA or Left Homology Arm or LeftHA, 1783 base pairs, bp); a PGK promoter (Phosphoglycerate Kinase promoter - PGK pmt); a puromycin resistance gene; the Enhanced Green Fluorescent Protein (EGFP) gene, a polyadenylation sequence (Poly-A or pA); a numeromer; and a 3' or Right homology arm (3'HA or Ríght Homology Arm or Ríght HA, 1590 bp).
[00115] No âmbito da presente descrição designa-se por "plasmídeo p925", ou apenas p925, um plasmídeo que expressa resistência à ampicilina para a seleção dos clones perfeitamente editados, assim como uma cassete que compreende o promotor PGK, o gene de Resistência à Puromicina e o gene da proteína EGFP e uma sequência Poli A. [00115] Within the scope of the present description, "plasmid p925", or simply p925, is a plasmid expressing ampicillin resistance for the selection of clones perfectly edited, as well as a cassette comprising the PGK promoter, the Puromycin Resistance gene and the EGFP protein gene and a Poly A sequence.
[00116] Numa forma de realização, foram utilizados diferentes iniciadores na preparação dos diversos componentes da Cassete 1/Gene BAG2: [00116] In one embodiment, different primers were used in preparing the various components of Cassette 1/Gene BAG2:
• Seq. ID N° 7 e Seq. ID N° 8 foram utilizadas como iniciadores para a síntese da sequência do Braço de• Seq. ID No. 7 and Seq. ID No. 8 were used as primers for the synthesis of the Sequence Arm of
Homologia 5', (1783pb), preparada para a subclonagemHomology 5', (1783bp), prepared for subcloning
(ou inserção) em sítios de reconhecimento para as endonucleases de restrição EcoRI e BamHI no plasmídeo p925, (PGK-PuroR-EGFP-2pA); (or insertion) into recognition sites for restriction endonucleases EcoRI and BamHI in plasmid p925, (PGK-PuroR-EGFP-2pA);
• Seq. ID N° 9 e Seq. ID N° 10 foram utilizadas como iniciadores para a síntese da sequência do Braço de• Seq. ID No. 9 and Seq. ID No. 10 were used as primers for the synthesis of the Sequence Arm of
Homologia 3', (1590pb), preparada para a subclonagemHomology 3', (1590bp), prepared for subcloning
(ou inserção) em sítios de reconhecimento para as endonucleases de restrição BamHI e Notl no plasmídeo (p925); (or insertion) into recognition sites for restriction endonucleases BamHI and NotI on the plasmid (p925);
• Iniciadores Seq. ID N° 11 e Seq. ID N° 12 foram utilizados para a síntese das sequências de poliadenilação e nulómero, preparadas para a subclonagem (ou inserção) em sítios de reconhecimento para as endonucleases de restrição Xbal e Notl no plasmídeo (p925). • Seq primers. ID No. 11 and Seq. ID No. 12 were used for the synthesis of the polyadenylation and nullomer sequences prepared for subcloning (or insertion) into recognition sites for the restriction endonucleases XbaI and NotI in the plasmid (p925).
[00117] O vetor de edição do gene BAG2 pode ser obtido de duas formas distintas. Numa forma de realização, é possível comprar à indústria o vetor completo, entregue sob a forma de plasmídeo em células competentes congeladas. A cultura dessas células competentes permite obter o vetor após o seu isolamento com recurso a sistemas de purificação tais como o Wizard™ Plus SV MINIPREPS DNA Purification System (Promega™) ou outro sistema equivalente. Numa outra forma de realização, pode ser feita a preparação do vetor por subclonagem dos seus diversos promotores e genes, quer num plasmideo virgem (do inglês: empty backbone plasmid), ou de outro mais completo e adequado. [00117] The BAG2 gene editing vector can be obtained in two different ways. In one embodiment, the entire vector can be purchased from industry, delivered as a plasmid in frozen competent cells. The culture of these competent cells makes it possible to obtain the vector after its isolation using purification systems such as the Wizard™ Plus SV MINIPREPS DNA Purification System (Promega™) or other equivalent system. In another embodiment, the vector can be prepared by subcloning its various promoters and genes, either into a virgin plasmid (empty backbone plasmid), or another more complete and suitable plasmid.
[00118] Na realização do presente exemplo, foi feita a subclonagem num plasmideo (p925), contendo o promotor PGK, o gene de resistência à puromicina (PuroR), o gene da proteína EGFP, e uma sequência Poli A, bem como um gene de resistência à Ampicilina para seleção das células competentes corretamente transformadas. [00118] In carrying out the present example, subcloning was performed into a plasmid (p925), containing the PGK promoter, the puromycin resistance gene (PuroR), the EGFP protein gene, and a Poly A sequence, as well as a gene of Ampicillin resistance for selection of correctly transformed competent cells.
[00119] Numa forma de realização, a subclonagem foi efetuada em três passos. [00119] In one embodiment, subcloning was performed in three steps.
[00120] O primeiro passo compreende a subclonagem da sequência Poli A + nulómero em sítios Xbal e Notl no plasmideo p925. As sequências Poli A e do nulómero foram subclonadas a jusante da sequência do gene EGFP, para permitir simultaneamente a seleção dos clones de células off-the-shelf corretamente editadas bem como, numa fase posterior à de substituição cromossómica, a eliminação dos clones de células com manutenção da expressão do gene PuroR e do gene EGFP. (Figura 4). Os iniciadores Seq. ID N° 11 e Seq. ID N° 12 foram utilizados na reação em cadeia da polimerase (PCR, do inglês polymerase chain reactíon) para a síntese das sequências Poli A + nulómero preparadas para a sua subclonagem (ligação) nos sítios Xbal e Notl adequados no plasmideo p925. [00120] The first step comprises subcloning the Poly A + nullomer sequence into XbaI and NotI sites in plasmid p925. The Poly A and nucleomer sequences were subcloned downstream of the EGFP gene sequence, to allow both the selection of correctly edited off-the-shelf cell clones and, at a stage after the chromosomal replacement, the elimination of the cell clones. with maintenance of the expression of the PuroR gene and the EGFP gene. (Figure 4). Seq primers. ID No. 11 and Seq. ID No. 12 were used in polymerase chain reaction (PCR) for the synthesis of the Poly A + nucleomer sequences prepared for their subcloning (ligation) into the appropriate XbaI and NotI sites on plasmid p925.
[00121] Numa forma de realização, para a reação da PCR foi preparada uma solução com a mistura dos vários reagentes (MIX), de acordo com a Tabela 1. Após preparação, foram misturados 47,5uL da MIX obtida a 2,5uL/40ng p925ADN [20ng/uL]. A reação PCR prosseguiu num termociclador (Applied Systems Thermocycler™), nas condições indicadas na Tabela 2. [00121] In one embodiment, for the PCR reaction, a solution was prepared by mixing the various reagents (MIX), according to Table 1. After preparation, 47.5uL of the obtained MIX was mixed at 2.5uL/40ng p925DNA [20ng/uL]. The PCR reaction proceeded in a thermocycler (Applied Systems Thermocycler™), under the conditions indicated in Table 2.
Tabela 1: Concentração de reagentes utilizados na reação PCR.
Figure imgf000041_0001
Table 1: Concentration of reagents used in the PCR reaction.
Figure imgf000041_0001
Tabela 2: Condições da PCR num termociclador.
Figure imgf000041_0002
Table 2: PCR conditions in a thermal cycler.
Figure imgf000041_0002
[00122] Após a PCR, foi feita eletroforese em gel de agarose a 0,8% de uma amostra de 5uL do produto da PCR, para confirmar a presença de uma banda de 470bp, que contem o produto da PCR composto pela sequência de Poliadenilação e nulómero. Verificando-se a presença da banda, procedeu-se à electroforese de todo o restante produto da PCR num gel de agarose a 0,8%. A banda de 470bp foi extraída do gel, com um kit apropriado, (p. ex. Wizard™ SV Gel and PCR clean-up System - PROMEGA™), seguindo as instruções do fabricante. O produto obtido (PCR ADN) foi ressuspendido em 20uL de PCRH2O, isto é, água livre das enzimas ARNase e ADNase. [00122] After PCR, 0.8% agarose gel electrophoresis was performed on a 5uL sample of the PCR product, to confirm the presence of a 470bp band, which contains the PCR product composed of the Polyadenylation sequence and numeromer. After verifying the presence of the band, the electrophoresis of all remaining PCR product on a 0.8% agarose gel. The 470bp band was extracted from the gel, with an appropriate kit, (eg Wizard™ SV Gel and PCR clean-up System - PROMEGA™), following the manufacturer's instructions. The product obtained (PCR DNA) was resuspended in 20uL of PCRH2O, that is, water free of RNase and DNase enzymes.
[00123] O produto obtido, e já ressuspendido, foi submetido à dupla digestão das endonucleases de restrição Xbal + NotIHF, utilizando as condições mencionadas na Tabela 3, durante 2 horas a 37°C. [00123] The product obtained, and already resuspended, was subjected to double digestion of restriction endonucleases XbaI + NotIHF, using the conditions mentioned in Table 3, for 2 hours at 37°C.
Tabela 3 : Condições utilizadas na digestão do ADN obtido na reação PCR pelas enzimas de restrição Xbal e NotIHF.
Figure imgf000042_0001
Table 3: Conditions used in the digestion of DNA obtained in the PCR reaction by restriction enzymes XbaI and NotIHF.
Figure imgf000042_0001
[00124] Após a dupla digestão Xbal + Notl, o produto obtido foi purificado com uma mistura de fenol/clorofórmio/álcool isoamilico, precipitado em etanol e ressuspendido em 20uL de PCR H2O. Após purificação e precipitação, o inserto obtido contém a sequência Poli A + Nulómero, pronta a ligar ao vector p925. [00124] After XbaI + NotI double digestion, the product obtained was purified with a mixture of phenol/chloroform/isoamyl alcohol, precipitated in ethanol and resuspended in 20uL of PCR H2O. After purification and precipitation, the obtained insert contains the Poly A + Nunomer sequence, ready to be ligated into the p925 vector.
[00125] Numa forma de realização, o vector p925 é preparado para a ligação ao inserto obtido. Para tal, foi feita uma dupla digestão com endonucleases de restrição Xbal e Notl, e uma defosforilação. [00126] A dupla digestão com Xbal e Notl foi feita com uma amostra de 5ug de ADN do plasmídeo p925, usando as condições descritas na Tabela 4. Após a preparação da solução, a amostra foi incubada 2 horas a 37°C [00125] In one embodiment, the p925 vector is prepared for ligation to the obtained insert. For this, a double digestion with restriction endonucleases XbaI and NotI, and a dephosphorylation was performed. [00126] XbaI and NotI double digestion was performed with a 5ug sample of p925 plasmid DNA, using the conditions described in Table 4. After the solution was prepared, the sample was incubated for 2 hours at 37°C.
Tabela 4 : Condições experimentais para a dupla digestão por endonucleases Xbal e NotIHF do plasmídeo p925.
Figure imgf000043_0001
Table 4: Experimental conditions for XbaI and NotIHF double digestion of plasmid p925.
Figure imgf000043_0001
[00127] Após a dupla digestão Xbal + NotIHF, uma amostra de 5uL do produto obtido foi submetido a uma electroforese num gel de agarose a 0,8%, para confirmar a presença da banda correta. Após confirmação, o ADN do produto foi purificado com fenol/clorofórmio/álcool isoamilico, precipitado em etanol, e ressuspendido em 20uL de PCRH2O [00127] After XbaI + NotIHF double digestion, a 5uL sample of the obtained product was subjected to electrophoresis on a 0.8% agarose gel to confirm the presence of the correct band. After confirmation, the product DNA was purified with phenol/chloroform/isoamyl alcohol, ethanol precipitated, and resuspended in 20uL PCRH2O
[00128] Após purificação, precipitação e ressuspensão do produto de ADN, este foi defosforilado, utilizando as condições descritas na Tabela 5. A reação foi feita a 37°C durante 10 minutos, sendo terminada por aumento da temperatura (5 minutos a 75°C). Após a reação, foi feita nova purificação com fenol/clorofórmio/álcool isoamilico e precipitação com etanol. O produto obtido (vetor p925/duplamente digerido Xbal+ NotI/Defosforilado) está pronto para a subclonagem (ligação) com o inserto (Poli A + Nulómero), e pode ser conservado a -20°C para uso futuro. Tabela 5: Condições experimentais para a desfosforilação.
Figure imgf000044_0001
[00128] After purification, precipitation and resuspension of the DNA product, it was dephosphorylated using the conditions described in Table 5. The reaction was carried out at 37°C for 10 minutes, being terminated by increasing the temperature (5 minutes at 75° Ç). After the reaction, further purification was performed with phenol/chloroform/isoamyl alcohol and precipitation with ethanol. The product obtained (p925 vector/double digested Xbal+ NotI/Dephosphorylated) is ready for subcloning (ligation) with the insert (Poly A + Nunomer), and can be stored at -20°C for future use. Table 5: Experimental conditions for dephosphorylation.
Figure imgf000044_0001
[00129] Antes de proceder à ligação do vetor p925/duplamente digerido Xbal + NotI/Defosforilado com o inserto (Poli A + nulómero), foi feita uma electroforese em gel de agarose a 0,8% de uma amostra de luL do primeiro e de 2uL do segundo para avaliar as suas concentrações relativas. Foi obtido um rácio de 1:5 entre o vetor e o inserto, respetivamente . [00129] Prior to the ligation of the p925/double digested Xbal + NotI/Dephosphorylated vector with the insert (Poly A + nullomer), a 0.8% agarose gel electrophoresis was performed on a luL sample from the first and of 2uL of the second to assess their relative concentrations. A 1:5 ratio between vector and insert, respectively, was obtained.
[00130] A ligação (subclonagem) do vetor p925/duplamente digerido Xbal+ NotI/Defosforilado com o inserto (Poli A + nulómero) foi realizada de acordo com a Tabela 6. [00130] The ligation (subcloning) of the p925/double digested Xbal+ NotI/Dephosphorylated vector with the insert (Poly A + nullomer) was performed according to Table 6.
Tabela 6: Mistura de reagentes de ligação.
Figure imgf000044_0002
Table 6: Mixture of Binding Reagents.
Figure imgf000044_0002
[00131] Numa forma de realização, foram pipetados 8uL da mistura referida na Tabela 6 e 2uL do inserto (Poli A + nulómero) para um tubo de fundo redondo (Tubo 1). Num outro tubo de fundo redondo (Tubo 2, controlo negativo), foram pipetados 8uL da mistura e 2uL de PCR H2O. Ambos os tubos foram incubados a 15°C durante pelo menos duas 2 horas, de preferência durante uma noite. Após incubação, foi feita a transformação de células competentes E. coli (adquiridas à indústria) com 5uL do conteúdo de cada Tubo 1 e Tubo 2. A existência de colónias corretamente inseridas foi confirmada por sequenciação, cujas amostras foram conservadas a -80°C em glicerol. (Sigma Aldrich® - 66DE65 and 66DE66). A partir de uma colónia corretamente inserida, foi feita uma inoculação em lOOmL de solução LB ampicilina 100 num frasco Erlenmeyer, que foi incubado toda a noite num agitador horizontal. Após a incubação, foi feita uma extração de uma MIDI ou MAXI-prep (p.ex. QIAGEN™ Midi ou Maxi Kit), para a obtenção de uma quantidade suficiente de plasmideo, a ser utilizado nos passos seguintes. [00131] In one embodiment, 8uL of the mixture referred to in Table 6 and 2uL of the insert (Poly A + nucleomer) into a round-bottomed tube (Tube 1). In another round bottom tube (Tube 2, negative control), 8uL of the mixture and 2uL of PCR H2O were pipetted. Both tubes were incubated at 15°C for at least two hours, preferably overnight. After incubation, competent E. coli cells (purchased from the industry) were transformed with 5uL of the contents of each Tube 1 and Tube 2. The existence of correctly inserted colonies was confirmed by sequencing, whose samples were stored at -80°C in glycerol. (Sigma Aldrich® - 66DE65 and 66DE66). From a correctly inserted colony, 100ml of LB ampicillin 100 solution was inoculated into an Erlenmeyer flask, which was incubated overnight on a horizontal shaker. After incubation, a MIDI or MAXI-prep (eg QIAGEN™ Midi or Maxi Kit) was extracted to obtain a sufficient amount of plasmid to be used in the following steps.
[00132] Numa forma de realização, o método descrito na presente divulgação compreende um segundo passo de subclonagem, para a ligação do Braço de Homologia 5' ou Esquerdo, a montante do promotor PGK no plasmideo p925/PoliA+nulómero) . O Braço de Homologia 5' ou Esquerdo (1873bp), foi ligado (subclonado) num sitio EcoRI imediatamente a montante do promotor PGK no plasmideo p925/PoliA+nulómero . Foi providenciada a presença de um sitio BamHI na extremidade 5' da sequência para permitir o isolamento do vetor completo para a edição do gene BAG2. [00132] In one embodiment, the method described in the present disclosure comprises a second subcloning step, for ligating the 5' or Left Homology Arm, upstream of the PGK promoter on plasmid p925/PolyA+nulomer). The 5' or Left Homology Arm (1873bp), was ligated (subcloned) into an EcoRI site immediately upstream of the PGK promoter on plasmid p925/PolyA+nulomer. The presence of a BamHI site at the 5' end of the sequence was provided to allow isolation of the complete vector for editing the BAG2 gene.
[00133] Numa forma de realização, o produto da PCR foi obtido a partir de ADN genómico (do inglês: genomíc DNA - gDNA) de células HEK293T, pela utilização do Kit NZY Tissue gDNA Isolation Kit (NZYtech®), seguindo as instruções do fabricante. Utilizando os iniciadores Seq. ID N° 7 e Seq. ID N° 8, foi preparada uma solução com a mistura dos vários reagentes (MIX), de acordo com a Tabela 7, sendo depois misturados 45uL da MIX obtida a 5uL (200ng) de gDNA [41ng/uL] isolado das HEK293T. A reação de PCR prosseguiu num termociclador (Applied Systems Thermocycler™), nas condições indicadas na Tabela 8. [00133] In one embodiment, the PCR product was obtained from genomic DNA (gDNA) from HEK293T cells, using the NZY Tissue gDNA Isolation Kit (NZYtech®), following the instructions of the manufacturer. Using Seq primers. ID No. 7 and Seq. ID No. 8, a solution was prepared with the mixture of the various reagents (MIX), according to Table 7, and then 45uL of the MIX obtained was mixed with 5uL (200ng) of gDNA [41ng/uL] isolated from the HEK293T. The PCR reaction proceeded in a thermocycler (Applied Systems Thermocycler™), under the conditions indicated in Table 8.
Tabela 7: Concentração de reagentes utilizados na reação PCR.
Figure imgf000046_0001
Table 7: Concentration of reagents used in the PCR reaction.
Figure imgf000046_0001
Tabela 8: Condições da PCR no termociclador.
Figure imgf000046_0002
[00134] Após a PCR, foi feita eletroforese em gel de agarose a 0,8% de uma amostra de 2uL do produto da PCR, para confirmar a presença de uma banda de 1783pb correspondente ao fragmento de ADN que contém a sequência do Braço de Homologia Esquerdo da Cassete 1. Verificando-se a presença da banda, o restante ADN obtido na reação de PCR foi purificado por fenol/clorofórmio/álcool isoamilico, e digerido com a enzima de restrição EcoRI, de acordo com a Tabela 9, durante 2 horas a 37°C. Table 8: PCR conditions in the thermal cycler.
Figure imgf000046_0002
[00134] After PCR, 0.8% agarose gel electrophoresis was performed on a 2uL sample of the PCR product, to confirm the presence of a band of 1783bp corresponding to the DNA fragment that contains the sequence of the Arm of Cassette Left Homology 1. Checking for the presence of the band, the remaining DNA obtained in the PCR reaction was purified by phenol/chloroform/isoamyl alcohol, and digested with the restriction enzyme EcoRI, according to Table 9, for 2 hours at 37°C.
Tabela 9 : Condições experimentais para a digestão do produto da PCR (Braço de Homologia Esquerdo BAG2) pela enzima EcoRI.
Figure imgf000047_0001
Table 9: Experimental conditions for digestion of PCR product (Left Homology Arm BAG2) by EcoRI enzyme.
Figure imgf000047_0001
[00135] Após a digestão, foi feita eletroforese em gel de agarose a 0,8% de uma amostra de 2uL do produto da digestão, para confirmar a presença de uma banda de 1783pb correspondente ao fragmento de ADN produzido por PCR e que contem o Braço de Homologia Esquerdo da Cassete 1. Verificando-se a presença da banda, o produto digerido remanescente foi isolado com recurso à extração e purificação da banda do gel de agarose a 0,8% utilizando o sistema Wizard™ SV Gel and PCR clean-up System - PROMEGA®, seguindo as instruções do fabricante. O produto obtido (Braço de Homologia Esquerdo BAG2 digerido) foi depois purificado por fenol/clorofórmio/álcool isoamilico, precipitado em etanol e ressuspendido em 20uL PCR H20. [00136] De seguida, foi feita a ligação do vetor p925/PoliA+nulómero ao inserto Braço de Homologia Esquerdo BAG2, digerido. Numa forma de realização, o plasmideo p925/PoliA+nulómero foi digerido com a enzima de restrição EcoRI (Tabela 10), para preparar o vetor para a ligação (subclonagem) com o produto de PCR Braço de Homologia Esquerdo também digerido com EcoRI, previamente isolado. A digestão ocorreu durante 2 horas, a 37°C. [00135] After digestion, 0.8% agarose gel electrophoresis was performed on a 2uL sample of the digestion product, to confirm the presence of a 1783bp band corresponding to the DNA fragment produced by PCR and containing the Cassette Left Homology Arm 1. Checking for the presence of the band, the remaining digested product was isolated using the band extraction and purification from the 0.8% agarose gel using the Wizard™ SV Gel and PCR clean- up System - PROMEGA®, following the manufacturer's instructions. The obtained product (Digested BAG2 Left Homology Arm) was then purified by phenol/chloroform/isoamyl alcohol, ethanol precipitated and resuspended in 20uL PCR H 2 0. [00136] Then, the p925/PolyA+nulomer vector was ligated to the digested BAG2 Left Homology Arm insert. In one embodiment, the p925/PolyA+nunomer plasmid was digested with the restriction enzyme EcoRI (Table 10), to prepare the vector for ligation (subcloning) with the PCR product Left Homology Arm also digested with EcoRI, previously isolated. Digestion took place for 2 hours at 37°C.
Tabela 10: Condições experimentais para a digestão do vetor plasmideo p925/PoliA+nulómero pela enzima EcoRI.
Figure imgf000048_0001
Table 10: Experimental conditions for digestion of p925/PolyA+nulomer plasmid vector by EcoRI enzyme.
Figure imgf000048_0001
[00137] A digestão completa foi confirmada através de electroforese em gel de agarose a 0,8% de 5ul do produto digerido. Verificando-se a digestão completa, o produto digerido foi purificado por fenol/clorofórmio/álcool isoamilico e precipitado em etanol. Por fim, o produto foi desfosforilado, seguindo o protocolo referido na Tabela 11. Após incubação durante 10 minutos a 37°C em banho de água, a reação foi terminada incubando 5 minutos a 75°C. O produto obtido foi purificado por fenol/clorofórmio/álcool isoamilico e precipitado em etanol. Tabela 11: Protocolo de desfosforilação.
Figure imgf000049_0001
[00137] Complete digestion was confirmed by 0.8% agarose gel electrophoresis of 5ul of digested product. Upon complete digestion, the digested product was purified by phenol/chloroform/isoamyl alcohol and precipitated in ethanol. Finally, the product was dephosphorylated, following the protocol mentioned in Table 11. After incubation for 10 minutes at 37°C in a water bath, the reaction was terminated by incubating 5 minutes at 75°C. The product obtained was purified by phenol/chloroform/isoamyl alcohol and precipitated in ethanol. Table 11: Dephosphorylation protocol.
Figure imgf000049_0001
[00138] Numa forma de realização, o vetor p925/PoliA digerido e desfosforilado foi ligado ao inserto Braço de Homologia Esquerdo BAG2, também já digerido por EcoRI, utilizando as condições descritas na Tabela 12. [00138] In one embodiment, the digested and dephosphorylated p925/PolyA vector was ligated to the BAG2 Left Homology Arm insert, also already digested by EcoRI, using the conditions described in Table 12.
Tabela 12: Preparação da solução de reagentes (MIX) para ligação do vetor p925/PoliA digerido e desfosforilado ao inserto Braço de Homologia Esquerdo BAG2.
Figure imgf000049_0002
Table 12: Preparation of reagent solution (MIX) for binding the digested and dephosphorylated p925/PolyA vector to the BAG2 Left Homology Arm insert.
Figure imgf000049_0002
[00139] Numa forma de realização, foram pipetados para um tubo de fundo redondo (Tubo 1) 4uL da MIX e 6uL do inserto (Braço de Homologia Esquerdo BAG2 digerido). Num outro tubo (Tubo 2, tubo controlo negativo), foram pipetados 4uL da MIX e 6uL PCR H2O. Ambos os tubos foram incubados a 15°C durante pelo menos 2 horas, de preferência durante toda a noite. Após incubação, foi feita a transformação de células competentes E. coli (adquiridas à indústria) com 5uL do conteúdo de cada Tubo 1 e Tubo 2. Após a transformação, foi feita a extração dos plasmideos de várias colónias, usando o sistema Wizard™ Plus SV Minipreps DNA Purification System, 250 preps, PROMEGA™, e o ADN obtido quantificado, usando a Nanodrop™. A correta inserção dos plasmideos foi confirmada através de enzimas de restrição e/ou sequenciação, usando métodos amplamente conhecidos no estado da arte. [00139] In one embodiment, 4uL of the MIX and 6uL of the insert (Digested BAG2 Left Homology Arm) were pipetted into a round bottom tube (Tube 1). In another tube (Tube 2, negative control tube), 4uL of the MIX was pipetted and 6uL PCR H2O. Both tubes were incubated at 15°C for at least 2 hours, preferably overnight. After incubation, E. coli competent cells (purchased from the industry) were transformed with 5uL of the contents of each Tube 1 and Tube 2. After transformation, plasmids were extracted from several colonies, using the Wizard™ Plus system. SV Minipreps DNA Purification System, 250 preps, PROMEGA™, and the DNA obtained quantified using the Nanodrop™. The correct insertion of the plasmids was confirmed by restriction enzymes and/or sequencing, using methods widely known in the state of the art.
[00140] A partir de uma colónia corretamente inserida, foi feita uma inoculação em lOOmL de solução LB ampicilina 100 num frasco Erlenmeyer, que foi incubado toda a noite num agitador horizontal.Após a incubação, foi feita uma extração de uma MIDI ou MAXI-prep (p.ex. QIAGEN™ Midi ou Maxi Kit), para a obtenção de uma quantidade suficiente de plasmideo, a ser utilizado nos passos seguintes. [00140] From a correctly inserted colony, an inoculation was made in 100mL of LB ampicillin 100 solution in an Erlenmeyer flask, which was incubated overnight on a horizontal shaker. prep (e.g. QIAGEN™ Midi or Maxi Kit), to obtain a sufficient amount of plasmid, to be used in the following steps.
[00141] Numa forma de realização, após o isolamento do plasmideo p925/PoliA+nulómero/Braço de Homologia Esquerdo corretamente inserido, um terceiro passo de subclonagem foi iniciado, para efetuar a ligação do Braço de Homologia Direito a jusante do nulómero ao vetor p925/PoliA+ Nulómero/Braço de Homologia Esquerdo. [00141] In one embodiment, after the isolation of the correctly inserted p925/PolyA+nulomer/Left Homology Arm plasmid, a third subcloning step was initiated, to effect binding of the Right Homology Arm downstream of the nullomer to the p925 vector /PolyA+ Nunomer/Left Homology Arm.
[00142] O Braço de Homologia 3' ou Direito, (1590bp), foi ligado (subclonado), num sitio Notl imediatamente a jusante do nulómero no plasmideo p925/PoliA+Nulómero/Braço de Homologia Esquerdo. Foi providenciada a presença de um sitio BamHI na extremidade 3' da sequência para permitir futuramente o isolamento do vetor completo para a edição do gene BAG2. [00143] O produto da PCR foi obtido de ADN genómico de células HEK293T pela utilização do kit NZY Tissue gDNA Isolation Kit (NZYtech®), seguindo as instruções do fabricante. Utilizando os iniciadores Seq. ID N° 9 e Seq. ID N° 10, foi preparada uma solução com a mistura dos vários reagentes (MIX), de acordo com a Tabela 13, sendo depois misturados 45uL da MIX obtida a 5uL (200ng) de gDNA [41ng/uL] isolado das HEK293T. A reação PCR prosseguiu num termociclador (Applied Systems Thermocycler™), nas condições indicadas na Tabela 14. [00142] The 3' or Right Homology Arm, (1590bp), was ligated (subcloned) at a NotI site immediately downstream of the nullomer on plasmid p925/PolyA+Nulomer/Left Homology Arm. The presence of a BamHI site at the 3' end of the sequence was provided to allow future isolation of the complete vector for editing the BAG2 gene. [00143] The PCR product was obtained from genomic DNA from HEK293T cells using the NZY Tissue gDNA Isolation Kit (NZYtech®) following the manufacturer's instructions. Using Seq primers. ID No. 9 and Seq. ID No. 10, a solution was prepared with the mixture of the various reagents (MIX), according to Table 13, and then 45uL of the obtained MIX was mixed with 5uL (200ng) of gDNA [41ng/uL] isolated from the HEK293T. The PCR reaction proceeded in a thermocycler (Applied Systems Thermocycler™), under the conditions indicated in Table 14.
Tabela 13: Concentração de reagentes utilizados na reaçãoTable 13: Concentration of reagents used in the reaction
PCR.
Figure imgf000051_0001
PCR
Figure imgf000051_0001
Tabela 14: Condições da PCR no termociclador.
Figure imgf000052_0001
Table 14: PCR conditions in the thermal cycler.
Figure imgf000052_0001
[00144] Após a PCR, foi feita eletroforese em gel de agarose a 0,8% de uma amostra de 2uL do produto da PCR, para confirmar a presença de uma banda de 1590pb que corresponde ao fragmento de DNA produzido por PCR e que contém o Braço de Homologia Direito. Verificando-se a presença da banda, o restante ADN obtido na reação de PCR foi purificado por fenol/clorofórmio/álcool isoamilico, precipitado em etanol, e digerido com a enzima de restrição Notl, de acordo com a Tabela 15, durante 2 horas a 37°C. [00144] After PCR, 0.8% agarose gel electrophoresis was performed on a 2uL sample of the PCR product, to confirm the presence of a 1590bp band that corresponds to the DNA fragment produced by PCR and that contains the Right Homology Arm. Checking for the presence of the band, the remaining DNA obtained in the PCR reaction was purified by phenol/chloroform/isoamyl alcohol, precipitated in ethanol, and digested with the restriction enzyme Notl, according to Table 15, for 2 hours at 37°C.
Tabela 15: Condições experimentais para a digestão do Braço de Homologia Direito BAG2 pela enzima Notl.
Figure imgf000052_0002
[00145] A digestão completa foi confirmada através de eletroforese em gel de agarose a 0,8% do produto digerido. Verificando-se a digestão completa, o produto digerido foi isolado e purificado por extração do gel após electroforese em gel de agarose a 0,8% usando o sistema Wizard™ SV Gel and PCR clean-up System - PROMEGA™, seguindo as instruções do fabricante. De seguida, foi feita a purificação do produto obtido por fenol/clorofórmio/álcool isoamilico, seguindo-se de precipitação em etanol. Por fim, o precipitado obtido, que contém o Braço de Homologia Direito BAG 2 (Cassete 1) pronto para ser ligado (subclonado), foi ressuspendido em 2OuL PCR H20. Table 15: Experimental conditions for digestion of the Right Homology Arm BAG2 by Notl enzyme.
Figure imgf000052_0002
[00145] Complete digestion was confirmed by electrophoresis on agarose gel at 0.8% of the digested product. Upon complete digestion, the digested product was isolated and purified by gel extraction after electrophoresis on 0.8% agarose gel using the Wizard™ SV Gel and PCR clean-up System - PROMEGA™, following the instructions of the manufacturer. Then, the product obtained was purified by phenol/chloroform/isoamyl alcohol, followed by precipitation in ethanol. Finally, the obtained precipitate, which contains the Right Homology Arm BAG 2 (Cassette 1) ready to be ligated (subcloned), was resuspended in 20uL PCR H 2 0.
[00146] Antes de ser feita a ligação do inserto Braço de Homologia Direito BAG2, foi necessário preparar o vetor p925/PoliA+nulómero/Braço de Homologia Esquerdo BAG2, por digestão com a enzima NotIHF. Para isso, o vetor foi tratado com Plasmid-Safe™ ATP-Dependent DNase (EPICENTRE®), utilizando os reagentes descritos na Tabela 16. A mistura foi incubada a 37°C durante 30 minutos, e após este período a enzima Plasmid-Safe™ DNAse foi inativada por incubação a 70°C, por 30 minutos, parando a reação. [00146] Before ligation of the BAG2 Right Homology Arm insert, it was necessary to prepare the p925/PolyA+nulomer/BAG2 Left Homology Arm vector, by digestion with the NotIHF enzyme. For this, the vector was treated with Plasmid-Safe™ ATP-Dependent DNase (EPICENTRE®), using the reagents described in Table 16. The mixture was incubated at 37°C for 30 minutes, after which the Plasmid-Safe enzyme ™ DNAse was inactivated by incubation at 70°C for 30 minutes, stopping the reaction.
Tabela 16: Tratamento do vetor p925/PoliA+nulómero/Braço de Homologia Esquerdo BAG2 com Plasmid-Safe™ ATP-Dependent DNase (EPICENTRE®).
Figure imgf000054_0001
Table 16: Treatment of p925/PolyA+nulomer/Left Homology Arm BAG2 vector with Plasmid-Safe™ ATP-Dependent DNase (EPICENTRE®).
Figure imgf000054_0001
[00147] O produto da reação foi purificado por fenol/clorofórmio/álcool isoamilico, precipitado com etanol e ressuspendido em 20ul PCR H2O. [00147] The reaction product was purified by phenol/chloroform/isoamyl alcohol, ethanol precipitated and resuspended in 20ul PCR H2O.
[00148] Após o tratamento com Plasmid-Safe™, o plasmideo p925/PoliA+nulómero/Braço de Homologia Esquerdo foi digerido com a enzima de restrição NotIHF durante 2 horas a 37°C em banho de água, segundo o protocolo indicado na Tabela 17. [00148] After treatment with Plasmid-Safe™, plasmid p925/PolyA+nunomer/Left Homology Arm was digested with restriction enzyme NotIHF for 2 hours at 37°C in a water bath, according to the protocol indicated in the Table 17.
Tabela 17: Condições utilizadas na digestão do plasmideo p925/PoliA+nulómero/Braço de Homologia Esquerdo pela enzima de restrição NotIHF
Figure imgf000054_0002
[00149] Após digestão completa, confirmada por eletroforese em gel de agarose a 0,8%, o plasmideo resultante foi purificado por fenol/clorof órmio/álcool isoamilico, precipitado com etanol, e ressuspendido em 20ul PCR H2O. Por fim, o produto foi desfosforilado, seguindo o protocolo referido na Tabela 18. Após incubação durante 10 minutos a 37°C em banho de água, a reação foi terminada incubando 5 minutos a 75°C. O produto obtido foi purificado por fenol/clorofórmio/álcool isoamilico, precipitado em etanol, e ressuspendido em 50uL PCR H2O. Table 17: Conditions used in the digestion of plasmid p925/PolyA+nulomer/Left Homology Arm by the restriction enzyme NotIHF
Figure imgf000054_0002
[00149] After complete digestion, confirmed by 0.8% agarose gel electrophoresis, the resulting plasmid was purified by phenol/chloroform/isoamyl alcohol, ethanol precipitated, and resuspended in 20ul PCR H2O. Finally, the product was dephosphorylated, following the protocol mentioned in Table 18. After incubation for 10 minutes at 37°C in a water bath, the reaction was terminated by incubating 5 minutes at 75°C. The product obtained was purified by phenol/chloroform/isoamyl alcohol, precipitated in ethanol, and resuspended in 50uL PCR H2O.
Tabela 18: Protocolo de desfosforilação do vetor p925/PoliA+nulómero/Braço de Homologia Esquerdo tratado e digerido.
Figure imgf000055_0001
Table 18: Dephosphorylation protocol of treated and digested p925/PolyA+nulomer/Left Homology Arm vector.
Figure imgf000055_0001
[00150] Numa forma de realização, o vetor p925/PoliA+nulómero/Braço de Homologia Esquerdo digerido e desfosforilado foi ligado ao inserto Braço de Homologia Direito BAG2, também já digerido por Notl, utilizando as condições descritas na Tabela 19. Tabela 19: Preparação da solução MIX para ligação do vetor PoliA+nulómero/Braço de Homologia Esquerdo digerido e desfosforilado ao inserto Braço de Homologia direito BAG2.
Figure imgf000056_0001
[00150] In one embodiment, the digested and dephosphorylated p925/PolyA+nulomer/Left Homology Arm vector was ligated to the BAG2 Right Homology Arm insert, also already digested by Notl, using the conditions described in Table 19. Table 19: Preparation of MIX solution for binding the digested and dephosphorylated PolyA+nulomer/Left Homology Arm vector to the BAG2 Right Homology Arm insert.
Figure imgf000056_0001
[00151] Numa forma de realização, foram pipetados para um tubo de fundo redondo (Tubo 1) 4uL da MIX e 6,4uL do inserto (Braço de Homologia Direito BAG2 digerido). Num outro tubo (Tubo 2, tubo controlo negativo), foram pipetados 4uL da MIX e 6uL PCR H2O. Ambos os tubos foram incubados a 15°C durante pelo menos 2 horas, de preferência durante toda a noite. Após incubação, foi feita a transformação de células competentes E. coli (adquiridas à indústria) com 5uL do conteúdo de cada Tubo 1 e Tubo 2. Após a transformação, foi feita a extração dos plasmideos de várias colónias, usando o sistema Wizard™ Plus SV Minipreps DNA Purification System, 250 preps, PROMEGA™, e o ADN obtido quantificado, usando a Nanodrop™. A correta inserção dos plasmideos foi confirmada através de enzimas de restrição e/ou sequenciação, usando métodos amplamente conhecidos no estado da arte. [00151] In one embodiment, 4uL of the MIX and 6.4uL of the insert (Digested BAG2 Right Homology Arm) were pipetted into a round bottom tube (Tube 1). In another tube (Tube 2, negative control tube), 4uL of the MIX and 6uL of PCR H2O were pipetted. Both tubes were incubated at 15°C for at least 2 hours, preferably overnight. After incubation, E. coli competent cells (purchased from the industry) were transformed with 5uL of the contents of each Tube 1 and Tube 2. After transformation, plasmids were extracted from several colonies, using the Wizard™ Plus system. SV Minipreps DNA Purification System, 250 preps, PROMEGA™, and the DNA obtained quantified using the Nanodrop™. The correct insertion of the plasmids was confirmed by restriction enzymes and/or sequencing, using methods widely known in the state of the art.
[00152] A partir de uma colónia corretamente inserida, foi feita uma inoculação em lOOmL de solução LB ampicilina 100 num frasco Erlenmeyer, que foi incubado toda a noite num agitador horizontal.Após a incubação, foi feita uma extração de uma MIDI ou MAXI-prep (p.ex. QIAGEN™ Midi ou Maxi Kit), para a obtenção de uma quantidade suficiente de plasmideo, a ser utilizado nos passos seguintes. [00152] From a correctly inserted colony, an inoculation was made in 100mL of LB ampicillin 100 solution in an Erlenmeyer flask, which was incubated overnight on a horizontal shaker. from a MIDI or MAXI-prep (eg QIAGEN™ Midi or Maxi Kit) to obtain a sufficient amount of plasmid to be used in the following steps.
[00153] Depois do isolamento do plasmideo p925/poliA+nulómero/Braço de Homologia Esquerdo/Braço de Homologia Direito corretamente inserido, designado por R52, o construto (do inglês construct) do vetor completo, para a edição do Gene BAG2 no braço longo do cromossoma 6 humano, ficou concluído (Figura 2), (Seq. ID N° 43). [00153] After isolation of correctly inserted p925/polyA+nulomer/Left Homology Arm/Right Homology Arm, designated R52, the complete vector construct, for editing the BAG2 Gene in the long arm of human chromosome 6, was concluded (Figure 2), (Seq. ID No. 43).
[00154] Numa forma de realização, o construto R52, preparado para a edição do gene BAG2, está flanqueado por dois sítios de reconhecimento para a enzima de restrição BamHI. Tirando vantagem desta característica intencional, o vetor para a edição do gene BAG2 foi preparado pela sua excisão por digestão com BamHI, através da reação descrita na Tabela 20. [00154] In one embodiment, the R52 construct, prepared for editing the BAG2 gene, is flanked by two recognition sites for the restriction enzyme BamHI. Taking advantage of this intentional feature, the vector for editing the BAG2 gene was prepared by excision by digestion with BamHI, through the reaction described in Table 20.
Tabela 20: Mistura de reagentes preparada para a digestão do construto R52 pela enzima de restrição BamHI
Figure imgf000057_0001
Table 20: Mixture of reagents prepared for the digestion of the construct R52 by the restriction enzyme BamHI
Figure imgf000057_0001
[00155] Numa forma de realização, foram pipetados 94uL da MIX e 6uL de ADN plasmidico R52 para um microtubo, que foi depois incubado durante 2 horas a 37°C num banho de água. Depois da digestão com BamHI, o ADN foi purificado por fenol/clorofórmio/álcool isoamilico, seguindo-se uma precipitação em etanol. Após a precipitação, o construto R52 ficou pronto para a transfeção celular, em que transfeção designa o procedimento em que ADN é levado a entrar dentro das células com o objetivo de conseguir alterações no seu genoma. [00155] In one embodiment, 94uL of the MIX and 6uL of R52 plasmid DNA were pipetted into a microtube, which was then incubated for 2 hours at 37°C in a water bath. After digestion with BamHI, the DNA was purified by phenol/chloroform/isoamyl alcohol, followed by precipitation in ethanol. After precipitation, the R52 construct was ready for cell transfection, where transfection designates the procedure in which DNA is made to enter cells in order to achieve changes in their genome.
[00156] Após a preparação do construto, foi feita a transfeção de células estaminais pluripotentes humanas (hiPSCs) no gene BAG2, com o vetor R52 que contém a Cassete[00156] After the preparation of the construct, human pluripotent stem cells (hiPSCs) were transfected into the BAG2 gene, with the R52 vector that contains the Cassette
1. 1.
- Cultura de hiPSCs: - Culture of hiPSCs:
[00157] A cultura de células hiPSCs imediatamente após descongelamento foi feita em placas de 6 poços para cultura de células aderentes, previamente revestidas com Matrigel™ Corning™, seguindo as instruções do fabricante. As hiPSCs foram semeadas em cada um dos poços, na presença de meio de cultura mTeSR™l STEMCELL Technologies™ suplementado a 1% com uma solução de Penicilina 10.000UI/mL/Estreptomicina lO.OOOug/mL (Sigma®), sendo depois incubadas a 37°C em atmosfera húmida de 5%CC>2. O meio de cultura foi trocado diariamente. [00157] The culture of hiPSC cells immediately after thawing was done in 6-well adherent cell culture plates, previously coated with Matrigel™ Corning™, following the manufacturer's instructions. The hiPSCs were seeded in each well, in the presence of mTeSR™l STEMCELL Technologies™ culture medium supplemented at 1% with a solution of Penicillin 10,000IU/mL/10.000ug/mL Streptomycin (Sigma®), and then incubated at 37°C in a humidified atmosphere of 5% CC> 2 . The culture medium was changed daily.
[00158] Após três passagens, as células foram semeadas em placas de 12 ou de 24 poços de fundo plano, previamente revestidos com Matrigel™ Corning™, seguindo as instruções do fabricante. O meio de cultura, regime de mudança de meio e as condições de incubação foram mantidas. [00158] After three passages, cells were seeded into 12- or 24-well flat-bottomed plates, previously coated with Matrigel™ Corning™, following the manufacturer's instructions. The culture medium, medium change regime and incubation conditions were maintained.
- Transfeção das hiPSCs em cultura: - Transfection of hiPSCs in culture:
[00159] Quando as células atingiram ~10% de confluência, o meio de cultura foi suplementado com Y-27632 ROCK inhibitor STEMCELL Technologies™ (Rocki), seguindo-se uma incubação durante 2 horas previamente à introdução dos materiais de transfeção. [00159] When the cells reached ~10% confluence, the culture medium was supplemented with Y-27632 ROCK inhibitor STEMCELL Technologies™ (Rocki), followed by an incubation for 2 hours prior to the introduction of the transfection materials.
[00160] A edição do gene BAG2 deu-se pela via da reparação do ADN celular designada por Reparação Dirigida por Homologia (HDR). Para isso, foi necessária a transfeção simultânea de CRISPR-Cas9/BAG2gARN, para ser feita uma clivagem da dupla cadeia do ADN (DBS) num local escolhido, e de um construto com a sequência do ADN (inserto) a incorporar no local da clivagem. Esta introdução simultânea de dois elementos diferentes na célula com um objetivo comum designa-se por co-transfeção . [00160] Editing of the BAG2 gene took place via the cellular DNA repair pathway called Homology Directed Repair (HDR). For this, the simultaneous transfection of CRISPR-Cas9/BAG2gRNA was necessary, in order to cleave the DNA double strand (DBS) at a chosen site, and a construct with the DNA sequence (insert) to be incorporated at the cleavage site. . This simultaneous introduction of two different elements into the cell with a common objective is called co-transfection.
[00161] Numa forma de realização, foi efetuada a co- transfeção de CRISPR-Cas9/BAG2gARN (~250ng ADN/poço) com o construto R52 (~250ng ADN/poço) em placas de 24 poços, utilizando as células previamente preparadas nos referidos poços ao atingirem 10% de confluência. A co-transfeção foi conseguida por lipofeção com recurso a Lipofectamine™ 3000 Transfection Reagent, Thermo Fisher Scientific, seguindo as instruções do fabricante. Na Figura 5 é apresentado o resultado das reações PCR obtidas em amostras de ADN genómico de hiPSCs VIRGENS (WT) como controlo negativo, e ADN genómico de hiPSCs da mesma linha das WT mas co-transfetadas, com recurso aos iniciadores SEQ. ID N° 15, SEQ. ID N°16, SEQ. ID N° 17, SEQ ID N° 18, SEQ. ID N°19, SEQ. ID N° 20 e SEQ ID N° 21, as quais permitiram avaliar a presença de células corretamente editadas. As condições para as PCRs encontram- se listadas na Tabela 21 e foram preparadas com os tubos de todos os reagentes colocados em gelo. Tabela 21: Concentração de reagentes utilizados nas reações de PCR.
Figure imgf000060_0001
[00161] In one embodiment, CRISPR-Cas9/BAG2gRNA (~250ng DNA/well) was co-transfected with the R52 construct (~250ng DNA/well) in 24-well plates, using the cells previously prepared in the referred wells when reaching 10% confluence. Co-transfection was achieved by lipofection using Lipofectamine™ 3000 Transfection Reagent, Thermo Fisher Scientific, following the manufacturer's instructions. Figure 5 shows the results of PCR reactions obtained on samples of genomic DNA from VIRGIN hiPSCs (WT) as a negative control, and genomic DNA from hiPSCs of the same line as WT but co-transfected, using SEQ primers. ID NO: 15, SEQ. ID NO:16, SEQ. ID NO: 17, SEQ ID NO: 18, SEQ. ID NO:19, SEQ. ID No. 20 and SEQ ID No. 21, which allowed to assess the presence of correctly edited cells. Conditions for PCRs are listed in Table 21 and were prepared with tubes of all reagents placed on ice. Table 21: Concentration of reagents used in PCR reactions.
Figure imgf000060_0001
[00162] Numa forma de realização, foram pipetados 279uL de MIX para cada um de 2 microtudos separados (microtubo 1 e microtubo 2). [00162] In one embodiment, 279uL of MIX was pipetted into each of 2 separate microtubes (microtube 1 and microtube 2).
A- Ao microtubo 1 foram adicionados 30uL do ADN genómico WT= Virgem; A- 30uL of WT=Virgin genomic DNA was added to microtube 1;
B- Ao microtubo 2 foram adicionados 30uL do ADN genómico das hiPSCs co-transfetadas; B- 30uL of genomic DNA from the co-transfected hiPSCs were added to microtube 2;
C- Foram preparados 5 microtubos marcados com os algarismos correspondentes às respetivas SEQ. ID N° 15 a 21: SEQ. ID N° 15 + SEQ. ID N° 16 = 5'fora do construto até ao promotor PGK (PGKpmt); SEQ. ID N° 15 + SEQ. ID N° 17 = 5'fora do construto até ao Braço de Homologia 5'(5'BH)(~500bp); SEQ. ID N° 20 + SEQ. ID N° 21 = 3'fora do construto até ao Braço de Homologia 3' (3'BH)(~900bp); SEQ. ID N° 16 + SEQ. ID N° 18 = Braço de Homologia 5'(5'BH) até ao promotor PGK (PGKpmt); SEQ. ID N° 19 + SEQ. ID N° 20 = 3 fora do construto até ao Nulómero, para as 5 diferentes reações de PCR de controlo negativo (ADN genómico WT=Virgem); C- 5 microtubes marked with the numbers corresponding to the respective SEQs were prepared. ID NO: 15 to 21: SEQ. ID NO: 15 + SEQ. ID No. 16 = 5' outside the construct to the PGK promoter (PGKpmt); SEQ ID NO: 15 + SEQ. ID No. 17 = 5' out of construct to Homology Arm 5'(5'BH)(~500bp); SEQ ID NO: 20 + SEQ. ID No. 21 = 3' out of the construct to the 3' Homology Arm (3'BH)(~900bp); SEQ ID NO: 16 + SEQ. ID No. 18 = Homology Arm 5'(5'BH) to PGK promoter (PGKpmt); SEQ ID NO: 19 + SEQ. ID No. 20 = 3 out of the construct to the Nunomer, for the 5 different negative control PCR reactions (WT=Virgin genomic DNA);
D- Foram preparados outros 5 microtubos marcados com os algarismos correspondentes às respetivas Seq. ID N° 15 a 21: SEQ. ID N° 15 + SEQ. ID N° 16 = 5'fora do construto até ao promotor PGK (PGKpmt); SEQ. ID N° 15 + SEQ. ID N° 17 = 5'fora do construto até ao Braço de Homologia 5'(5'BH)(~500bp); SEQ. ID N° 20 + SEQ. ID N° 21= 3'fora do construto até ao Braço de Homologia 3'(3'BH)(~900bp); SEQ. ID N° 16 + SEQ. ID N° 18 = Braço de Homologia 5'(5'BH) até ao promotor PGK (PGKpmt); SEQ. ID N° 19 + SEQ. ID N° 20 = 3' fora do construto até ao Nulómero, para as 5 diferentes reações de PCR efetuadas com ADN genómico das células co-transfetadas. D- Another 5 microtubes marked with the numerals corresponding to the respective Seq. ID NO: 15 to 21: SEQ. ID NO: 15 + SEQ. ID N° 16 = 5' out of the construct to the PGK promoter (PGKpmt); SEQ ID NO: 15 + SEQ. ID No. 17 = 5' out of construct to Homology Arm 5'(5'BH)(~500bp); SEQ ID NO: 20 + SEQ. ID No. 21= 3'out of construct to Homology Arm 3'(3'BH)(~900bp); SEQ ID NO: 16 + SEQ. ID No. 18 = Homology Arm 5'(5'BH) to PGK promoter (PGKpmt); SEQ ID NO: 19 + SEQ. ID No. 20 = 3' off the construct to the Nunomer, for the 5 different PCR reactions performed with genomic DNA from the co-transfected cells.
E- 46uL da mistura de reagentes do microtubo 1 (A-), foram pipetados para cada um dos 5 microtubos (C-) de reação de PCR (genómico WT) adicionados de 2uL de cada um dos iniciadores FWD e REV marcados (SEQ. ID N° 15 a 21), e E-46uL of the reagent mixture from microtube 1 (A-), was pipetted into each of 5 PCR reaction microtubes (C-) (WT genomic) added 2uL of each of labeled FWD and REV primers (SEQ. ID No. 15 to 21), and
F- 46uL da mistura de reagentes do microtubo 2 (B-), foram pipetados para cada um dos 5 microtubos (D-) de reação de PCR (co-transfetadas) adicionados de 2uL de cada um dos iniciadores FWD e REV marcados (SEQ. ID N° 15 a 21). F-46uL of reagent mixture from microtube 2 (B-), was pipetted into each of 5 PCR reaction microtubes (D-) (co-transfected) with 2uL of each of labeled FWD and REV primers added (SEQ ID No. 15 to 21).
[00163] De imediato foram iniciadas num termociclador as reações de PCR realizadas nas condições referidas na Tabela 22. [00163] The PCR reactions performed under the conditions mentioned in Table 22 were immediately started in a thermocycler.
Tabela 22: Condições das PCR no termociclador.
Figure imgf000061_0001
[00164] Após a PCR, foi feita electroforese de uma amostra de 3uL de cada um dos microtubos, num gel de agarose a 0,8%, (Figura 5). Table 22: PCR conditions in the thermocycler.
Figure imgf000061_0001
[00164] After PCR, a 3uL sample from each of the microtubes was electrophoresed on a 0.8% agarose gel (Figure 5).
[00165] Na Figura 6 são apresentados os resultados da avaliação por Citometria de Fluxo (Citómetro Becton Dickinson FACS Calibur®) das percentagens de células que expressam a Proteína Fluorescente Verde (GFP) em quatro amostras diferentes de uma linha de Células Estaminais Pluripotentes Humanas induzidas (hiPSCs), (linha TCLab, Passagem 37), co-transfetadas de acordo com a presente divulgação. A análise foi realizada com recurso ao programa informático Flowing Software. [00165] Figure 6 shows the results of the evaluation by Flow Cytometry (Becton Dickinson FACS Calibur® Cytometer) of the percentages of cells expressing Green Fluorescent Protein (GFP) in four different samples of an induced Human Pluripotent Stem Cell line. (hiPSCs), (TCLab line, Passage 37), co-transfected in accordance with the present disclosure. The analysis was performed using the Flowing Software software.
[00166] Na Figura 7 são apresentados os resultados da avaliação por Citometria de Fluxo (Citómetro Becton Dickinson FACS Calibur®) das percentagens de células que expressam a Proteína Fluorescente Verde (GFP) em duas amostras diferentes de uma outra linha de Células Estaminais Pluripotentes Humanas induzidas (hiPSCs), (Linha GEpi/ GIBCO, Passagem 50). Esta avaliação foi realizada após marcação das células não viáveis com recurso ao Kit: LIVE/DEAD™Fixable Dead Cell Stain Kit, ThermoFisher Scientific™. Esta marcação das células não viáveis permite aumentar a certeza em relação às células transfetadas e expressadoras de GFP que podem ser expandidas em cultura (viáveis). A análise foi realizada com recurso ao programa informático Flowing software®. [00166] Figure 7 shows the results of the evaluation by Flow Cytometry (Becton Dickinson FACS Calibur® Cytometer) of the percentages of cells expressing Green Fluorescent Protein (GFP) in two different samples from another line of Human Pluripotent Stem Cells (hiPSCs), (GEpi/GIBCO Line, Passage 50). This evaluation was performed after labeling non-viable cells using the Kit: LIVE/DEAD™Fixable Dead Cell Stain Kit, ThermoFisher Scientific™. This labeling of non-viable cells allows for increased certainty regarding transfected and GFP-expressing cells that can be expanded in culture (viable). The analysis was performed using the Flowing software® software.
[00167] Os valores das percentagens obtidas em ambas as linhas celulares e com os dois métodos diferentes estão dentro dos limites referidos nos trabalhos de vários autores. [00167] The values of the percentages obtained in both cell lines and with the two different methods are within the limits referred to in the works of several authors.
- Seleção das hiPSCs em cultura, resistentes à puromicina: [00168] Após 48 horas do processo transfeção, foi iniciada a seleção das células editadas. Para tal, o meio de cultura foi suplementado com puromicina (0,1 ug/mL). Numa forma de realização, a seleção de clones GFP+/Resistentes à puromicina foi feita através do aumento da concentração da puromicina no meio de cultura, com incrementos de 0,025ug/mL/dia até um máximo de 0,2ug/mL (Figura 6 e Figura7). Após 15 dias de seleção com puromicina, células GFP+ são selecionadas por Fluorescence Associated Cell Sorting (FACS). Após a sua expansão em cultura, e por método de diluição seriada, clones derivados de células únicas são isolados e expandidos. Após a sua expansão, os clones corretamente editados são selecionados de acordo com o teste para a seleção de clones de hiPSCs descrito na presente realização. - Selection of puromycin-resistant hiPSCs in culture: [00168] After 48 hours of the transfection process, the selection of edited cells was started. For this, the culture medium was supplemented with puromycin (0.1 ug/ml). In one embodiment, selection of GFP+/Puromycin-Resistant clones was done by increasing the concentration of puromycin in the culture medium, in increments of 0.025ug/mL/day to a maximum of 0.2ug/mL (Figure 6 and Figure 7). After 15 days of puromycin selection, GFP+ cells are sorted by Fluorescence Associated Cell Sorting (FACS). After their expansion in culture, and by serial dilution method, clones derived from single cells are isolated and expanded. After their expansion, the correctly edited clones are selected according to the test for the selection of hiPSC clones described in the present embodiment.
- Teste para a seleção dos Clones de hiPSCs corretamente editados: - Test for the selection of correctly edited hiPSC Clones:
[00169] Uma vez obtida a cultura de clones, é feita a seleção dos clones corretamente editados. Para tal, são realizadas reações PCR seguidas de sequenciação do Gene BAG2 nos clones cultivados, utilizando iniciadores amplificadores de sequências de fora para dentro dos construtos do Gene BAG2 (Cassete 1) e do Gene LGSN (cassete2) em ambas as extremidades 5' e 3'. É também feita a confirmação de que as sequências inseridas estão corretas, através da amplificação de sequências dentro dos insertos. Para a avaliação da correta inserção no Gene BAG2 foram utilizados os iniciadores Seq. ID N° 15, Seq. ID N° 16, Seq. ID N° 17, Seq. ID N° 18, Seq. ID N° 19, Seq. ID N° 20, Seq. ID N° 21 (Figura 5). Após a identificação dos clones corretamente editados no Gene BAG2, é feita a sua cultura e expansão, de forma a construir um banco destas células. As células corretamente editadas são submetidas a uma nova edição, no Gene LGSN com a Cassete 2 (Figura 3). [00169] Once the clone culture is obtained, the correctly edited clones are selected. For this, PCR reactions followed by BAG2 Gene sequencing are performed in the cultivated clones, using primers amplifying sequences from outside to inside the BAG2 Gene (Cassette 1) and the LGSN Gene (Cassette 2) constructs at both 5' and 3' ends. '. Confirmation that the inserted sequences are correct is also performed by amplifying sequences within the inserts. To evaluate the correct insertion in the BAG2 Gene, Seq primers were used. ID No. 15, Seq. ID No. 16, Seq. ID No. 17, Seq. ID No. 18, Seq. ID No. 19, Seq. ID No. 20, Seq. ID No. 21 (Figure 5). After identifying the clones correctly edited in the BAG2 Gene, they are cultured and expanded in order to build a bank of these cells. Correctly edited cells are submitted to a new edition, in Gene LGSN with Cassette 2 (Figure 3).
- Edição do Gene LGSN por HDR com a Cassete 2 (Figura 3), (Seq. ID N° 44): - Editing the LGSN Gene by HDR with Cassette 2 (Figure 3), (Seq. ID No. 44):
[00170] Para a edição do Gene LGSN, são preparados dois vetores, a fim de serem transfectados nas células da linha de hiPSCs (já corretamente editadas no Gene BAG2). O primeiro vetor, o vetor CRISPR-Cas9 nuclease e LGSNgARN para realizar DSBs no Gene LGSN, é preparado a partir da nuclease CRISPR- Cas9 obtida comercialmente, (e.g. TrueCut Cas9 Protein v2® ThermoFisher®), e LGSNgARN sintético (TrueGuide Synthetic gRNA® ThermoFisher®), seguindo as instruções do fabricante. A Seq.ID N° 2 foi a guia escolhida após ensaio com T7E1, (EnGen® Mutation Detection Kit, NewEngland Biolabs, Inc.), para a inserção do inserto da cassete 2 no Gene LGSN. [00170] For the editing of the LGSN Gene, two vectors are prepared in order to be transfected into the cells of the hiPSCs line (already correctly edited in the BAG2 Gene). The first vector, the CRISPR-Cas9 nuclease and LGSNgARN vector to perform DSBs on the LGSN Gene, is prepared from commercially obtained CRISPR-Cas9 nuclease, (e.g. TrueCut Cas9 Protein v2® ThermoFisher®), and synthetic LGSNgARN (TrueGuide Synthetic gRNA® ThermoFisher®), following the manufacturer's instructions. Seq.ID No. 2 was the guide chosen after testing with T7E1, (EnGen® Mutation Detection Kit, NewEngland Biolabs, Inc.), for inserting cassette 2 insert into Gene LGSN.
[00171] Numa forma de realização, o segundo vetor, o vetor Cassete 2 para o gene LGSN (Figura 3, Seq. ID N° 44) é adquirido a fornecedores externos. [00171] In one embodiment, the second vector, the Cassette 2 vector for the LGSN gene (Figure 3, Seq. ID No. 44) is purchased from external suppliers.
[00172] Após a preparação dos vetores de acordo com as indicações dos fabricantes, é feita a edição HDR do gene LGSN com a Cassete 2, em clones de hiPSCs corretamente editadas com a Cassete 1 no Gene BAG2. [00172] After preparing the vectors according to the manufacturers' instructions, HDR editing of the LGSN gene is performed with Cassette 2, in clones of hiPSCs correctly edited with Cassette 1 in Gene BAG2.
- Cultura de hiPSCs/BAG2-Cassete 1 editadas: - Culture of edited hiPSCs/BAG2-Cassette 1:
[00173] Numa forma de realização, a cultura de hiPSCs/BAG2-Cassete 1 é feita em placas de 6 poços para cultura de células aderentes, previamente revestidas com Matrigel™ Corning™, seguindo as instruções do fabricante. As células hiPSCs são semeadas em cada um dos poços, na presença de meio de cultura mTeSR™! STEMCELL Technologies™ suplementado a 1% com uma solução de Penicilina 10.000UI/mL/Estreptomicina lO.OOOug/mL (Sigma®), sendo depois incubadas a 37°C em atmosfera húmida de 5%CC>2. O meio de cultura foi trocado diariamente. [00173] In one embodiment, the culture of hiPSCs/BAG2-Cassette 1 is done in 6-well adherent cell culture plates, previously coated with Matrigel™ Corning™, following the manufacturer's instructions. The hiPSC cells are seeded in each of the wells, in the presence of mTeSR™ culture medium! STEMCELL Technologies™ supplemented at 1% with a solution of Penicillin 10,000IU/mL/Streptomycin 10,000ug/mL (Sigma®), then incubated at 37°C in a humidified atmosphere of 5% CC> 2 . The culture medium was changed daily.
[00174] Três passagens após descongelamento, as células são semeadas em placas de 12 ou 24 poços de fundo plano, previamente revestidos com Matrigel™ Corning™, seguindo as instruções do fabricante. O meio de cultura, regime de mudança de meio e as condições de incubação foram mantidas. [00174] Three passages after thawing, cells are seeded into 12 or 24-well flat-bottomed plates, pre-coated with Matrigel™ Corning™, following the manufacturer's instructions. The culture medium, medium change regime and incubation conditions were maintained.
- Transfeção das hiPSCs/BAG2-Cassete 1 editadas, em cultura: - Transfection of edited hiPSCs/BAG2-Cassette 1, in culture:
[00175] Numa forma de realização. quando as células atingiram ~10% de confluência, o meio de cultura foi suplementado com Y-27632 ROCK inhibitor STEMCELL Technologies™ (Rocki), seguindo-se uma incubação durante 2 horas previamente à realização da co-transfeção. [00175] In one embodiment. when the cells reached ~10% confluency, the culture medium was supplemented with Y-27632 ROCK inhibitor STEMCELL Technologies™ (Rocki), followed by an incubation for 2 hours prior to co-transfection.
[00176] A edição correta do gene LSGN é feita por HDR, com a transfeção simultânea do vetor CRISPR-eCas9/LGSNgARN (~250ng ADN/poço) e o construto LSGN (~250ng ADN/poço), em placas de 24 poços com hiPSCs a ~10% de confluência, com recurso a Lipofeção por Lipofectamine™ 3000 Transfection Reagent, ThermoFisher Scientific, seguindo as instruções do fabricante . [00176] Correct editing of the LSGN gene is performed by HDR, with the simultaneous transfection of the CRISPR-eCas9/LGSNgARN vector (~250ng DNA/well) and the LSGN construct (~250ng DNA/well), in 24-well plates with ~10% confluency hiPSCs using Lipofectamine™ 3000 Transfection Reagent, ThermoFisher Scientific, following manufacturer's instructions.
- Seleção das hiPSCs em cultura resistentes à puromicina e neomicina: - Selection of puromycin- and neomycin-resistant hiPSCs in culture:
[00177] Numa realização, após 48 horas do processo de transfeção, é iniciada a seleção das células editadas resistentes à puromicina e neomicina. Numa forma de realização, a seleção de clones mCherry+/resistentes à neomicina, é feita através do aumento da concentração da neomicina no meio de cultura em incrementos 10ug/mL no meio de cultura iniciando com uma concentração de 40ug/mL até 200ug/mL dependendo da linha de hiPSCs utilizada. Após 15 dias de seleção com neomicina, as células em cultura são selecionadas com recurso a Fluorescence Associated Cell Sorting (FACS), para a expressão dupla de Proteína Fluorescente Verde (GFP) e Proteína Fluorescente Vermelha (mCherry). Estas células são depois cultivadas e expandidas. Após uma passagem, clones derivados de células únicas são selecionados com recurso a um processo de diluição seriada. Os clones resultantes são avaliados quanto à sua correta edição. [00177] In one embodiment, after 48 hours of the transfection process, selection of the edited cells resistant to puromycin and neomycin is initiated. In one embodiment, selection of mCherry+/neomycin-resistant clones is done by increasing the concentration of the neomycin in the culture medium in 10ug/mL increments in the culture medium starting with a concentration of 40ug/mL up to 200ug/mL depending on the hiPSC line used. After 15 days of neomycin selection, cells in culture are sorted using Fluorescence Associated Cell Sorting (FACS) for dual expression of Green Fluorescent Protein (GFP) and Red Fluorescent Protein (mCherry). These cells are then cultured and expanded. After one passage, clones derived from single cells are selected using a serial dilution procedure. The resulting clones are evaluated for their correct editing.
- Seleção dos Clones de hiPSCs corretamente editados nos Genes BAG2 e LGSN: - Selection of Clones of hiPSCs correctly edited in Genes BAG2 and LGSN:
[00178] Numa forma de realização, uma vez obtida a cultura de clones, é feita a seleção dos clones corretamente editados. Para tal, são realizadas reações PCR, seguida de sequenciação do Gene BAG2 e do Gene LGSN dos clones cultivados utilizando iniciadores amplificadores de sequências de fora para dentro dos construtos do Gene BAG2 (Cassete 1) e do Gene LGSN (Cassete 2), em ambas as extremidades 5' e 3'. É também feita a confirmação de que as sequências inseridas estão corretas, a partir da amplificação por PCR de sequências dentro dos insertos. Para a confirmação da correta inserção do inserto no Gene BAG2 podem ser utilizados os iniciadores com as sequências: Seq. ID N° 13, Seq. ID N° 14, Seq. ID N° 15, Seq. ID N° 16, Seq. ID N° 17, Seq. ID N° 18, Seq. ID N° 19, Seq. ID N° 20 e Seq. ID N° 21. Para a confirmação da correta inserção do inserto do Gene LGSN podem ser utilizados os correspondentes iniciadores: Seq. ID N° 45, Seq. ID N° 46, Seq. ID N° 47, Seq. ID N°48, Seq ID N° 49, seq ID N° 50, Seq. ID N° 51, Seq. ID N° 52, Seq ID n°53 e Seq. ID N° 54. [00178] In one embodiment, once the clone culture is obtained, the correctly edited clones are selected. To this end, PCR reactions are performed, followed by sequencing the Gene BAG2 and Gene LGSN of the cultivated clones using primers amplifying sequences from outside to inside the constructs of Gene BAG2 (Cassette 1) and Gene LGSN (Cassette 2), in both the 5' and 3' ends. Confirmation is also made that the inserted sequences are correct, from the PCR amplification of sequences within the inserts. To confirm the correct insertion of the insert in the Gene BAG2, primers with the following sequences can be used: Seq. ID No. 13, Seq. ID No. 14, Seq. ID No. 15, Seq. ID No. 16, Seq. ID No. 17, Seq. ID No. 18, Seq. ID No. 19, Seq. ID No. 20 and Seq. ID N° 21. To confirm the correct insertion of the LGSN Gene insert, the corresponding primers can be used: Seq. ID No. 45, Seq. ID No. 46, Seq. ID No. 47, Seq. ID No. 48, Seq ID No. 49, seq ID No. 50, Seq. ID No. 51, Seq. ID No. 52, Seq ID No. 53 and Seq. ID No. 54.
- Linha de hiPSC Off-the-shelf: - Off-the-shelf hiPSC line:
[00179] Após a identificação dos clones corretamente editados, é feita a sua cultura e expansão de forma a construir um banco destas células. As células corretamente editadas em ambos os genes BAG2 e LSGN constituem o produto off-the-shelf de hiPSCs preparadas para perderem o seu par de cromossomas 6. (Figura 4) [00179] After identifying the correctly edited clones, they are cultured and expanded in order to build a bank of these cells. Correctly edited cells in both the BAG2 and LSGN genes constitute the off-the-shelf product of hiPSCs primed to lose their chromosome 6 pair. (Figure 4)
[00180] Numa forma de realização, estas células permitem a substituição do seu par de cromossomas 6 indígena, pelo par de cromossomas 6 de um paciente e a obtenção de células saudáveis, (não portadoras de eventuais mutações presentes no genoma do paciente) e com o exato e completo conjunto de recetores HLA do paciente. Por isso, estas células off-the- shelf têm grande relevância, no campo da transplantação, visto que o problema de rejeição por incompatibilidade HLA é ultrapassado. [00180] In one embodiment, these cells allow the replacement of their indigenous pair of chromosomes 6, by the pair of chromosomes 6 of a patient and the obtaining of healthy cells, (not carrying any mutations present in the patient's genome) and with the patient's exact and complete set of HLA receptors. Therefore, these off-the-shelf cells are of great relevance in the field of transplantation, as the problem of rejection due to HLA incompatibility is overcome.
- Preparação do vetor CRISPR-Cas9/NulómerogARN para a perda do par de cromossomas 6 das hiPSCs/editadas nos genes BAG2 e LSGN: - Preparation of the CRISPR-Cas9/NulómergRNA vector for the loss of the chromosome 6 pair of hiPSCs/edited in the BAG2 and LSGN genes:
[00181] Numa forma de realização, para se conseguir a perda dirigida do par de cromossomas 6 das hiPSCs off-the-shelf, foi preparado um vetor CRISPR-Cas9/nulómerogARN. Este vetor compreende uma nucleasse CRISPR-Cas9 e nulómerogARN, que determina a clivagem do par de cromossomas 6 das hiPSCs/BAG2- LSGN editadas, flanqueando os centrómeros. A Seq. ID N° 42 foi utilizada como nulómero para a construção do vetor, e as Seq. ID N° 22 Seq. ID N° 23 como iniciadores para a modificação do plasmídio ADDGENE™ #71814 eCas9, capaz de produzir nuclease CRISPR/Cas9 e o sgRNA correspondente ao nulómero . [00181] In one embodiment, to achieve targeted loss of chromosome 6 pair of off-the-shelf hiPSCs, a CRISPR-Cas9/gRNA-nulomer vector was prepared. This vector comprises a CRISPR-Cas9 nuclease and gRNA nullomer, which cleaves chromosome 6 pair of edited hiPSCs/BAG2-LSGN, flanking the centromeres. The Seq. ID No. 42 was used as the nullomer for vector construction, and Seq. ID No. 22 Seq. ID No. 23 as primers for the modification of the ADDGENE™ plasmid #71814 eCas9, capable of produce CRISPR/Cas9 nuclease and the sgRNA corresponding to the nullomer.
[00182] Numa outra forma de realização, foram adquiridos a partir da indústria a nuclease CRISPR-Cas9,[00182] In another embodiment, CRISPR-Cas9 nuclease,
(ThermoFisher®) nuclease Cas9 (TrueCut Cas9 Protein v2® ThermoFisher®) e o nulómerogARN sintético (TrueGuide Synthetic gRNA® ThermoFisher®). (ThermoFisher®) Cas9 nuclease (TrueCut Cas9 Protein v2® ThermoFisher®) and the synthetic gRNA nullomer (TrueGuide Synthetic gRNA® ThermoFisher®).
[00183] Numa forma de realização, podem ser utilizadas as seguintes sequências de nulómeros, suas misturas, múltiplos ou frações, complementadas por uma sequência PAM do tipo NGG ou NGA 5' para CRISPR/Cas9: Seq. ID N° 24, Seq. ID N° 25, Seq. ID N° 26, Seq. ID N° 27, Seq. ID N° 28, Seq. ID N° 29, Seq. ID N° 30, Seq. ID N° 31, Seq. ID N° 32, Seq. ID N° 33, Seq. ID N° 34, Seq. ID N° 35, Seq. ID N° 36, Seq. ID N° 37, Seq. ID N° 38, Seq. ID N° 39, Seq. ID N° 40, Seq. ID N° 41, Seq. ID N° 42. Para outras nucleases do sistema CRISPR, a sequência PAM e a sua respetiva posição são variáveis, o que deve ser tomado em conta, se estiverem a ser utilizadas em vez da nuclease CRISPR/Cas9. [00183] In one embodiment, the following sequences of nucleomers, mixtures, multiples or fractions thereof, complemented by a PAM-like NGG or NGA 5' sequence for CRISPR/Cas9 can be used: Seq. ID No. 24, Seq. ID No. 25, Seq. ID No. 26, Seq. ID No. 27, Seq. ID No. 28, Seq. ID No. 29, Seq. ID No. 30, Seq. ID No. 31, Seq. ID No. 32, Seq. ID No. 33, Seq. ID No. 34, Seq. ID No. 35, Seq. ID No. 36, Seq. ID No. 37, Seq. ID No. 38, Seq. ID No. 39, Seq. ID No. 40, Seq. ID No. 41, Seq. ID No. 42. For other nucleases of the CRISPR system, the PAM sequence and its respective position are variable, which should be taken into account if they are being used instead of the CRISPR/Cas9 nuclease.
[00184] Numa forma de realização, a cassete 1 com o promotor PGK, plasmideo R52, compreende a Seq. ID N° 43, um construto com origem em ADN sintético. [00184] In one embodiment, cassette 1 with the PGK promoter, plasmid R52, comprises Seq. ID No. 43, a construct originating from synthetic DNA.
[00185] Numa outra forma de realização, a cassete 2 compreende a Seq.ID N° 44, que é um construto com origem em ADN sintético. [00185] In another embodiment, cassette 2 comprises Seq.ID No. 44, which is a construct originating from synthetic DNA.
Lista de Sequências: Sequence List:
Tabela 23: Sequências relativas ao gARN (ADN complementar)
Figure imgf000068_0001
Tabela 24: Sequências de iniciadores e Nulómeros.
Figure imgf000069_0001
Figure imgf000070_0001
Table 23: Sequences related to gRNA (complementary DNA)
Figure imgf000068_0001
Table 24: Sequences of Primers and Numomers.
Figure imgf000069_0001
Figure imgf000070_0001
SEQ ID N° 43 - SEQUÊNCIA DO VECTOR CASSETE 1 para a edição do Gene BAG2: cggatcctat agggttgaag ctttgagaga agcagcaact gctgttgagc aagagaaaga aatccttctg gaaatgatcc acagtatcca aaatagccag gacatgaggc agatcagtga cggtgagagc catctccaca gaaggggctc atcttttaca ctcctttaaa gagtttatga taagtgtggg tcaatttttg cttgactttt ggtatgcgta caccaggcca cagcatttaa tgtgagccac agagagcagc tgcagctgtc agttcattac agcagccact tactcagaga tttcttccag catgctctct accccaagcc cctggctagg agagatagga aaaagcctgc aagctcagaa aagctggccc caagcagtaa ggccatgtgt tttatcccag gttcacagtc ctaacttcag ttcctcacaa gttactggac tgcaagagat taaaaaagca gaggaagagg gtgagagagt ggaagcagag aagtagagac caaaaaaaaa aaaaaaaaaa agatgaattg aggtacaaca taaagagaag ggacaaaatg cagagaaaat gaggaaactt gtaggaccaa taaaaggcat gaatgtaaaa atgtgctgtc attttctttt ttcttttttt gagatggagt tttactcgtc acccaggctg gagtgcaatg gcgcgatctt ggctcactgc aacctctgcc tcctgggttg aagcgattct cctgcctcag cctcccgagt agctgggatt acaggcacgt gccaccacgc ctggctaatt tttatatttt tagtaaagac agggtttcac catgttggcc aagctggtct caaactcctg acctcaggtg atccgcctgc ctcagcctcc caaagtgctg ggattacagg catgtgacat ctgcctaacc tgtgctgtca ttttcatgtg aagaacatga aataggaata ggtcacttct gattttcttt tattggcact ggacttccta cttgggctcc tttggtgcca cacaggccac atggtactgg gccaagtggc tggtgaggta agtaagcaac ctaaatccag atcaaaactg ttacacatct ataggtaggc catgatctag tctctacgct atcctgccca aagatcaatg aattaggaag ttacagaagc aggagcttgc tgactttctc atataaataa tgaaatgttg atagagctcc tcgtgctggc caagacttta gaccactgtc ctgcttctat ctggagaagg atgaagtcag tgcctagtat tgacgacaga agacagctta tcttctatcg tggctacatt ttctcaacca aaatttgggg cattgtattc tgataaatgc acatgtcgtt ctggagagat ctgggacatc tgatctctgg agctctaccc agcataaacc ctatatgtgc ttactgctca cagtgttaag acttgatctc catcccagtt acaaatgcat acctacccaa acttttgcag acatgccaat ttgagaagtc atctggtatc ccatatatgc agattgtcag gaagtggtat tctacaacct ctttctgtaa tctaatacat ttcatcagtt aatcttacat gctatagttt cagcttttat ctctaaaaaa atttcatggc agaggtaaag aaaataataa atttagtcac aaacaaatgc cttaatgttt ttgacacaga taagtttaca tctcatattg atttttagga gaaagagaag aattaaatct gactgcaaac cgttgaattc gtcgacctcg aaattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg acctgcatcc atctagatcc gtcgacaaaa tctatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaatat ctagagcggg atgcagaaat tgatgatcta ttaaacaata aagatgtcca ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag agtacctaca ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag ataaatgcct gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt tggatatcat aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca tgatctatag atctatagat ctctcgtggg atcattgttt ttctcttgat tcccactttg tggttctaag tactgtggtt tccaaatgtg tcagtttcat agcctgaaga acgagatcag cagcctctgt tccacataca cttcattctc agtattgttt tgccaagttc taattccatc agaagctggg tacgctcgga ccgacgtatc ggatgcggcc gctgatggga agaactctca ccgttgaagt gtcagtagaa acaattagaa acccccagca gcaagaatcc ctaaagcatg ccacaaggat tattgatgag gtggtcaata agtttctgga tgatttggga aatgccaaga gtcatttaat gtcgctctac agtgcatgtt catctgaggt gccacatggg ccagttgatc agaagtttca atccatagta attggctgtg ctcttgaaga tcagaagaaa attaagagaa gattagagac tctgcttaga aatattgaaa actctgacaa ggccatcaag ctattagagc attctaaagg agctggttcc aaaactctgc aacaaaatgc tgaaagcaga ttcaattagt cttcaaacct aagagcattt acacaataca caaggtgtaa aaatgataaa atactatttt aattgataac tagttctttg ttaggtataa ccacttagtt gacactgata gttgtttcag atgaggaaaa tattccatca agtatcttca gttttgtgaa taacaaaact agcaatattt taattatcta tctagagatt ttttagattg aattcttgtc ttgtactagg atctagcata tttcactatt ctgtggatga atacatagtt tgtggggaaa acaaacgttc agctaggggc aaaaagcatg actgcttttt cctgtctggc atggaatcac gcagtcacct tgggcattta gtttactaga aattctttac cttaagcagc acacacattt actacacaca cagtgttaac aaagcactgt gcttagaggg taaaaaggaa tcacaaaaca agaatctttc caaagttgtc tcattcagca atgttaaggc atctgtatca aattattttg gatgtaaaga ttcctgtgtc tcataatatg aatgtatttt ttgatataca agaaactgac ataaaatgtg agaaaaccac ctataattta ccactgtgaa caattatata tctatctgct tcatcttttc tcaaatgcat caattctcta aaattcctat attgtaactt gccttttttt aaaaaagtta gatgctgata taaagtctgc ttaattgtca acttaatgag ctctattttg tgtagttata tctttatcca ttcctctttt atggacattt aggttgtttc caacttgttg ctattactgc aacatatttt tgtacacagg actttttcct tctttcattt ttgtttttct ctgtataaag gcccagcagt gaattatatt gggtcaaagg atatagacgt tttcatggcc tcacacatat taaacttttt ttgtataaag gttacagcaa tttatacttt tttcagtaat taaaatatag ctatttcact gaaatatttc cagcactgag cattaatacc tagtttgcat tttgtttact aaaaaggttg accagtgtgt tgattcttct tttatctgat aaagtgtgaa gcaactagag aacacttatt tgttcaaagt aactagtcct attgatatac aaaaaccaca acaacttccc tggatactat tttg SEQ ID N° 43 - CASSETTE 1 VECTOR SEQUENCE for BAG2 Gene Edition: cggatcctat agggttgaag ctttgagaga agcagcaact gctgttgagc aagagaaaga aatccttctg gaaatgatcc acagtatcca aaatagccag gacatgaggc agatcagtga cggtgagagc catctccaca gaaggggctc atcttttaca ctcctttaaa gagtttatga taagtgtggg tcaatttttg cttgactttt ggtatgcgta caccaggcca cagcatttaa tgtgagccac agagagcagc tgcagctgtc agttcattac agcagccact tactcagaga tttcttccag catgctctct accccaagcc cctggctagg agagatagga aaaagcctgc aagctcagaa aagctggccc caagcagtaa ggccatgtgt tttatcccag gttcacagtc ctaacttcag ttcctcacaa gttactggac tgcaagagat taaaaaagca gaggaagagg gtgagagagt ggaagcagag aagtagagac caaaaaaaaa aaaaaaaaaa agatgaattg aggtacaaca taaagagaag ggacaaaatg cagagaaaat gaggaaactt gtaggaccaa taaaaggcat gaatgtaaaa atgtgctgtc attttctttt ttcttttttt gagatggagt tttactcgtc acccaggctg gagtgcaatg gcgcgatctt ggctcactgc aacctctgcc tcctgggttg aagcgattct cctgcctcag cctcccgagt agctgggatt acaggcacgt gccaccacgc ctggctaatt tttatatttt tagtaaagac agggtttcac catgttggcc aagctggtct caaactcctg acctcaggtg atccgcctgc ctcagcctcc caaagtgctg ggattacagg catgtgacat ctgcctaacc tgtgctgtca ttttcatgtg aagaacatga aataggaata ggtcacttct gattttcttt tattggcact ggacttccta cttgggctcc tttggtgcca cacaggccac atggtactgg gccaagtggc tggtgaggta agtaagcaac ctaaatccag atcaaaactg ttacacatct ataggtaggc catgatctag tctctacgct atcctgccca aagatcaatg aattaggaag ttacagaagc aggagcttgc tgactttctc atataaataa tgaaatgttg atagagctcc tcgtgctggc caagacttta gaccactgtc ctgcttctat ctggagaagg atgaagtcag tgcctagtat tgacgacaga agacagctta tcttctatcg tggctacatt ttctcaacca aaatttgggg cattgtattc tgataaatgc acatgtcgtt ctggagagat ctgggacatc tgatctctgg agctctaccc agcataaacc ctatatgtgc ttactgctca cagtgttaag acttgatctc catcccagtt acaaatgcat acctacccaa acttttgcag acatgccaat ttgagaagtc atctggtatc ccatatatgc agattgtcag gaagtggtat tctacaacct ctttctgtaa tctaatacat ttcatcagtt aatcttacat gctatagttt cagcttttat ctctaaaaaa atttcatggc agaggtaaag aaaataataa atttagtcac aaacaaatgc cttaatgttt ttgacacaga taagtttaca tctcatattg atttttagga gaaagagaag aattaaatct gactgcaaac cgttgaattc gtcgacctcg aaattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg acctgcatcc atctagatcc gtcgacaaaa tctatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaatat ctagagcggg atgcagaaat tgatgatcta ttaaacaata aagatgtcca ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag agtacctaca ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag ataaatgcct gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt tggatatcat aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca tgatctatag atctatagat ctctcgtggg atcattgttt ttctcttgat tcccactttg tggttctaag tactgtggtt tccaaatgtg tcagtttcat agcctgaaga acgagatcag cagcctctgt tccacataca cttcattctc agtattgttt tgccaagttc taattccatc agaagctggg tacgctcgga ccgacgtatc ggatgcggcc gctgatggga agaactctca ccgttgaagt gtcagtagaa acaattagaa acccccagca gcaagaatcc ctaaagcatg ccacaaggat tattgatgag gtggtcaata agtttctgga tgatttggga aatgccaaga gtcatttaat gtcgctctac agtgcatgtt catctgaggt gccacatggg ccagttgatc agaagtttca atccatagta attggctgtg ctcttgaaga tcagaagaaa attaagagaa gattagagac tctgcttaga aatattgaaa actctgacaa ggccatcaag ctattagagc attctaaagg agctggttcc aaaactctgc aacaaaatgc tgaaagcaga ttcaattagt cttcaaacct aagagcattt acacaataca caaggtgtaa aaatgataaa atactatttt aattgataac tagttctttg ttaggtataa ccacttagtt gacactgata gttgtttcag atgaggaaaa tattccatca agtatcttca gttttgtgaa taacaaaact agcaatattt taattatcta tctagagatt ttttagattg aattcttgtc ttgtactagg atctagcata tttcactatt ctgtggatga atacatagtt tgtggggaaa acaaacgttc agctaggggc aaaaagcatg actgcttttt cctgtctggc atggaatcac gcagtcacct tgggcattta gtttactaga aattctttac cttaagcagc acacacattt actacacaca cagtgttaac aaagcactgt gcttagaggg taaaaaggaa tcacaaaaca agaatctttc caaagttgtc tcattcagca atgttaaggc atctgtatca aattattttg gatgtaaaga ttcctgtgtc tcataatatg aatgtatttt ttgatataca agaaactgac ataaaatgtg agaaaaccac ctataattta ccactgtgaa caattatata tctatctgct tcatcttttc tcaaatgcat caattctcta aaattcctat attgtaactt gccttttttt aaaaaagtta gatgctgata taaagtctgc ttaattgtca acttaatgag ctctattttg tgtagttata tctttatcca ttcctctttt atggacattt aggttgtttc caacttgttg ctattactgc aacatatttt tgtacacagg actttttcct tctttcattt ttgtttttct ctgtataaag gcccagcagt gaattatatt gggtcaaagg atatagacgt tttcatggcc tcacacatat taaacttttt ttgtataaag gttacagcaa tttatacttt tttcagtaat taaaatatag ctatttcact gaaatatttc cagcactgag cattaatacc tagtttgcat tttgtttact aaaaaggttg accagtgtgt tgattcttct tttatctgat aaagtgtgaa gcaactagag aacacttatt tgttcaaagt aactagtcct attgatatac aaaaaccaca acaacttccc tggatactat tttg
SEQ ID N° 44 - SEQUENCIA DO VECTOR CASSETE 2, para edição do GENE LGSN: agtcactggc caagaaagtg cattgtttat agagaattta aaaacaggcc aggtgaggtg gctcatgtct gtagcctgta atcccagcac tttggaaggc tgaggcaggt ggatcatttg aggtcaggag tttgagacca gaccggctaa catggtgaaa ccccatctct actagaaata taaaaaatta gcgtggcatg gtggcatgcc ctgtaaaccc agctactcag gaggctgagg caggaggatc acttgagccc aggaggcaga gattgcagtg agccaagatc atgccactgc actccagcct gggtgacaga gtgagactct gtctcaaaaa aataaattaa ttaattaaaa ataaataaat aaataataaa atctactaaa atctgactgc tttttattgt aaccatatgc tgggaattcc tactgtcctg ccttaggaca caatgattcc taggattgag cctgttgccc aaattaactt ctagttttag cagtgtctca catctgttga aatctagttg gagttccaaa atgtgttatg aaaacacctt ccataccttt cttatggccc ctcaaatcct ctgtaatgat ttttgtattc attttcagag cagaaaactg tctctcttcc tactcataca ttcctctgtt tgttcttttt tttttctact actgtcatcc taattctaac cctctttaca ttgtaaatat tttgattggt tatttttata tatttacgtt gtaagcttat atttgccatc ttttatgaca cttatgtagg aataactttt atttgtcctt ttgcttctcc caaaaaagaa atgaagatgt tttatgaaaa gaaagaccaa atggagattg cctctgacca caccattttt gaggactcag agtagttaga taccatttga attggacata tccgtttctc ccacttcagt tgaacaaaca tatggtttag tgactttctt ccttgtcctt cttaatgtgt tcatgctgtt ggcttcagtc tcattgcctt caggatcctc cctttatcca gccctcactc cttctctagg cgccggaatt cccagtccaa gctaggcagg ttcccctcta attaatgcag agactctaaa agaatttccc gggctcgggc agccattgtg atgcatatag gattattcac gtggtaatga gcacagtcga cagttcttgc tctcgagtag aatcgaattc ccagtccaag ctaggcaggt tcccctctaa ttaatgcaga gactctaaaa gaatttcccg ggctcgggca gccattgtga tgcatatagg attattcacg tggtaatgag cacagtcgac agttcttgct ctcgagtaga atcgaattcc cagtccaagc taggcaggtt cccctctaat taatgcagag actctaaaag aatttcccgg gctcgggcag ccattgtgat gcatatagga ttattcacgt ggtaatgagc acagtcgaca gttcttgctc tcgagtagaa tcgaattccc agtccaagct aggcaggttc ccctctaatt aatgcagaga ctctaaaaga atttcccggg ctcgggcagc cattgtgatg catataggat tattcacgtg gtaatgagca cagtcgacag ttcttgctct cgagtagaat cgaattctcc cctcccccag cctgaaacct gcttgctcag gggtggagct tcctgctcat tcgttctgcc acgcccactg ctggaacctg cggagccaca cccgtgcacc tttctactgg accagagatt attcggcggg aatcgggtcc cctccccctt ccttcataac tagtgtcgca actataaaat ttgagccttg atcagagtaa ctgtcttggc tacattcttt cttccgcccc gtctagattc ctctcttaca gctcgagcgg ccttctcagt cgaaccgttc acgttgcgag ctgctggcgg ccgcaacaag atctgcgatc taagtaagct tggcattccg gtactgttgg taaagccacc atgcccacgc tactgcgggt ttatatagac ggtccccacg ggatggggaa aaccaccacc acgcaactgc tggtggccct gggttcgcgc gacgatatcg tctacgtacc cgagccgatg acttactggc gggtgctggg ggcttccgag acaatcgcga acatctacac cacacaacac cgccttgacc agggtgagat atcggccggg gacgcggcgg tggtaatgac aagcgcccag ataacaatgg gaatgcctta tgccgtgacc gacgccgttc tggctcctca tatcgggggg gaggctggga gctcacatgc cccgcccccg gccctcaccc tcatcttcga ccgccatccc atcgccgccc tcctgtgcta cccggccgcg cgatacctta tgggcagcat gaccccccag gccgtgctgg cgttcgtggc cctcatcccg ccgaccttgc ccggcacaaa catcgtgttg ggggcccttc cggaggacag acacatcgac cgcctggcca aacgccagcg ccccggcgag cggcttgacc tggctatgct ggccgcgatt cgccgcgttt acgggctgct tgccaatacg gtgcggtatc tgcagggcgg cgggtcgtgg cgggaggatt ggggacagct ttcggggacg gccgtgccgc cccagggtgc cgagccccag agcaacgcgg gcccacgacc ccatatcggg gacacgttat ttaccctgtt tcgggccccc gagttgctgg cccccaacgg cgacctgtac aacgtgtttg cctgggcctt ggacgtcttg gccaaacgcc tccgtcccat gcacgtcttt atcctggatt acgaccaatc gcccgccggc tgccgggacg ccctgctgca acttacctcc gggatgatcc agacccacgt caccacccca ggctccatac cgacgatctg cgacctggcg cgcacgtttg cgcgggagat gggggaggct aactgacccg gggcatgcgc ggccgcaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttagt acgctcggac cgacgttatc gggggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggaattgc caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca acggagccgg agccattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc aatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggatc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgactcg aggggcgcgc ccccagctgg ttctttccgc ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtctcttg ttgagtcctg ttgataatta ataaacaaaa agccaaataa cagattattc aaacaatgat atttggatgt cgaaagagta tatttgaata gctcaatttt ttaatgagca tagaagactt ttataatgat gaaaattttg tcacttccac actcacctga ataactcaca atttaaaagc ccagcgttgt gctgataggc cagatagtct ctatctcctt ttatatgtga tacagaaaat atcattaagt aaattaaaat cgtgtctaat tactcaagat ttctatgttt attttttact ttgggaattt tatattattt attcctcaat aattttgcaa aacctatttt aactaaaaag tttacttttt atgatttcta ttttttagga tttctaacct ttttgtctga aggcataagc atctgaagaa ttgtattctc tgctctcttt ttccttatga aaaaggaaaa aattaataat gcaattaata attgcatcaa tatttacttc acttcaaaaa agtacttgaa ttcataaagt gctattttgt gactgtattt tatatgtgta aaacaaacat catatccaaa ccattttata gcttaagaaa atctaaggaa atgcattcaa aacaaaataa cctaactttc acaatctagt tataatcctt gacatgacag gatatgccca gcagggagct aggcagtgag gcacaggctg gtgctgggga gctcaggaat ctcagcacta acagtgcaca gagtcacccc tgcagtccca agacacaaaa gctgtcactg aaaaaaaata tgaaaaattt tgaaaaagct gacactggta tttttaaggt tttttacaaa cccctcatag tggtttgagg agagaggaaa gtgacagggt cagggtcaaa aagtatgctg taaacacttt gagcttagta tctttttgaa aagcaggctc cgagaggttt cacatctgtg ggcaagttat atgaatatat gttgagtaac tgctggggag cagctgtatc acttcatcta taaaaccaat tcattctccc aa [00186] A presente divulgação não é, naturalmente, de modo algum restrita às realizações descritas neste documento e uma pessoa com conhecimentos médios da área poderá prever muitas possibilidades de modificação da mesma e de substituições de caracteristicas técnicas por outras equivalentes, dependendo dos requisitos de cada situação, tal como definido nas reivindicações anexas. SEQ ID N° 44 - SEQUENCIA DO VECTOR CASSETE 2, para edição do GENE LGSN: agtcactggc caagaaagtg cattgtttat agagaattta aaaacaggcc aggtgaggtg gctcatgtct gtagcctgta atcccagcac tttggaaggc tgaggcaggt ggatcatttg aggtcaggag tttgagacca gaccggctaa catggtgaaa ccccatctct actagaaata taaaaaatta gcgtggcatg gtggcatgcc ctgtaaaccc agctactcag gaggctgagg caggaggatc acttgagccc aggaggcaga gattgcagtg agccaagatc atgccactgc actccagcct gggtgacaga gtgagactct gtctcaaaaa aataaattaa ttaattaaaa ataaataaat aaataataaa atctactaaa atctgactgc tttttattgt aaccatatgc tgggaattcc tactgtcctg ccttaggaca caatgattcc taggattgag cctgttgccc aaattaactt ctagttttag cagtgtctca catctgttga aatctagttg gagttccaaa atgtgttatg aaaacacctt ccataccttt cttatggccc ctcaaatcct ctgtaatgat ttttgtattc attttcagag cagaaaactg tctctcttcc tactcataca ttcctctgtt tgttcttttt tttttctact actgtcatcc taattctaac cctctttaca ttgtaaatat tttgattggt tatttttata tatttacgtt gtaagcttat atttgccatc ttttatgaca cttatgtagg aataactttt atttgtcctt ttgcttctcc caaaaaagaa atgaagatgt tttatgaaaa gaaagaccaa atggagattg cctctgacca caccattttt gaggactcag agtagttaga taccatttga attggacata tccgtttctc ccacttcagt tgaacaaaca tatggtttag tgactttctt ccttgtcctt cttaatgtgt tcatgctgtt ggcttcagtc tcattgcctt caggatcctc cctttatcca gccctcactc cttctctagg cgccggaatt cccagtccaa gctaggcagg ttcccctcta attaatgcag agactctaaa agaatttccc gggctcgggc agccattgtg atgcatatag gattattcac gtggtaatga gcacagtcga cagttcttgc tctcgagtag aatcgaattc ccagtccaag ctaggcaggt tcccctctaa ttaatgcaga gactctaaaa gaatttcccg ggctcgggca gccattgtga tgcatatagg attattcacg tggtaatgag cacagtcgac agttcttgct ctcgagtaga atcgaattcc cagtccaagc taggcaggtt cccctctaat taatgcagag actctaaaag aatttcccgg gctcgggcag ccattgtgat gcatatagga ttattcacgt ggtaatgagc acagtcgaca gttcttgctc tcgagtagaa tcgaattccc agtccaagct aggcaggttc ccctctaatt aatgcagaga ctctaaaaga atttcccggg ctcgggcagc cattgtgatg catataggat tattcacgtg gtaatgagca cagtcgacag ttcttgctct cgagtagaat cgaattctcc cctcccccag cctgaaacct gcttgctcag gggtggagct tcctgctcat tcgttctgcc acgcccactg ctggaacctg cggagccaca cccgtgcacc tttctactgg accagagatt attcggcggg aatcgggtcc cctccccctt ccttcataac tagtgtcgca actataaaat ttgagccttg atcagagtaa ctgtcttggc tacattcttt cttccgcccc gtctagattc ctctcttaca gctcgagcgg ccttctcagt cgaaccgttc acgttgcgag ctgctggcgg ccgcaacaag atctgcgatc taagtaagct tggcattccg gtactgttgg taaagccacc atgcccacgc tactgcgggt ttatatagac ggtccccacg ggatggggaa aaccaccacc acgcaactgc tggtggccct gggttcgcgc gacgatatcg tctacgtacc cgagccgatg acttactggc gggtgctggg ggcttccgag acaatcgcga acatctacac cacacaacac cgccttgacc agggtgagat atcggccggg gacgcggcgg tggtaatgac aagcgcccag ataacaatgg gaatgcctta tgccgtgacc gacgccgttc tggctcctca tatcgggggg gaggctggga gctcacatgc cccgcccccg gccctcaccc tcatcttcga ccgccatccc atcgccgccc tcctgtgcta cccggccgcg cgatacctta tgggcagcat gaccccccag gccgtgctgg cgttcgtggc cctcatcccg ccgaccttgc ccggcacaaa catcgtgttg ggggcccttc cggaggacag acacatcgac cgcctggcca aacgccagcg ccccggcgag cggcttgacc tggctatgct ggccgcgatt cgccgcgttt acgggctgct tgccaatacg gtgcggtatc tgcagggcgg cgggtcgtgg cgggaggatt ggggacagct ttcggggacg gccgtgccgc cccagggtgc cgagccccag agcaacgcgg gcccacgacc ccatatcggg gacacgttat ttaccctgtt tcgggccccc gagttgctgg cccccaacgg cgacctgtac aacgtgtttg cctgggcctt ggacgtcttg gccaaacgcc tccgtcccat gcacgtcttt atcctggatt acgaccaatc gcccgccggc tgccgggacg ccctgctgca acttacctcc gggatgatcc agacccacgt caccacccca ggctccatac cgacgatctg cgacctggcg cgcacgtttg cgcgggagat gggggaggct aactgacccg gggcatgcgc ggccgcaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttagt acgctcggac cgacgttatc gggggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggaattgc caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca acggagccgg agccattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc aatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggatc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgactcg aggggcgcgc ccccagctgg ttctttccgc ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtctcttg ttgagtcctg ttgataatta ataaacaaaa agccaaataa cagattattc aaacaatgat atttggatgt cgaaagagta tatttgaata gctcaatttt ttaatgagca tagaagactt ttataatgat gaaaattttg tcacttccac actcacctga ataactcaca atttaaaagc ccagcgttgt gctgataggc cagatagtct ctatctcctt ttatatgtga tacagaaaat atcattaagt aaattaaaat cgtgtctaat tactcaagat ttctatgttt attttttact ttgggaattt tatattattt attcctcaat aattttgcaa aacctatttt aactaaaaag tttacttttt atgatttcta ttttttagga tttctaacct ttttgtctga aggcataagc atctgaagaa ttgtattctc tgctctcttt ttccttatga aaaaggaaaa aattaataat gcaattaata attgcatcaa tatttacttc acttcaaaaa agtacttgaa ttcataaagt gctattttgt gactgtattt tatatgtgta aaacaaacat catatccaaa ccattttata gcttaagaaa atctaaggaa atgcattcaa aacaaaataa cctaactttc acaatctagt tataatcctt gacatgacag gatatgccca gcagggagct aggcagtgag gcacaggctg gtgctgggga gctcaggaat ctcagcacta acagtgcaca gagtcacccc tgcagtccca agacacaaaa gctgtcactg aaaaaaaata tgaaaaattt tgaaaaagct gacactggta tttttaaggt tttttacaaa cccctcatag tggtttgagg agagaggaaa gtgacagggt cagggtcaaa aagtatgctg taaacacttt gagcttagta tctttttgaa aagcaggctc cgagaggttt cacatctgtg ggcaagttat atgaatatat gttgagtaac tgctggggag cagctgtatc acttcatcta taaaaat tcattctccc aa [00186] The present disclosure is, of course, in no way restricted to the embodiments described in this document and a person with average knowledge of the area will be able to foresee many possibilities of modification of the same and of substitutions of technical characteristics for other equivalents, depending on the requirements of each situation, as defined in the appended claims.
[00187] O termo "compreende" ou "compreendendo" quando utilizado neste documento destina-se a indicar a presença das caracteristicas, elementos, inteiros, passos e componentes mencionados, mas não impede a presença ou a adição de uma ou mais outras caracteristicas, elementos, inteiros, passos e componentes, ou grupos dos mesmos. [00187] The term "comprises" or "comprising" when used in this document is intended to indicate the presence of the features, elements, integers, steps and components mentioned, but does not preclude the presence or addition of one or more other features, elements, integers, steps and components, or groups thereof.
[00188] As seguintes reivindicações definem realizações adicionais da presente descrição. [00188] The following claims define further embodiments of the present description.
Referências References
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• Acquisti C, Poste G, Curtiss D, Kumar S (2007) Nullomers: Really a Matter of Natural Selection, PLoS ONE 2(10): el022. doi:10.1371/journal.pone.0001022• Acquisti C, Poste G, Curtiss D, Kumar S (2007) Nullomers: Really a Matter of Natural Selection, PLoS ONE 2(10): el022. doi:10.1371/journal.pone.0001022
• Vergni D, Santoni D (2016) Nullomers and High Order Nullomers in Genomic Sequences, PLoS ONE 11(12): e0164540. doi:10.1371/journal.pone.0164540 • Vergni D, Santoni D (2016) Nullomers and High Order Nullomers in Genomic Sequences, PLoS ONE 11(12): e0164540. doi:10.1371/journal.pone.0164540
• B. Roberts, A. Haupt, et al. Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. Molecular Biology of the Cell Volume 28 October 15, 2017 • B. Roberts, A. Haupt, et al. Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. Molecular Biology of the Cell Volume 28 October 15, 2017
• Haupt, A., Grancharova, T., Arakaki, J., Fuqua, M.A.,• Haupt, A., Grancharova, T., Arakaki, J., Fuqua, M.A.,
Roberts, B., Gunawardane, R.N. Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9. J. Vis. Exp . (138), e58130, doi:10.3791/58130 (2018). Roberts, B., Gunawardane, R.N. Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9. J. Vis. Exp. (138), e58130, doi:10.3791/58130 (2018).
• R. E. K. FOURNIER AND F. H. RUDDLE, Microcell-mediated transfer of murine chromosomes into mouse, Chinese- hamster, and human somatic cells (somatic cell genetics/gene mapping) Proc. Nati. Acad. Sci. USA Vol. 74, No. 1, pp. 319-323, January 1977 Genetics • R. E. K. FOURNIER AND F. H. RUDDLE, Microcell-mediated transfer of murine chromosomes into mouse, Chinese-hamster, and human somatic cells (somatic cell genetics/gene mapping) Proc. native academy Sci. USA Vol. 74, No. 1, pp. 319-323, January 1977 Genetics
• Molecular Cloning: a laboratory manual/ 4th Edition; Tom Maniatis; 2012 Cold Spring Harbor Laboratory Press. • Molecular Cloning: a laboratory manual/ 4th Edition; Tom Maniatis; 2012 Cold Spring Harbor Laboratory Press.
• (2007) ABSENT SEQUENCES: NULLOMERS AND PRIMES, - GREG HAMPIKIAN and TIM ANDERSEN - Pacific Symposium on Biocomputing 12:355-366(2007) • (2007) ABSENT SEQUENCES: NULLOMERS AND PRIMES, - GREG HAMPIKIAN and TIM ANDERSEN - Pacific Symposium on Biocomputing 12:355-366(2007)
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DOI 10.1186/sl3059-017-1354-4 DOI 10.1186/sl3059-017-1354-4
• METHOD FOR REMOVING DESIRED CHROMOSOME AND TALOR-MADE MEDI CAL TREATMENT UTILIZING THE SAME; Patente N° US 2009/0264312 A1 de 22 de Outubro de 2009, inventores: Takashi Tada, Kyoto (JP); Norio Nakatsuji, Kyoto (JP); Hiroyuki Matsumura, Kyoto (JP); Masako Tada, Tokyo (JP) • METHOD FOR REMOVING DESIRED CHROMOSOME AND TALOR-MADE MEDI CAL TREATMENT UTILIZING THE SAME; US Patent No. 2009/0264312 A1 of October 22, 2009, inventors: Takashi Tada, Kyoto (JP); Norio Nakatsuji, Kyoto (JP); Hiroyuki Matsumura, Kyoto (JP); Masako Tada, Tokyo (JP)

Claims

R E I V I N D I C A Ç Õ E S
1. Método para a edição do genoma de células estaminais caracterizado por compreender os seguintes passos: preparar pelo menos uma cassete genética, em que a cassete genética compreende: um promotor; um marcador; e, uma sequência genética ausente do genoma das células estaminais a editar; em que o marcador compreende pelo menos um gene codificador de uma proteína de: fluorescência, resistência a antibiótico, sensibilidade a drogas, ou suas combinações; e em que a sequência genética ausente do genoma das células estaminais a editar é clivável por pelo menos um sistema de nucleases de edição genómica, de preferência o sistema de repetições palindrómicas curtas agrupadas e regularmente interespaçadas, o dedo de zinco, nucleases efetoras semelhantes a ativadores de transcrição, ou as suas misturas; inserir pelo menos uma cassete genética num cromossoma indígena da célula a editar por meio de clivagens da cadeia dupla do ADN perto e de cada lado do centrómero do cromossoma indígena da célula a editar; selecionar as células editadas com o marcador introduzido na referida cassete genética. 1. Method for editing the stem cell genome, characterized in that it comprises the following steps: preparing at least one gene cassette, wherein the gene cassette comprises: a promoter; a marker; and, a genetic sequence absent from the stem cell genome to be edited; wherein the marker comprises at least one gene encoding a protein of: fluorescence, antibiotic resistance, drug sensitivity, or combinations thereof; and wherein the genetic sequence absent from the genome of the stem cells to be edited is cleavable by at least one genomic editing nuclease system, preferably the system of regularly interspaced clustered short palindromic repeats, the zinc finger, activator-like effector nucleases transcription, or mixtures thereof; inserting at least one genetic cassette into an indigenous chromosome of the cell to be edited by means of double-stranded DNA cleavages near and on either side of the centromere of the indigenous chromosome of the cell to be edited; select cells edited with the marker introduced in said genetic cassette.
2. Método de acordo com a reivindicação anterior em que a sequência genética ausente do genoma das células estaminais a editar compreende um nulómero. A method according to the preceding claim, wherein the genetic sequence absent from the genome of the stem cells to be edited comprises a nullomer.
1 1
3. Método de acordo com a reivindicação anterior em que o nulómero compreende pelo menos uma sequência com pelo menos 90% de identidade das seguintes sequências: Seq. ID N° 24, Seq. ID N° 25, Seq. ID N° 26, Seq. ID N° 27, Seq. ID N° 28, Seq. ID N° 29, Seq. ID N° 30, Seq. ID N° 31, Seq. ID N° 32, Seq. ID N° 33, Seq. ID N° 34, Seq. ID N°A method according to the preceding claim wherein the nullomer comprises at least one sequence having at least 90% identity of the following sequences: Seq. ID No. 24, Seq. ID No. 25, Seq. ID No. 26, Seq. ID No. 27, Seq. ID No. 28, Seq. ID No. 29, Seq. ID No. 30, Seq. ID No. 31, Seq. ID No. 32, Seq. ID No. 33, Seq. ID No. 34, Seq. ID No.
35, Seq. ID N° 36, Seq. ID N° 37, Seq. ID N° 38, Seq. ID35, Seq. ID No. 36, Seq. ID No. 37, Seq. ID No. 38, Seq. ID
N° 39, Seq. ID N° 40, Seq. ID N° 41, Seq. ID N° 42 ou suas misturas, múltiplos ou frações. No. 39, Seq. ID No. 40, Seq. ID No. 41, Seq. ID No. 42 or mixtures, multiples or fractions thereof.
4. Método de acordo com qualquer uma das reivindicações anteriores 2-3 em que o nulómero compreende 95% de identidade com uma das seguintes sequências: Seq. ID N° 24, Seq. ID N° 25, Seq. ID N° 26, Seq. ID N° 27, Seq. IDA method according to any one of the preceding claims 2-3 wherein the numeromer comprises 95% identity to one of the following sequences: Seq. ID No. 24, Seq. ID No. 25, Seq. ID No. 26, Seq. ID No. 27, Seq. ID
N° 28, Seq. ID N° 29, Seq. ID N° 30, Seq. ID N° 31, Seq.No. 28, Seq. ID No. 29, Seq. ID No. 30, Seq. ID No. 31, Seq.
ID N° 32, Seq. ID N° 33, Seq. ID N° 34, Seq. ID N° 35, Seq. ID N° 36, Seq. ID N° 37, Seq. ID N° 38, Seq. ID N° 39, Seq. ID N° 40, Seq. ID N° 41, Seq. ID N° 42 ou suas misturas, múltiplos ou frações, de preferência 96%, 97%, 98%, 99% ou idênticas. ID No. 32, Seq. ID No. 33, Seq. ID No. 34, Seq. ID No. 35, Seq. ID No. 36, Seq. ID No. 37, Seq. ID No. 38, Seq. ID No. 39, Seq. ID No. 40, Seq. ID No. 41, Seq. ID No. 42 or mixtures thereof, multiples or fractions, preferably 96%, 97%, 98%, 99% or the like.
5. Método de acordo com qualquer uma das reivindicações anteriores, que compreende adicionalmente um passo de substituir o cromossoma indígena que compreende a cassete genética por um cromossoma equivalente exógeno não editado. A method according to any one of the preceding claims, further comprising a step of replacing the indigenous chromosome comprising the gene cassette with an equivalent unedited exogenous chromosome.
6. Método de acordo com qualquer uma das reivindicações anteriores, em que a substituição do cromossoma que compreende a cassete genética por um cromossoma equivalente não editado é feita por transferência cromossómica mediada por microcélulas. A method according to any one of the preceding claims, wherein the replacement of the chromosome comprising the gene cassette with an equivalent unedited chromosome is by microcell-mediated chromosomal transfer.
2 two
7. Método de acordo com qualquer uma das reivindicações anteriores em que o marcador codifica para uma proteína que confere sensibilidade a drogas, de preferência a tamoxifeno, aciclovir ou suas combinações. A method according to any one of the preceding claims wherein the marker encodes a protein which confers sensitivity to drugs, preferably to tamoxifen, acyclovir or combinations thereof.
8. Método de acordo com qualquer uma das reivindicações anteriores em que a edição do genoma da célula a editar compreende a deleção total de pelo menos um par de cromossomas. A method according to any one of the preceding claims, wherein editing the genome of the cell to be edited comprises the total deletion of at least one pair of chromosomes.
9. Método de acordo com qualquer uma das reivindicações anteriores, em que o cromossoma da célula a editar é o cromossoma 6. A method according to any one of the preceding claims, wherein the chromosome of the cell to be edited is chromosome 6.
10. Método de acordo com qualquer uma das reivindicações anteriores, em que as clivagens da cadeia dupla do ADN ocorrem pela adição de nucleases, de preferência nucleases do sistema de repetições palindrómicas curtas agrupadas e regularmente interespaçadas, dedo de zinco, nucleases efetoras semelhantes a ativadores de transcrição (TALENs), transposases, recombinases sítio- específicas de tirosina (T-SSRs), ou recombinases sítio- específicas de serina (S-SSRs). A method according to any one of the preceding claims, wherein the DNA double strand cleavages occur by the addition of nucleases, preferably clustered and regularly interspaced clustered short palindromic repeat system nucleases, zinc finger, activator-like effector nucleases (TALENs), transposases, site-specific tyrosine recombinases (T-SSRs), or site-specific serine recombinases (S-SSRs).
11. Cassete genética caracterizada por compreender pelo menos um promotor, um marcador, e uma sequência genética ausente do genoma das células estaminais a editar, em que o marcador compreende pelo menos um gene codificador de uma proteína de fluorescência, de resistência a antibiótico, de sensibilidade a drogas, ou suas combinações; e em que a sequência genética ausente do genoma das células estaminais a editar é clivável por pelo menos um sistema de nucleases de edição genómica, de preferência o sistema de repetições palindrómicas curtas 11. Genetic cassette characterized in that it comprises at least a promoter, a marker, and a genetic sequence absent from the genome of the stem cells to be edited, wherein the marker comprises at least one gene coding for a fluorescence, antibiotic resistance, drug sensitivity or combinations thereof; and wherein the genetic sequence absent from the genome of the stem cells to be edited is cleavable by at least one genomic editing nuclease system, preferably the short palindromic repeat system
3 agrupadas e regularmente interespaçadas, o dedo de zinco, nucleases efetoras semelhantes a ativadores de transcrição (TALENs), ou as suas misturas. 3 grouped and regularly interspaced, the zinc finger, transcriptional activator-like effector nucleases (TALENs), or mixtures thereof.
12. Cassete genética de acordo com a reivindicação anterior em que a sequência genética ausente do genoma das células estaminais a editar compreende um nulómero. A genetic cassette according to the preceding claim, wherein the genetic sequence absent from the stem cell genome to be edited comprises a nullomer.
13. Cassete genética de acordo com a reivindicação anterior em que o nulómero compreende pelo menos uma sequência com pelo menos 90% de identidade com as sequências:, Seq. ID N° 24, Seq. ID N° 25, Seq. ID N° 26, Seq. ID N° 27, Seq. ID N° 28, Seq. ID N° 29, Seq. ID N° 30, Seq. ID N° 31, Seq. ID N° 32, Seq. ID N° 33, Seq. ID N° 34, Seq. ID N° 35, Seq. ID N° 36, Seq. ID N° 37, Seq. ID N° 38, Seq. ID N° 39, Seq. ID N° 40, Seq. ID N° 41, Seq. ID N° 42 ou suas misturas, múltiplos ou frações. A genetic cassette according to the preceding claim wherein the nullomer comprises at least one sequence having at least 90% sequence identity:, Seq. ID No. 24, Seq. ID No. 25, Seq. ID No. 26, Seq. ID No. 27, Seq. ID No. 28, Seq. ID No. 29, Seq. ID No. 30, Seq. ID No. 31, Seq. ID No. 32, Seq. ID No. 33, Seq. ID No. 34, Seq. ID No. 35, Seq. ID No. 36, Seq. ID No. 37, Seq. ID No. 38, Seq. ID No. 39, Seq. ID No. 40, Seq. ID No. 41, Seq. ID No. 42 or mixtures, multiples or fractions thereof.
14. Cassete genética descrita em qualquer uma das reivindicações anteriores para uso em medicina ou medicina veterinária. The genetic cassette described in any one of the preceding claims for use in medicine or veterinary medicine.
15. Cassete genética de acordo com a reivindicação anterior para uso no tratamento de doenças genéticas, de preferência doenças monogénicas ou multigénicas, de maior preferência hemofilia, drepanocitose, talassémia, anemia de Fanconi, síndroma de Alagille, doença congénita da glicosilação, ou retinite pigmentosa. A genetic cassette according to the preceding claim for use in the treatment of genetic disorders, preferably monogenic or multigenic disorders, most preferably hemophilia, sickle cell disease, thalassemia, Fanconi anemia, Alagille syndrome, congenital glycosylation disease, or retinitis pigmentosa. .
16. Cassete genética de acordo com qualquer uma das reivindicações anteriores compreendendo pelo menos 90% de identidade com a Seq. ID N° 43 ou Seq. ID N° 44. A genetic cassette according to any preceding claim comprising at least 90% identity to Seq. ID No. 43 or Seq. ID No. 44.
4 4
17. Cassete genética de acordo com qualquer uma das reivindicações anteriores compreendendo pelo menos 95% de identidade com Seq. ID N° 43 ou Seq. ID N° 44. A genetic cassette according to any preceding claim comprising at least 95% identity to Seq. ID No. 43 or Seq. ID No. 44.
18. Cassete genética de acordo com qualquer uma das reivindicações anteriores compreendendo pelo menos 99% de identidade com Seq. ID N° 43 ou Seq. ID N° 44. A genetic cassette according to any preceding claim comprising at least 99% identity to Seq. ID No. 43 or Seq. ID No. 44.
19. Vetor caracterizado por compreender pelo menos uma cassete genética descrita em qualquer uma das reivindicações anteriores. 19. Vector characterized in that it comprises at least one genetic cassette described in any one of the preceding claims.
20. Células estaminais editadas obtidas pelo método descrito nas reivindicações 1-10 que compreendem a cassete genética descrita nas reivindicações 11-18. Edited stem cells obtained by the method described in claims 1-10 which comprise the genetic cassette described in claims 11-18.
21. Células de acordo com qualquer uma das reivindicações anteriores que compreendem pelo menos a substituição de um cromossoma indígena por um cromossoma exógeno, sem compreender resíduos do cromossoma indígena. Cells according to any one of the preceding claims which comprise at least the replacement of an indigenous chromosome with an exogenous chromosome, not comprising residues from the indigenous chromosome.
22. Célula, micro-tecido, tecido ou órgão obtido a partir da diferenciação das células estaminais editadas descritas em qualquer uma das reivindicações anteriores 20-21. 22. Cell, microtissue, tissue or organ obtained from the differentiation of the edited stem cells described in any one of the preceding claims 20-21.
23. Célula, micro-tecido, tecido ou órgão de acordo com a reivindicação anterior para o uso em medicina ou medicina veterinária. A cell, microtissue, tissue or organ according to the preceding claim for use in medicine or veterinary medicine.
24. Célula, micro-tecido, tecido ou órgão de acordo com qualquer uma das reivindicações 22-23 para uso no tratamento de doenças genéticas, de preferência doenças monogénicas ou multigénicas, de maior preferência hemofilia, drepanocitose, talassémia, anemia de Fanconi, A cell, microtissue, tissue or organ according to any one of claims 22-23 for use in the treatment of genetic diseases, preferably monogenic or multigenic diseases, most preferably hemophilia, sickle cell disease, thalassemia, Fanconi anemia,
5 síndroma de Alagille, doença congénita da glicosilação, ou retinite pigmentosa. 5 Alagille syndrome, congenital glycosylation disease, or retinitis pigmentosa.
25. Célula, micro-tecido, tecido ou órgão de acordo com qualquer uma das reivindicações 22-23 para uso no tratamento de doenças genéticas, de preferência nos equinos, Hemofilia A, Trombastenia de Glanzmann, Trombastenia Atípica Equina, Doença de von Willebrand, Deficiência de pre-calicreína, Miopatia do armazenamento de polissacaridos Tipo 1 e Tipo2, Deficiência da Enzima de Ramificação do Glicogénio, ou Miopatia das cadeias pesadas 2X da miosina, em equinos; Deficiência da beta- manosidase, Deficiência da adesão leucocitária bovina, Deficiência hereditária do Zinco, ou Citrulémia, em bovinos; Hemofilia A e Doença de von Willebrand, em caninos. A cell, microtissue, tissue or organ according to any one of claims 22-23 for use in the treatment of genetic diseases, preferably in horses, Hemophilia A, Glanzmann's Thrombasthenia, Equine Atypical Thrombasthenia, von Willebrand's Disease, Prekallikrein Deficiency, Type 1 and Type 2 Polysaccharide Storage Myopathy, Glycogen Branching Enzyme Deficiency, or Myosin 2X heavy chain myopathy, in horses; beta-mannosidase deficiency, bovine leukocyte adhesion deficiency, hereditary zinc deficiency, or citrulemia in cattle; Hemophilia A and von Willebrand Disease, in canines.
6 6
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