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EP-4476353-B1 - METHOD FOR THE MANUFACTURE OF A VIRAL SYSTEM, A VECTOR SYSTEM OR ANY TRANSPORT SYSTEM FOR CANCER-SPECIFIC CRISPR COMPLEXES

EP4476353B1EP 4476353 B1EP4476353 B1EP 4476353B1EP-4476353-B1

Inventors

  • WILKENS, BODO
  • PATNAIK, Sarita
  • HESHMATPOUR, Najmeh
  • Macarrón Palacios, Arturo
  • KORUS, Patrick

Dates

Publication Date
20260506
Application Date
20230324

Claims (9)

  1. Method for the manufacture of individualized CRISPR/Cas complexes comprising the steps a) identifying in a tumor specimen of a human cancer patient at least one of the following cancer specific mutations: mutation at position 127736999 on chromosome 8, mutation at position 37020625 on chromosome 9 and mutation at position 36840626 on chromosome 9 b) preparing for the at least one mutation identified in a), preferably for two or all mutations identified in a), an individualized CRISPR/Cas complex, wherein each individualized CRISPR/Cas complex comprises a guide RNA and a Cas endonuclease, wherein the guide RNA for the individualized CRISPR/Cas complex for the mutation at position 127736999 on chromosome 8 comprises a crRNA according to SEQ ID NO 37 and a tracrRNA, wherein the guide RNA for the individualized CRISPR/Cas complex for the mutation at position 37020625 on chromosome 9 comprises a crRNA according to SEQ ID NO 38 and a tracrRNA, and wherein the guide RNA for the individualized CRISPR/Cas complex for the mutation at position 36840626 on chromosome 9 comprises a crRNA according to SEQ ID NO 39 and a tracrRNA, wherein the tumor specimen is from B-cell-lymphocytes.
  2. Method according to claim 1, wherein two or all of the mutations according to a) are identified and wherein two or all individualized CRISPR/Cas complexes according to b) are prepared.
  3. Method according to claim 1 or 2, wherein each guide RNA is directed against the nucleic acid sequence that is specific for the mutation in cancerous or pre-cancerous cells.
  4. Method according to any of the preceding claims, wherein the Cas endonuclease is a type I, type II or type III endonuclease, preferably a type I endonuclease, most preferably a CRISPR/Cas9 endonuclease.
  5. In-vitro method for inducing cell death in cancerous or pre-cancerous cells of B-cell-lymphocytes, comprising the steps: a) isolation of B-cell-lymphocytes from one or more specimen obtained from a human cancer patient or a human subject supposed to have or to develop cancer, whereby non-cancerous B-cell-lymphocytes and cancerous or pre-cancerous B-cell-lymphocytes are obtained; b) cultivation, preferably in-vitro cultivation, of the B-cell-lymphocytes from the one or more specimen obtained in a); c) identifying in the cancerous or pre-cancerous B-cell-lymphocytes at least one of the following cancer specific mutations: mutation at position 127736999 on chromosome 8, mutation at position 37020625 on chromosome 9 and mutation at position 36840626 on chromosome 9 d) preparing an individualized CRISPR/Cas complex for each of the cancer specific mutations identified in c) according to step b) of a method for the manufacture of individualized CRISPR/Cas complexes according to at least one of claims 1 to 4, e) adding the one ore more prepared individualized CRISPR/Cas complexes to the cultivated B-cell-lymphocytes obtained in step b), and f) optionally, determining if cell death is induced in the cancerous or pre-cancerous B-cell-lymphocytes of said cultivated B-cell-lymphocytes and preferably determining if cell death is not induced in the non-cancerous B-cell-lymphocytes of said cultivated B-cell-lymphocytes.
  6. Composition comprising one or more individualized CRISPR/Cas complexes manufactured according to the method of any of claims 1 to 4.
  7. Individualized CRISPR/Cas complex comprising a guide RNA and a Cas endonuclease, wherein the guide RNA comprises a crRNA according to SEQ ID NO 37 and a tracrRNA, wherein the guide RNA comprises a crRNA according to SEQ ID NO 38 and a tracrRNA, or wherein the guide RNA comprises a crRNA according to SEQ ID NO 39 and a tracrRNA.
  8. An individualized CRISPR/Cas complex manufactured according to the method of any of claims 1 to 4, a composition according to claim 6 or a CRISPR/Cas complex according to claim 7, for use in the treatment of B-cell-lymphoma.
  9. An individualized CRISPR/Cas complex manufactured according to the method of any of claims 1 to 4, a composition according to claim 6 or a CRISPR/Cas complex according to claim 7, for use in inducing cell death in cancerous or pre-cancerous B-cell-lymphocytes.

Description

BACKGROUND The present invention relates to a method for the manufacture of individualized CRISPR/Cas complexes according to the claims comprising the steps a) identifying in a tumor specimen of a human cancer patient at least one of the following cancer specific mutations: mutation at position 127736999 on chromosome 8, mutation at position 37020625 on chromosome 9 and mutation at position 36840626 on chromosome 9; b) preparing for the at least one mutation identified in a), preferably for two or all mutations identified in a), an individualized CRISPR/Cas complex, wherein each individualized CRISPR/Cas complex comprises a guide RNA and a Cas endonuclease, wherein the guide RNA for the individualized CRISPR/Cas complex for the mutation at position 127736999 on chromosome 8 comprises a crRNA according to SEQ ID NO 37 and a tracrRNA, wherein the guide RNA for the individualized CRISPR/Cas complex for the mutation at position 37020625 on chromosome 9 comprises a crRNA according to SEQ ID NO 38 and a tracrRNA, and wherein the guide RNA for the individualized CRISPR/Cas complex for the mutation at position 36840626 on chromosome 9 comprises a crRNA according to SEQ ID NO 39 and a tracrRNA; an in-vitro method, for inducing cell death in cancerous or pre-cancerous cells of B-cell-lymphocytes, a composition related thereto and individualized CRISPR/Cas complexes preferably for use in the treatment of B-cell-lymphoma or for use in inducing cell death in cancerous or pre-cancerous B-cell-lymphocytes. According to the World Health Organization (WHO), cancer represents nowadays the second leading cause of death globally. Despite improving comprehensive understanding of the pathomechanisms and the rapid advances in treatment over the last decades, cancer is with roughly 19.3 million new cancer cases and almost 10.0 million cancer-related deaths in 2020, the second leading cause of mortality and morbidity globally after cardiovascular diseases (Sung et al., 2021). Considering the rising world population and the annual cost of cancer care, cancer represents an evident health burden. Cancer is a highly heterogeneous disease generally based on a complex process of progressive accumulation of genetic and molecular changes that result in abnormal growth of cells. During tumorigenesis, healthy cells successively acquire hallmark features including resistance to cell death, sustained proliferative signaling, evasion of growth suppressors, replicative immortality, induction of angiogenesis, and activation of invasion and metastasis (Hanahan & Weinberg, 2011). Depending on their origin, most cancer types can be broadly classified in mesenchymal, epithelial, neuroectodermal, and hematopoietic. Hematological neoplasms may be further divided into lymphomas, leukemias, and myelomas, whereupon plenty further subtypes differing in cellular lineage, characteristics, response rate to therapy, and prognosis have been identified (Weinberg RA, 2014). Within the plethora of genetic alterations known to be associated with hematological malignancies, few aberrations in particular genomic regions have been identified to be recurrent among multiple cancer subtypes. B cell lymphomas, a major subclass of lymphoma, represent around 5% of all newly diagnosed malignancies. This disease comprises a large variety of subtypes, including mantle cell lymphoma, Burkitt's lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, or multiple myeloma among others, depending on the stage of development of the B cell. Current standard therapies against lymphomas and several other cancers comprise a variety of available treatments such as surgery, chemotherapy, radiation therapy, drug treatment therapy or immunotherapy among others. In general, remarkable advancements have been made during the last decades in the management of hematological malignancies. In spite of varying effectivity depending on the cancer subtype, large responsiveness has been observed for several lymphomas and leukemias upon combination of bone marrow transplantation and targeted chemotherapy. Notwithstanding initially high response rates, many patients treated with conventional strategies suffer disease relapse upon treatment interruption. This may be due to incomplete cancer remission, whereat remaining malignant cells proliferate after time, thus leading to relapse, resistance and poor prognosis (Shankland et al., 2012). In addition, conventional therapies are often accompanied by severe side effects and physical pain derived from induction of high levels of cytotoxicity in healthy, mostly rapidly proliferating cells such as cells of the digestive tract, hair follicles, and bone marrow. Alternative therapies to chemotherapy such as targeted immunotherapy have rapidly expanded since the development of the hybridoma technology in 1975. Immunotherapy takes advantage of specific cellular receptors that are overexpressed on the surface of cancer cells. Antibodies significantly en