Patent Application
20220244244
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 18, 2020, is named 56322-701_601_SL.txt and is 732,058 bytes in size.
Human cancers are extraordinarily heterogeneous, differing in DNA sequence, epigenomic landscape, RNA expression, and protein levels, resulting in vast combinatorial complexity in cell behavior. Despite impressive advances in our armamentarium of molecularly targeted anti-cancer therapies, the extreme molecular complexity underlying cancer cell behavior has led to dramatic shortfalls in our ability to predict which patients will benefit from any particular therapy. The lack of an effective means of predicting patient response directly leads to cycles of futile therapy, at enormous opportunity cost to patients and economic cost to both patients and healthcare payers.
The presently disclosed methods seek to provide a rational and personalized selection of therapeutics by determining which molecularly targeted therapy would be effective for a particular patient's disease. In one aspect, the methods comprise determining the functional susceptibility of a patient's cancer cells to a library of perturbagens which model the action of a library of known oncology drugs. Representative perturbagens include components of a gene editing or silencing system capable of knocking out, or knocking down, the genes encoding for the protein targets of the known oncology drugs. For instance, the perturbagens may include gene modulatory reagents such as guide RNA sequences for CRISPR-based gene editing, or RNAi for gene silencing. Accordingly, an exemplary method of functional susceptibility profiling comprises modifying a patient's cancer cells with a library of gene modulatory reagents capable of knocking down, or knocking out, the function of the genes encoding for protein targets of a library of known oncology drugs. In some methods the functionality of all such genes is knocked down or knocked out such that the susceptibility of a patient's cancer to all available molecularly targeted therapies may be interrogated. The modified cancer cells may be screened by next-generation sequencing to determine the effect of the individual genetic perturbations on the viability of the patient's cancer cells. Oncology drugs associated with the perturbagens that reduce viability of the cancer cells may be selected as a putative therapeutic, allowing for personalized selection of a cancer therapeutic.
In one aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutic molecule selected from a library of therapeutic molecules; wherein the therapeutic molecule has been selected by a method comprising: modifying cancer cells from the subject to knock down or knock out the function of a plurality of genes, each gene in the plurality of genes encoding for a protein target of a therapeutic molecule in the library of therapeutic molecules, whereby the therapeutic molecule has been selected if knocking down or knocking out the function of the gene that encodes for the protein target of the selected therapeutic molecule impairs cancer cell viability. In some embodiments, the library of therapeutic molecules comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 therapeutic agents of Table 2. In some embodiments, the library of therapeutic molecules comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 therapeutic agents of Table 3. In some embodiments, one or more of the plurality of genes encode for a protein of Table 5B. In some embodiments, one or more of the plurality of genes encode for a protein of Table 5A. In some embodiments, one or more of the plurality of genes encode for a protein of Table 5C. In some embodiments, one or more of the plurality of genes encode for a protein of Table 5D. In some embodiments, one or more of the plurality of genes encode for a protein of Table 4. In some embodiments, one or more of the plurality of genes encode for a protein of Table 3.
In another aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutic molecule selected from a library of therapeutic molecules; wherein the cancer of the subject has been determined to be susceptible to the selected therapeutic molecule by a method comprising: (a) contacting a sample of cancer cells from the subject with a library of gene modulatory reagents to generate a plurality of modified cancer cells, wherein each modified cancer cell harbors one or more of the gene modulatory reagents, and each gene modulatory reagent is capable of knocking down or knocking out the function of a gene that encodes a protein target of a therapeutic molecule in the library of therapeutic molecules, and (b) sequencing the plurality of modified cancer cells, wherein a gene modulatory reagent that impairs cell viability will have fewer sequence reads than a gene modulatory reagent that does not impair cell viability, and the gene that is knocked down or knocked out by the gene modulatory reagent that impairs cell viability encodes for the protein targeted by the selected therapeutic molecule. In some embodiments, prior to sequencing, one or more of the plurality of modified cancer cells have been propagated. In some embodiments, propagation comprises maintenance of the modified cancer cells in a 2D in vitro culture. In some embodiments, propagation comprises maintenance of the modified cancer cells in a 3D in vitro culture. In some embodiments, propagation comprises maintenance of the modified cancer cells in vivo. In some embodiments, propagation occurs within an animal model. In some embodiments, the animal is a rodent. In some embodiments, the cancer cells are primary cancer cells.
In some embodiments, contacting comprises introducing the one or more gene modulatory reagents into each cancer cell by a viral or non-viral delivery method. In some embodiments, one or more of the gene modulatory reagents in the library are encoded on a viral vector. In some embodiments, the viral vector comprises a lentiviral vector, adenoviral vector, or adeno-associated viral vector. In some embodiments, the non-viral delivery method comprises transposase-mediated transposition.
In some embodiments, the library comprises from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different gene modulatory reagents. In some embodiments, one or more gene modulatory reagents from the library of gene modulatory reagents comprises a nucleic acid sequence homologous to at least about 15 contiguous nucleotides of a gene encoding a protein of Tables 3-5D. In some embodiments, one or more gene modulatory reagents from the library of gene modulatory reagents comprises a nucleic acid sequence homologous to at least about 15 contiguous nucleotides of a gene encoding a protein of Table 5B. In some embodiments, one or more gene modulatory reagents from the library of gene modulatory reagents comprises a nucleic acid sequence homologous to at least about 15 contiguous nucleotides of a gene encoding a protein of Table 5C. In some embodiments, one or more gene modulatory reagents from the library of gene modulatory reagents comprises a nucleic acid sequence homologous to at least about 15 contiguous nucleotides of a gene encoding a protein of Table 5D. In some embodiments, the homology is at least about 90% sequence homology. In some embodiments, the homology is at least about 90% sequence identity. In some embodiments, the library of therapeutic molecules comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 therapeutic agents of Table 2. In some embodiments, the library of therapeutic molecules comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 therapeutic agents of Table 3.
In some embodiments, the cancer comprises at least one cancer chosen from the group comprising acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, central nervous system cancers, basal cell carcinoma of the skin, bile duct cancer, bladder cancer, bone cancer, brain tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, cardiac tumors, central nervous system cancers, embryonal tumors, germ cell tumor, primary CNS lymphoma, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumors, central nervous system cancer, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumors, hepatocellular cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (non-small cell and small cell), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous neck cancer with occult primary, midline tract carcinoma with NUT gene changes, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, myelogenous leukemia, chronic (CML), myeloid leukemia, acute (AML), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, rhabdomyosarcoma, vascular tumors, osteosarcoma, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer, small intestine cancer, squamous cell carcinoma of the skin, squamous neck cancer with occult primary, stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, ureter and renal pelvis, urethral cancer, uterine cancer, endometrial, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, Wilms tumor, and other childhood kidney tumors.
In some embodiments, one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. In some embodiments, one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1526-2789. In some embodiments, one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 2980-3071. In some embodiments, the at least about 90% homology is at least about 90% identity. In some embodiments, one or more of the gene modulatory reagents comprise a guide RNA (gRNA) sequence comprising homology to at least a portion of the gene that encodes a protein target of a therapeutic molecule in the library of therapeutic molecules. In some embodiments, the gRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene. In some embodiments, the homology is at least about 90% sequence homology. In some embodiments, the homology is at least about 90% sequence identity. In some embodiments, the sample of cancer cells is contacted with an endonuclease. In some embodiments, the endonuclease comprises a Cas9 or Cas12a endonuclease. In some embodiments, the Cas9 or Cas12a endonuclease is selected from S. pyogenes Cas9 (SpCas9), SpCas9 D1135E variant, SpCas9 VRER variant, SpCas9 EQR variant, xCas9, SpCas9-NG, S. aureus Cas9 (SaCas9), Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LbCpfl), AsCpfl RR variant, LbCpfl RR variant, AsCpfl RVR variant, C. jejuni Cas9 (Cj Cas9), N meningitidis (NmCas9), S. thermophilus (StCas9), T. denticola (TdCas9), and Mad7. In some embodiments, the endonuclease does not comprise a Cas9 or Cas12a endonuclease. In some embodiments, the gRNA is positioned within a vector. In some embodiments, the vector further comprises genetic elements of a virus. In some embodiments, the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof In some embodiments, the vector further comprises an auxiliary nucleic acid sequence. In some embodiments, the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, and surface epitope expression cassette. In some embodiments, the marker is a fluorescent marker. In some embodiments, the auxiliary nucleic acid allows for the selection of cancer cells that have been modified to harbor the one or more gene modulatory reagents.
In some embodiments, one or more of the gene modulatory reagents comprise a short hairpin RNA (shRNA) sequence comprising homology to at least a portion of the gene that encodes a protein target of a therapeutic molecule in the library of therapeutic molecules. In some embodiments, the shRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene. In some embodiments, the homology is at least about 90% sequence homology. In some embodiments, the homology is at least about 90% sequence identity. In some embodiments, the shRNA is positioned within a vector. In some embodiments, the vector further comprises genetic elements of a virus. In some embodiments, the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof. In some embodiments, the vector further comprises an auxiliary nucleic acid sequence. In some embodiments, the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette. In some embodiments, the marker is a fluorescent marker. In some embodiments, the auxiliary nucleic acid allows for the selection of cancer cells that have been modified to harbor the one or more gene modulatory reagents.
In another aspect, provided herein is a method of generating a plurality of modified cancer cells from a subject having cancer, the method comprising delivering a library of gene modulatory reagents to a sample of cancer cells from the subject to generate the plurality of modified cancer cells; wherein each modified cancer cell harbors one or more of the gene modulatory reagents, and each gene modulatory reagent is capable of knocking down or knocking out the function of a gene that encodes a protein target from a library of protein targets. In some embodiments, one or more of the gene modulatory reagents comprises a guide RNA (gRNA) sequence comprising homology to at least a portion of the gene whose function is knocked down or knocked out in the modified cancer cell. In some embodiments, the gRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene. In some embodiments, the homology is at least about 90% sequence homology. In some embodiments, the homology is at least about 90% sequence identity. In some embodiments, one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. In some embodiments, one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1526-2789. In some embodiments, one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 2980-3071. In some embodiments, the homology is at least about 90% identity. In some embodiments, the sample of cancer cells is contacted with an endonuclease. In some embodiments, the endonuclease comprises a Cas9 or Cas12a endonuclease. In some embodiments, the Cas9 or Cas12a endonuclease is selected from S. pyogenes Cas9 (SpCas9), SpCas9 D1135E variant, SpCas9 VRER variant, SpCas9 EQR variant, xCas9, SpCas9-NG, S. aureus Cas9 (SaCas9), Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LbCpfl), AsCpfl RR variant, LbCpfl RR variant, AsCpfl RVR variant, C. jejuni Cas9 (Cj Cas9), N. meningitidis (NmCas9), S. thermophilus (StCas9), T. denticola (TdCas9), and Mad7. In some embodiments, the endonuclease does not comprise a Cas9 or Cas12a endonuclease. In some embodiments, the gRNA is positioned within a vector. In some embodiments, the vector further comprises genetic elements of a virus. In some embodiments, the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof In some embodiments, the vector further comprises an auxiliary nucleic acid sequence. In some embodiments, the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette. In some embodiments, the marker is a fluorescent marker. In some embodiments, the auxiliary nucleic acid allows for the selection of cancer cells that have been modified to harbor the one or more gene modulatory reagents.
In some embodiments, one or more of the gene modulatory reagents comprise a short hairpin RNA (shRNA) sequence comprising homology to at least a portion of the gene whose function is knocked down or knocked out in the modified cancer cell. In some embodiments, the shRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene. In some embodiments, the homology is at least about 90% sequence homology. In some embodiments, the homology is at least about 90% sequence identity. In some embodiments, the shRNA is positioned within a vector. In some embodiments, the vector further comprises genetic elements of a virus. In some embodiments, the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof. In some embodiments, the vector further comprises an auxiliary nucleic acid sequence. In some embodiments, the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette. In some embodiments, the marker is a fluorescent marker. In some embodiments, the auxiliary nucleic acid allows for the selection of cancer cells that have been modified to harbor the one or more gene modulatory reagents.
In some embodiments, delivery comprises transposase-mediated transposition. In some embodiments, the sample of cancer cells comprises primary cancer cells. In some embodiments, the sample of cancer cells comprises about 105 to about 108 cells. In some embodiments, the library comprises from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different gene modulatory reagents. In some embodiments, at least about 90% of the gene modulatory reagents are present in the library in a quantity within about 10% of the average gene modulatory reagent quantity. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5B. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5A. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5C. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5D. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 4. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 3. In some embodiments, the sample of cancer cells has been processed to preserve cell viability.
In some embodiments, the method further comprises preparing the sample of cancer cells to preserve cell viability prior to and/or after delivery of the library of gene modulatory reagents. In some embodiments, the method further comprises propagating the modified cancer cells. In some embodiments, propagation comprises maintenance of the modified cancer cells in a 2D in vitro culture. In some embodiments, propagation comprises maintenance of the modified cancer cells in a 3D in vitro culture. In some embodiments, propagation comprises maintenance of the modified cancer cells in vivo. In some embodiments, propagation occurs within an animal model. In some embodiments, the animal model is a rodent.
In another aspect, provided herein is a compilation comprising a plurality of modified cancer cells, wherein each modified cancer cell harbors one or more gene modulatory reagents, and each gene modulatory reagent is capable of knocking down or knocking out the function of a gene that encodes a protein target from a library of protein targets. In some embodiments, at least one of the one or more gene modulatory reagents comprises a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. In some embodiments, at least one of the one or more gene modulatory reagents comprises a sequence selected from SEQ ID NOS: 1526-2789. In some embodiments, at least one of the one or more gene modulatory reagents comprises a sequence selected from SEQ ID NOS: 2980-3071. In some embodiments, at least one of the one or more of gene modulatory reagents comprises a sequence at least about 90% homologous to a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. In some embodiments, at least one of the one or more of gene modulatory reagents comprises a sequence at least about 90% homologous to a sequence selected from SEQ ID NOS: 1526-2789. In some embodiments, at least one of the one or more of gene modulatory reagents comprises a sequence at least about 90% homologous to a sequence selected from SEQ ID NOS: 2980-3071. In some embodiments, the homology is 90% identity.
In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5B. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5A. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5C. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5D. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 4. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 3. In some embodiments, one of more of the gene modulatory reagents comprise a guide RNA (gRNA) sequence comprising homology to at least a portion of the gene whose function is knocked down or knocked out in the modified cancer cell. In some embodiments, the gRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene. In some embodiments, the homology is at least about 90% sequence homology. In some embodiments, the homology is at least about 90% sequence identity.
In some embodiments, the compilation comprises an endonuclease. In some embodiments, the endonuclease comprises a Cas9 or Cas12a endonuclease. In some embodiments, the Cas9 or Cas12a endonuclease is selected from S. pyogenes Cas9 (SpCas9), SpCas9 D1135E variant, SpCas9 VRER variant, SpCas9 EQR variant, xCas9, SpCas9-NG, S. aureus Cas9 (SaCas9), Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LbCpfl), AsCpfl RR variant, LbCpfl RR variant, AsCpfl RVR variant, C. jejuni Cas9 (CjCas9), N. meningitidis (NmCas9), S. thermophilus (StCas9), T. denticola (TdCas9), and Mad7. In some embodiments, the endonuclease does not comprise a Cas9 or Cas12a endonuclease.
In some embodiments, the gRNA is positioned within a vector. In some embodiments, the vector further comprises genetic elements of a virus. In some embodiments, the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof. In some embodiments, the vector further comprises an auxiliary nucleic acid sequence. In some embodiments, the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette. In some embodiments, the marker is a fluorescent marker. In some embodiments, the auxiliary nucleic acid allows for the selection of the modified cancer cells.
In some embodiments, one or more of the gene modulatory reagents comprise a short hairpin RNA (shRNA) sequence comprising homology to at least a portion of the gene whose function is knocked down or knocked out in the modified cancer cell. In some embodiments, the shRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene. In some embodiments, the homology is at least about 90% sequence homology. In some embodiments, the homology is at least about 90% sequence identity. In some embodiments, the shRNA is positioned within a vector. In some embodiments, the vector further comprises genetic elements of a virus. In some embodiments, the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof. In some embodiments, the vector further comprises an auxiliary nucleic acid sequence. In some embodiments, the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette. In some embodiments, the marker is a fluorescent marker. In some embodiments, the auxiliary nucleic acid allows for the selection of the modified cancer cells. In some embodiments, delivering comprising transposase-mediated transposition.
In some embodiments, the modified cancer cells are modified primary cancer cells. In some embodiments, the compilation comprises from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different gene modulatory reagents. In some embodiments, the compilation comprises from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different populations of modified cancer cells.
In another aspect, provided herein is a method of evaluating the functional effect of genetically modifying cancer cells from a subject, the method comprising: sequencing a plurality of modified cancer cells, wherein each modified cancer cell harbors one or more gene modulatory reagents, each gene modulatory reagent capable of knocking down or knocking out the function of a gene that encodes a protein target in a library of protein targets; and wherein a gene modulatory reagent that impairs cell viability will have fewer sequence reads than a gene modulatory reagent that does not impair cell viability. In some embodiments, the method comprises determining which gene modulatory regents have fewer than a threshold number of sequence reads. In some embodiments, the threshold number of sequence reads is an expected number of sequence reads if the gene modulatory reagent did not impair cell viability. In some embodiments, the threshold number of sequence reads is an average number of sequence reads for each gene modulatory reagent in the plurality of modified cancer cells.
In some embodiments, the method comprises correlating each gene modulatory reagent that has fewer than the threshold number of sequence reads to its corresponding protein target in the library of protein targets. In some embodiments, the method comprises correlating the corresponding protein target to a therapeutic molecule. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5B. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5A. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5C. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5D. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 4. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 3.
In some embodiments, one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. In some embodiments, one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1526-2789. In some embodiments, one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 2980-3071. In some embodiments, the at least about 90% homology is at least about 90% identity.
In another aspect, provided herein is a library comprising a plurality of gene modulatory reagents, each gene modulatory reagent capable of knocking down or knocking out the function of a gene that encodes a protein target from a library of protein targets. In some embodiments, the plurality of gene modulatory reagents is capable of knocking down or knocking out the function of at least about 50% of the genes that encode for the protein targets in the library. In some embodiments, the at least about 50% is at least about 60%. In some embodiments, the at least about 60% is at least about 70%. In some embodiments, the at least about 70% is at least about 80%. In some embodiments, the at least about 80% is at least about 90%. In some embodiments, the library of protein targets comprises all known proteins targeted by known drugs capable of treating a particular disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer comprises at least one cancer from the group comprising acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, central nervous system cancers, basal cell carcinoma of the skin, bile duct cancer, bladder cancer, bone cancer, brain tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, cardiac tumors, central nervous system cancers, embryonal tumors, germ cell tumor, primary CNS lymphoma, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumors, central nervous system cancer, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumors, hepatocellular cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (non-small cell and small cell), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous neck cancer with occult primary, midline tract carcinoma with NUT gene changes, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, myelogenous leukemia, chronic (CML), myeloid leukemia, acute (AML), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, rhabdomyosarcoma, vascular tumors, osteosarcoma, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer, small intestine cancer, squamous cell carcinoma of the skin, squamous neck cancer with occult primary, stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, ureter and renal pelvis, urethral cancer, uterine cancer, endometrial, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, Wilms tumor, and other childhood kidney tumors.
In some embodiments, the known drugs comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 drugs of Table 2. In some embodiments, the known drugs comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 drugs of Table 3. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5B. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5A. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5C. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5D. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 4. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 3. In some embodiments, one or more of the plurality of gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. In some embodiments, one or more of the plurality of gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1526-2789. In some embodiments, one or more of the plurality of gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 2980-3071. In some embodiments, the at least about 90% homology is at least about 90% identity.
In some embodiments, the plurality of gene modulatory reagents is capable of knocking down or knocking out the function of about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, about 50 to about 2,000, or about 100 to about 2,000 genes. In some embodiments, the library comprises about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, about 50 to about 2,000, or about 100 to about 2,000 gene modulatory reagents.
In some embodiments, at least one of the gene modulatory reagents is capable of knocking out the function of a gene. In some embodiments, at least one of the gene modulatory reagents comprise a gRNA sequence having homology to at least a portion of the gene whose function is knocked out by the gene modulatory reagent. In some embodiments, at least one of the gene modulatory reagents is capable of knocking down the function of a gene. In some embodiments, at least one of the gene modulatory reagents comprise a shRNA sequence having homology to at least a portion of the gene whose function is knocked down by the gene modulatory reagent. In some embodiments, the homology is at least about 90% sequence homology. In some embodiments, the homology is at least about 90% sequence identity. In some embodiments, the at least a portion is at least about 15 contiguous nucleotides.
In some embodiments, at least one of the gene modulatory reagents is positioned within a vector. In some embodiments, the vector comprises an adapter sequence. In some embodiments, the adapter sequence comprises a type IIS restriction enzyme cleavage sites. In some embodiments, the vector further comprises genetic elements of a virus. In some embodiments, the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof In some embodiments, the vector further comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette. In some embodiments, the marker is a fluorescent marker.
Most previous efforts in personalized cancer sensitivity testing have focused on treatment of tumor cells with proposed therapeutic small molecules in vitro. However, numerous studies have demonstrated that key differences exist between the behavior of cancer cells in vitro and their corresponding behavior in vivo, including the response of cancer cells to inhibition of various molecular pathways. Indeed, one of the most clinically successful approaches to date for chemosensitivity testing utilizes engraftment of primary patient-derived cancer cells into mice, followed by in vivo treatment of the animals with drugs. This approach is effective but extraordinarily slow (6-12 months), expensive (cost of goods related to compounds and animals; hands-on time for dosing, analysis), and non-comprehensive (i.e. only a few drugs can be tested). As a result, the approach is intrinsically non-scalable.
Over 300 molecularly targeted therapies are either approved or under study for the treatment of cancer. Each of these drugs (usually a low molecular weight compound, or in some cases an antibody) binds to and, in nearly all cases, inactivates the function of a particular protein target. While it is conceptually appealing to test each of these drugs on an individual patient's cancer cells in order to find effective therapies, this has proven over several decades to be an inherently limited and suboptimal process for a number of reasons: (1) The number of cells required to perform the test limits the number of therapies (drugs) which can be tested. (2) Testing can only be performed in vitro, which differs significantly from the in vivo context in which clinical therapy is performed. (3) Accurate testing depends on knowledge of in vitro drug stability and cellular exposure, which are usually not known. As a result, the data gathered from compound testing in vitro does not reflect achievable in vivo tissue exposure. (4) High cost of goods associated with maintaining validated and updated stocks of all drugs. (5) Testing cannot be performed in a pooled or multiplexed format, raising costs and limiting throughput. These processes are therefore in principle unable to be scaled to commercial levels. (6) Testing cannot be used to identify new targets, i.e. those for which drugs are not already available.
The presently described methods work by pivoting the currently available suite of anti-cancer therapies from ‘drug space’ to ‘gene space’. Specifically, the methods depend on the critical insight that each protein target of an existing therapy can also be inhibited indirectly via mutagenesis of the gene encoding that protein target, e.g. via gene editing. Thus, the ‘drug pharmacopeia’ can instead be represented by a ‘genetic pharmacopeia’. The genetic pharmacopeia can represent an entire targeted therapy landscape (e.g., for the oncology landscape, over 300 therapeutic molecules are represented), in a genetic format. This may be achieved by designing inhibitory genetic elements, for instance sgRNAs (CRISPR) or shRNAs (RNAi) corresponding to the gene or mRNA respectively of the protein target of each potential therapeutic. Accordingly, the genetic pharmacopeia allows for a genetic determination of the functional susceptibility of cancer cells to known oncology drugs, mitigating the shortcomings described above and as shown in Table 1.
In the same way that a chemical pharmacopeia reduces the vast potential drug space (i.e. all LMW chemical structures) to a size that is useful for the selection of therapies in actual practice, a genetic pharmacopeia reduces the complexity of the human genome to a scale suitable for practical use in personalized diagnostics. The limited availability of patient-derived cells, which are usually derived from scant biopsy or resection specimens, and the limited ability to propagate these cells in culture mandates this reduction in complexity, and makes the use of a genetic pharmacopeia indispensable for diagnostics applications. The use of larger (e.g. whole genome) libraries for personalized medicine is simply not feasible, which until now has precluded the use of these technologies for precision medicine.
In one aspect, provided herein are methods of determining the susceptibility of a disease or condition to a library of therapeutic agents represented by a library of perturbagens which model the action of those therapeutic agents. A clinical workflow of a functional susceptibility profiling method for a patient with cancer is shown in
In another aspect, the methods described herein facilitate the generation of a discovery panel 107, which may include newly discovered drug targets, e.g., to assist with drug development; newly discovered use(s) of a known drug (drug repurposing); and/or the functional correlation to the discoveries based on whole exome sequencing. These discoveries may be partnered with Biopharmaceutical companies 108 to assist with expansion of drug indications for known drugs; function-based clinical trials of known drugs; development of drugs against newly discovered targets; and/or improvement of sequence-based analyses via deorphanization of variants of unknown significance (VUS).
The method described in
The method described in
The library of gene modulatory reagents used in the functional analysis shown in
Accordingly, an exemplary method of the present disclosure may comprise one or more of the following steps: (1) Defining the full targeted pharmacologic landscape by generating a list of all targeted drugs for a disease or condition (drug library). (2) Determining the protein targets of these drugs, and the genes encoding those protein targets (genetic pharmacopeia). (3) Designing a library of gene modulatory reagents to target the genes encoding these proteins. (4) Constructing the library as well as any needed gene silencing/editing agents in a format compatible with use in cells, e.g., primary cancer cells. (5) Delivering the library and any needed gene silencing/editing agents into cells, e.g., primary, patient-derived cancer cells. (6) Propagating the edited cells. (7) Obtaining a readout of the effect of each perturbation, e.g., by next generation sequencing (NGS)-based methods. (8) Interpreting the resulting barcode distributions to determine the effect of individual perturbations on the viability of the patient's diseased cells. Although the methods have been exemplified with regard to personalized cancer treatment, these methods are also suitable for treatment of non-cancer based diseases or conditions.
A non-limiting exemplary generic flowchart for the identification of patient-specific tumor therapeutic vulnerabilities utilizing function genomics described herein is shown in
In the present description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the description and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising,” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It should also be noted that the term “or” includes “and/or” unless the context clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.
The terms “homologous,” “homology,” or “percent homology” when used herein to describe to an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J Mol Biol. 1990 Oct. 5; 215(3):403-10; Nucleic Acids Res. 1997 Sep. 1; 25(17):3389-402). Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application. Percent identity of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.
In one aspect, provided herein is a pharmacologic landscape comprising a library of therapeutic agents having known protein targets, referred to as a drug library. The drug library may include low molecular weight drugs (e.g., having a molecule weight less than about 1 kDa) and biologic drugs (e.g., proteins such as antibodies). The drug library may comprise drugs suitable for a patient's particular disease or condition, such as cancer or an autoimmune disease. In various embodiments, the drug library includes FDA-approved therapeutic agents and as such may be expanded as new drugs are developed. The drug library may include all or nearly all of the targeted drugs treating a particular class of disease, e.g., the drug library includes at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of known FDA-approved drugs for a particular disease class having a known protein target. Also provided herein are focused libraries for investigational therapies (e.g., those in Phase I-III clinical testing), and libraries of a particular target classes of interest (e.g., G-protein coupled receptors, kinases, etc.).
Drug Library for Cancer
In certain embodiments, a drug library is designed comprising two or more therapies shown to be efficacious for, and/or have received FDA approval for, treating cancer. In some embodiments, the drug library comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 therapeutic agents. In some embodiments, the drug library comprises up to about 100, up to about 200, up to about 300, up to about 400, up to about 500, or up to about 1000 therapeutic agents. One or more of the therapeutic agents may be selected from Table 2. One or more of the therapeutic agents may be selected from Table 3.
In certain embodiments, a drug library is designed comprising two or more cancer therapeutics specific for a certain type of cancer. As non-limiting examples, the drug library comprises two or more cancer therapeutics shown to be efficacious for, and/or have received FDA approved for, melanoma, thyroid, colorectal, endometrial, lung, pancreatic, breast, genitourinary, gastrointestinal, ovarian, or head and neck cancer, or any cancer listed herein or known in the art. In some embodiments, the cancer-specific drug library comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 therapeutic agents. In some embodiments, the cancer-specific drug library comprises up to about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 therapeutic agents. One or more of the therapeutic agents may be selected from Table 2. One or more of the therapeutic agents may be selected from Table 3.
In certain embodiments, the drug library comprises at least one cancer therapeutic agent chosen from Table 2. The drug library may include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, or at least 900 therapeutic agents chosen from Table 2. The drug library may comprise the at least one cancer therapeutic agent chosen from Table 2, and one or more additional FDA-approved therapeutic agent(s) for cancer. The drug library may comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of known FDA-approved molecularly targeted cancer drugs. The drug library may comprise the at least one cancer therapeutic agent chosen from Table 2, and one or more additional therapeutic agent(s) for cancer that is undergoing FDA-approval and/or is the subject of any current or completed clinical trial.
In certain embodiments, the drug library comprises at least one cancer therapeutic agent chosen from Table 3. The drug library may include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, or at least 900 therapeutic agents chosen from Table 3. The drug library may comprise the at least one cancer therapeutic agent chosen from Table 3, and one or more additional FDA-approved therapeutic agent(s) for cancer. The drug library may comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of known FDA-approved molecularly targeted cancer drugs. The drug library may comprise the at least one cancer therapeutic agent chosen from Table 3, and one or more additional therapeutic agent(s) for cancer that is undergoing FDA-approval and/or is the subject of any current or completed clinical trial.
Gene Target Libraries
Further provided herein is a library of genetic targets comprising the genes encoding the proteins targeted by the therapeutic agents in the drug library. For therapeutic agents that are non-specific inhibitors, such as multi-kinase inhibitors, the targets may include multiple gene targets. The number of targeted genes must be significantly smaller than the “whole genome,” generating a compact library amenable to both in vitro and in vivo analysis. Non-limiting examples of targeted genes are shown in Table 4. Non-limiting examples of targeted genes for oncology are shown in Tables 5A-6B, 6D. The targeted genes described herein may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 genes from Table 5A. The targeted genes described herein may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 genes from Table 5B. The targeted genes described herein may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 genes from Table 5C. The targeted genes described herein may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or all of the genes from Table 5D. The targeted genes described herein may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 genes from Table 6A. The targeted genes described herein may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 genes from Table 6B. The targeted genes described herein may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or all of the genes from Table 6D. In some embodiments, the library comprises one or more genes to validate successful gene editing. A non-limiting example utilized in experiments described herein is the B2M gene.
A non-limiting exemplary gene target library was constructed as further described in the examples and characterized in
Another non-limiting exemplary gene target library was constructed that targets 23 unique genes, as further described in the examples. The genes targeted by the library include those listed in Table 5D and B2M. Accordingly, provided herein is a library targeting one or more of the genes of Table 5D, e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or all of the genes of Table 5D. In some cases the gene target library comprises a gene for validation purposes, such as B2M.
In one aspect, provided herein is a library of genetic elements which represent a collection of existing drugs for a particular disease or condition. These genetic elements are capable of modifying a patient's cells to mimic the effect of the existing drugs on the patient, allowing for personalized comprehensive functional profiling. The profiling may be performed in a pooled screening format to allow for screening of the effects of the modifications in parallel. Such highly parallel functional genomics methodology is utilized in preclinical biology, but has not been applicable to personalized therapeutic sensitivity profiling. Additionally, this approach enables comprehensive assessment of the impact of therapeutic manipulations in an in vivo testing paradigm, of critical importance for the reasons previously indicated herein.
Accordingly, disclosed herein are methods for the design, construction, and use of a genetic pharmacopeia comprising a plurality of gene modulatory reagents capable of modifying a patient's cells to knock out, or knock down, function of genes encoding for protein targets of a collection of existing drugs. In some embodiments, a genetic pharmacopeia is designed using publicly available tools, e.g., publicly available methods and reagents for gene editing or gene silencing. In some embodiments, a subset of these reagents will work poorly, most will be acceptable, and a minority will demonstrate exceptional performance. Pre-selection of reagents that have been validated to work well will be advantageous both with regard to efficiency of delivery and production of a more “compact” library, both of which reduce the number of patient-derived cells needed and increase the quality of data produced. In some embodiments, the design includes selection of the most efficacious or advantageous modulatory mechanism (e.g., CRISPR, RNAi). For CRISPR-based methods, the design comprises selection of the most advantageous RNA-guided endonuclease (e.g., Cas9 vs. Cas12a vs. Mad7). The design may also include selection of the most efficacious guide or seed sequences. The design may also include multiple gene modulatory reagents expressed from a single vector as a single or multiple transcriptional units. For instance, multiplexed gRNAs may be constructed for use with a Cas12 based nuclease (e.g., Cpfl) to generate a highly compact library. The design may also include elements in the library that allow for the identification, selection, or enrichment of transduced cells (e.g., fluorescent markers, antibiotic resistance cassettes, surface epitope expression cassettes).
The genetic pharmacopeia may be constructed in a format that is compatible with use in patient derived cells, e.g., primary cancer cells. In some embodiments, a viral delivery method is chosen for introduction of the gene modulatory reagent (e.g., guide or seed sequence). Non-limiting examples of viruses include lentivirus, adenovirus, adeno-associated virus, and other viruses disclosed herein. In some embodiments, a non-viral delivery method is selected. As a non-limiting example, the delivery method is transposase-mediated transposition. The library may be constructed using a combination of gene synthesis and pooled molecular cloning techniques. The library may be subject to quality control analysis to ensure full and approximately equal representation of the desired sequences. In some embodiments of a viral delivery method, pooled high-titer virus is prepared. In other embodiments, the virus is delivered in an array to facilitate an arrayed screening format.
Library of Gene Modulatory Reagents
In one aspect, provided herein are libraries comprising a plurality of gene modulatory reagents, each gene modulatory reagent capable of knocking down or knocking out the function of a gene that encodes a protein target from a library of protein targets. The plurality of gene modulatory reagents may be capable of knocking down or knocking out the function of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the genes that encode for the protein targets in the library. In some cases, the library of protein targets comprises all known proteins targeted by known drugs capable of treating a particular disease or condition. An exemplary disease or condition is cancer, e.g., a cancer disclosed herein or otherwise known in the art.
In some embodiments, the library of gene modulatory reagents is capable of knocking down or knocking out the function of one or more genes encoding protein targets selected from Table 5B. In some cases, the protein targets comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5B. In some embodiments, the library of gene modulatory reagents is capable of knocking down or knocking out the function of one or more genes encoding protein targets selected from Table 5A. In some cases, the protein targets comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5A. In some embodiments, the library of gene modulatory reagents is capable of knocking down or knocking out the function of one or more genes encoding protein targets selected from Table 5C. In some cases, the protein targets comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5C. In some embodiments, the library of gene modulatory reagents is capable of knocking down or knocking out the function of one or more genes encoding protein targets selected from Table 5D. In some cases, the protein targets comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5D. In some cases, the library of gene modulatory reagents is capable of knocking down or knocking out the function of one or more genes encoding for protein targets of one or more known drugs selected from Table 2-3. In some cases, the one or more known drugs comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 drugs of Table 2-3. In some cases, the library of gene modulatory reagents is capable of knocking down or knocking out the function of one or more genes encoding for protein targets of one or more known drugs selected from Table 2. In some cases, the one or more known drugs comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 drugs of Table 2. The plurality of gene modulatory reagents may be capable of knocking down or knocking out the function of about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, about 50 to about 2,000, or about 100 to about 2,000 genes. The library may comprise about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, about 50 to about 2,000, or about 100 to about 2,000 gene modulatory reagents.
At least one of the gene modulatory reagents may be capable of knocking out the function of a gene. For instance, the at least one gene modulatory reagent is part of a CRISPR-based gene editing system. In some cases, one or more of the plurality of gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. In some cases, one or more of the plurality of gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 1526-2789. In some cases, one or more of the plurality of gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 2980-3071. In some cases, at least one of the gene modulatory reagents comprise a gRNA sequence having homology to at least a portion of the gene whose function is knocked out by the gene modulatory reagent. In some embodiments, the gene modulatory reagents comprise one or more control sequences. As a non-limiting example, the sequence is a gRNA control that does not have a target in the human genome. As another non-limiting example, the sequence is a gRNA control that targets a non-genetic region of the human genome. For instance, the library may comprise one or more of the sequences of SEQ ID NOS: 2790-2971 (Table 6C). The inclusion of targeting (e.g., CTRL-hg38 of Table 6C) and non-targeting (e.g., CTRL-non sequences of Table 6C) control gRNAs enables an estimate of the impact of dsDNA breaks in innocuous genome locations. In some embodiments, the gene modulatory reagents comprise a gRNA that targets a gene for validation of successful gene editing. For instance, as described in the examples and
At least one of the gene modulatory reagents may be capable of knocking down the function of a gene. For instance, the at least one gene modulatory reagent comprises an shRNA sequence having homology to at least a portion of the gene whose function is knocked down by the gene modulatory reagent. The homology may be at least about 90% sequence homology or identity. The at least a portion may be at least about 15 contiguous nucleotides.
Non-limiting exemplary libraries of gene modulatory reagents were prepared and characterized (
In some embodiments, a library of gene modulatory reagents comprises one or more gene modulatory reagents that target a gene of Table 5D. In some embodiments, the library comprises one or more gene modulatory reagents that target at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or all of the gene targets of Table 5D. In some embodiments, the library comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, or all of the gRNA of Table 6D.
In some embodiments, one or more of the gene modulatory reagents is designed to knock out or knock down the function of a positive control gene, such as a core essential gene for the cell. Such reagents may serve as a positive control for library functionality. In some embodiments, one or more of the gene modulatory reagents is designed to knock out or knock down the function of a non-targeting gene and/or a targeting and non-genic gene. Such gene modulatory reagents may serve as negative controls. Non-limiting control gene modulatory reagents are provided in Table 6C.
In some embodiments, one or more of the gene modulatory reagents is positioned within a vector. The vector may comprise an adapter sequence. The adapter sequence may comprise a type IIS restriction enzyme cleavage site, which may allow for GoldenGate assembly cloning. The adapter sequence may comprise homology arms compatible with a destination vector allowing for cloning by overhang homology based methods, such as Gibson assembly. The vector may also comprise genetic elements of a virus. Non-limiting examples of viruses include adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), and human immunodeficiency virus (HIV). The vector may also comprise a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette, or a combination thereof. The marker may be a fluorescent marker.
CRISPR
In one aspect, provided is a library comprising a plurality of gene modulatory reagents, wherein each modulatory reagent comprises a guide RNA (gRNA) homologous to a target gene. The target gene may encode for a protein targeted by a known therapeutic agent (e.g., a therapeutic agent from Tables 2-3). Non-limiting examples of target genes are listed in Tables 4-6B, 6D. In some embodiments, one or more of the gRNAs comprise a sequence at least about 85%, 90%, 95%, or 100% homologous to at least about 10, 15, or 20 contiguous nucleobases of a target gene. In some embodiments, one or more of the gRNAs comprise a sequence at least about 85%, 90%, 95%, or 100% homologous to at least about 10, 15, or 20 contiguous nucleobases of a target gene chosen from Tables 4-6B, 6D. In some embodiments, the library comprises one or a plurality of sequences selected from SEQ ID NOS: 1-2789, 2980-3071. In some embodiments, the library comprises one or a plurality of sequences having at least about 85%, 90%, 95%, or 100% homology to a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. In some embodiments, the library comprises one or a plurality of sequences selected from SEQ ID NOS: 1526-2789. In some embodiments, the library comprises one or a plurality of sequences having at least about 85%, 90%, 95%, or 100% homology to a sequence selected from SEQ ID NOS: 1526-2789. In some embodiments, the library comprises one or a plurality of sequences selected from SEQ ID NOS: 2790-2959. In some embodiments, the library comprises one or a plurality of sequences having at least about 85%, 90%, 95%, or 100% homology to a sequence selected from SEQ ID NOS: 2790-2959. In some embodiments, the library comprises one or a plurality of sequences selected from SEQ ID NOS: 1526-2790. In some embodiments, the library comprises one or a plurality of sequences having at least about 85%, 90%, 95%, or 100% homology to a sequence selected from SEQ ID NOS: 1526-2790. In some embodiments, the library comprises one or a plurality of sequences selected from SEQ ID NOS: 2980-3071. In some embodiments, the library comprises one or a plurality of sequences having at least about 85%, 90%, 95%, or 100% homology to a sequence selected from SEQ ID NOS: 2980-3071. The library may comprise from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, from about 200 to about 2,000, or from about 500 to about 2,000 different gRNA sequences. In some embodiments, one or more of the gRNA sequences is encoded on a vector.
In some embodiments, the library further comprises an RNA-guided endonuclease such as Cas9, Cas12, Cas12a (or Cpfl or Mad7), Cas12b (or C2c1 or Cpf2), Cas12c (C2c3), Cas12d (or CasY), Cas12e (or CasX), Cas13, Cas13a (or C2c2), Cas13b (or C2c6), Cas13c (or C2c7), Cas13d (or Casrx), Cas14, Cas14a, Cas14b, Cas14c, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Csnl, Csx12, Cas10, Cas10d, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cul966, or derivative thereof, variant thereof, fragment thereof, or any combination thereof. In some embodiments, the endonuclease is of the Cas9 or Cas12a family, which may include, but is not limited to, S. pyogenes Cas9 (SpCas9), SpCas9 D1135E variant, SpCas9 VRER variant, SpCas9 EQR variant, xCas9, SpCas9-NG, S. aureus Cas9 (SaCas9), Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LbCpfl), AsCpfl RR variant, LbCpfl RR variant, AsCpfl RVR variant, C. jejuni Cas9 (Cj Cas9), N. meningitidis (NmCas9), S. thermophilus (StCas9), T. denticola (TdCas9), and Mad7. Additionally, other RNA-guided endonucleases that are suitable for the library disclosed herein include zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), meganucleases, RNA-binding proteins (RBP), recombinases, flippases, transposases, Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)), and any functional fragment thereof, and any combination thereof.
In some cases, the gRNA and/or endonuclease is encoded on a vector. In some cases, a vector comprising gRNA and/or endonuclease comprises one or more features of a viral genome. As a non-limiting example, the viral vector includes retroviral vector, adenoviral vector, adeno-associated viral vector (AAV), pox vectors, parvoviral vectors, baculovirus vectors, measles viral vectors, or herpes simplex virus vector (HSV). In some instances, the retroviral vector includes gamma-retroviral vector, such as a vector derived from the Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or the Murine Stem cell Virus (MSCV) genome. In some instances, the retroviral vector comprises lentiviral vectors such as those derived from the human immunodeficiency virus (HIV) genome. In some instances, AAV vector comprises AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. In some instances, the viral vector is a chimeric viral vector, comprising viral portions from two or more viruses. In additional instances, the viral vector is a recombinant viral vector.
In some embodiments, the vector comprises a marker for selection, e.g., an antibiotic resistance cassette or surface epitope expression cassette. In some embodiments, the gene modulatory reagent and endonuclease are encoded by separate vectors. As a non-limiting example, the endonuclease is delivered via adenovirus, while the gRNA is delivered by lentivirus. In some embodiments, the endonuclease coding sequence may be split between two vectors. For instance, this method may be employed when constructing large endonucleases such as Cas9. In some embodiments, the gene modulatory reagent is encoded by a viral vector and the endonuclease is provided as a ribonuclear protein complex transfected into target cells, for instance using lipid or electroporation techniques.
RNAi
In one aspect, provided is a library comprising a plurality of gene modulatory reagents, wherein one or more of the modulatory reagents comprise a short hairpin RNA (shRNA) complementary to a target mRNA of a protein targeted by a known therapeutic agent (e.g., a therapeutic agent chosen from Tables 2-3). Non-limiting examples of target proteins include those encoded by the genes listed in Tables 4-6B, 6D. In some embodiments, one or more of the shRNA each comprise a sequence at least about 85%, 90%, 95%, or 100% complementary to at least about 10, 15, or 20 contiguous nucleobases of a target mRNA. In some embodiments, one or more of the shRNA each comprise a sequence at least about 85%, 90%, 95%, or 100% complementary to at least about 10, 15, or 20 contiguous nucleobases of a target mRNA encoding for a protein selected from Tables 4-6B, 6D. The library may comprise from about 10 to about 2,000, from about 50 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different shRNA sequences.
In some aspects of the disclosure, a library comprising a plurality of gene modulatory reagents is delivered to a sample of cells from a subject having a disease or condition to generate a plurality of modified cells. In exemplary embodiments, the subject has cancer and the sample of cells comprise primary cancer cells. For some embodiments involving cancer cells, tumor samples are processed in a manner that preserves cancer cell viability, while maximizing cellular yield. Non-limiting examples of delivery methods include viral methods (e.g., lentivirus, adenovirus, or adeno-associated virus) as well as non-viral methods (e.g., transposase-mediated transposition employing transposons such as piggybac or sleeping beauty, or integrases such as phi31). In some embodiments, delivery of viral particles to the cells is performed in a manner that ensures equal and adequate representation of clones, while minimizing multiplicity of infection. In particular, the number of times each clone is presented within the population (“representation”) may be a crucial factor which determines the power of the eventual analysis to sensitively and specifically detect changes in barcode abundance following in vitro or in in vivo propagation.
Methods of Genetic Modification
An exemplary method for generating a plurality of modified cancer cells from a subject comprises: delivering a library of gene modulatory reagents to a sample of cancer cells from the subject to generate the plurality of modified cancer cells, wherein each modified cancer cell harbors one or more of the gene modulatory reagents, and each gene modulatory reagent is capable of knocking down or knocking out the function of a gene that encodes a protein target from a library of protein targets.
In some embodiments, the method for generating the plurality of modified cancer cells comprises a CRISPR/endonuclease-based gene editing system. For instance, one or more of the gene modulatory reagents comprises a gRNA sequence comprising homology to at least a portion of the gene whose function is knocked out in the modified cancer cell. The gRNA may comprise homology to about 10 to about 50 contiguous nucleotides of the gene. The homology may be at least about 90% sequence homology or identity. In some embodiments, one or more of the gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. In some embodiments, one or more of the gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 1526-2789. In some embodiments, one or more of the gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 2790-2959. In some embodiments, one or more of the gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 2980-3071. The gRNA may be positioned within a vector, e.g., for viral delivery as discussed herein.
The method for generating modified cancer cells may further comprise contacting the cancer cells with an endonuclease. The endonuclease may comprise a Cas9 or Cas12a endonuclease. Non-limiting examples of Cas9 or Cas12a endonucleases include S. pyogenes Cas9 (SpCas9), SpCas9 D1135E variant, SpCas9 VRER variant, SpCas9 EQR variant, xCas9, SpCas9-NG, S. aureus Cas9 (SaCas9), Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LbCpfl), AsCpfl RR variant, LbCpfl RR variant, AsCpfl RVR variant, C. jejuni Cas9 (CjCas9), N. meningitidis (NmCas9), S. thermophilus (StCas9), T. denticola (TdCas9), and Mad7. In some cases, the endonuclease does not comprise a Cas9 or Cas12a endonuclease.
In some embodiments, the method for generating the plurality of modified cancer cells comprises an RNA interference (RNAi) gene silencing system. For instance, each gene modulatory reagent comprises a shRNA sequence targeting mRNA encoding for a protein target from the library of protein targets. The shRNA may have homology to about 10 to about 50 contiguous nucleotides of the gene. The homology may be at least about 90% sequence homology or identity. The shRNA may be positioned within a vector, e.g., for viral delivery as discussed herein.
In some embodiments, the library of gene modulatory reagents comprises from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different gene modulatory reagents. In some cases, at least about 90% of the gene modulatory reagents are present in the library in a quantity within about 10% of the average gene modulatory reagent quantity.
In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5B. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5A. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5C. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5D.
In some embodiments, the sample of cancer cells comprises primary cancer cells. The sample of cancer cells may comprise about 105 to about 108 cells. The sample of cancer cells may have been processed to preserve cell viability. The method may thus further comprise preparing the sample of cancer cells to preserve cell viability prior to and/or after delivery of the library of gene modulatory reagents. The method may also further comprise propagating the modified cancer cells. Propagation may comprise maintenance of the modified cancer cells in a 2D in vitro culture. Propagation may comprise maintenance of the modified cancer cells in a 3D in vitro culture. Propagation may comprise maintenance of the modified cancer cells in vivo. In some cases, propagation occurs within an animal model, e.g., in a rodent.
CRISPR Gene Editing
In some embodiments, a sample of cells is modified using a CRISPR-based gene editing method. The gene editing method may comprise contacting the sample of cells with a plurality of gRNA sequences, wherein one or more of the gRNAs have sequence homology to a target gene encoding a protein targeted by a therapeutic agent. Non-limiting examples of target genes are provided in Tables 4-6B, 6D. Non-limiting examples of therapeutic agents are provided in Tables 2-3. In some embodiments, the sample of cells is contacted with at least one or a plurality of gRNA sequences chosen from SEQ ID NOS: 1-2789, 2980-3071. In some embodiments, the sample of cells is contacted with at least one or a plurality of gRNA sequences, each having at least about 85% homology to a sequence chosen from SEQ ID NOS: 1-2789, 2980-3071. In some embodiments, the sample of cells is contacted with at least one or a plurality of gRNA sequences chosen from SEQ ID NOS: 2980-3071. In some embodiments, the sample of cells is contacted with at least one or a plurality of gRNA sequences, each having at least about 85% homology to a sequence chosen from SEQ ID NOS: 2980-3071. In some embodiments, the sample of cells is contacted with at least one or a plurality of gRNA sequences chosen from SEQ ID NOS: 1526-2789. In some embodiments, the sample of cells is contacted with at least one or a plurality of gRNA sequences, each having at least about 85% homology to a sequence chosen from SEQ ID NOS: 1526-2789. In some embodiments, the sample of cells is contacted with at least one or a plurality of gRNA sequences chosen from SEQ ID NOS: 1526-2959. In some embodiments, the sample of cells is contacted with at least one or a plurality of gRNA sequences, each having at least about 85% homology to a sequence chosen from SEQ ID NOS: 1526-2959. In some embodiments, the sample of cells is contacted with at least one or a plurality of gRNA sequences chosen from SEQ ID NOS: 2790-2959. In some embodiments, the sample of cells is contacted with at least one or a plurality of gRNA sequences, each having at least about 85% homology to a sequence chosen from SEQ ID NOS: 2790-2959. The sample of cells is also contacted with an RNA-guided endonuclease, e.g., Cas9, Cas12, Cas12a (or Cpfl or Mad7), Cas12b (or C2c1 or Cpf2), Cas12c (C2c3), Cas12d (or CasY), Cas12e (or CasX), Cas13, Cas13a (or C2c2), Cas13b (or C2c6), Cas13c (or C2c7), Cas13d (or Casrx), Cas14, Cas14a, Cas14b, Cas14c, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Csn1, Csx12, Cas10, Cas10d, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx15, Csf1, Csf2, Csf3, Csf4, Cul966, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), meganucleases, RNA-binding proteins (RBP), recombinases, flippases, transposases, Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), or eukaryotic Argonaute (eAgo)), or derivative thereof, variant thereof, fragment thereof, or combination thereof.
RNAi
In some embodiments, a sample of cells is modified using an RNAi method. In some embodiments, the sample of cells is contacted with a plurality of shRNA sequences, each shRNA sequence complementary to a target mRNA of a protein targeted by a therapeutic agent. Non-limiting examples of target proteins include those encoded by the genes listed in Tables 4-6B, 6D. Non-limiting examples of therapeutic agents are provided in Tables 2-3.
Compilation of Modified Cancer Cells
Further provided herein are compilations of modified cancer cells. An exemplary compilation comprises a plurality of modified cancer cells, wherein each modified cancer cell harbors one or more gene modulatory reagents, and each gene modulatory reagent is capable of knocking down or knocking out the function of a gene that encodes a protein target from a library of protein targets. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5B. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5C. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5D. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5A. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 3. In some embodiments, the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 4. In some embodiments, the modified cancer cells are modified primary cancer cells. The modified cancer cells may comprise from about 10 to about 2,000, from about 50 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 100 to about 2,000, or from about 500 to about 2,000 different populations of modified cancer cells.
The modified cancer cells may comprise from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different gene modulatory reagents. At least one of the one or more gene modulatory reagents may comprise a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. At least one of the one or more of gene modulatory reagents may comprise a sequence at least about 90% homologous or identical to a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. At least one of the one or more gene modulatory reagents may comprise a sequence selected from SEQ ID NOS: 2980-3071. At least one of the one or more of gene modulatory reagents may comprise a sequence at least about 90% homologous or identical to a sequence selected from SEQ ID NOS: 2980-3071. At least one of the one or more gene modulatory reagents may comprise a sequence selected from SEQ ID NOS: 1526-2789. At least one of the one or more of gene modulatory reagents may comprise a sequence at least about 90% homologous or identical to a sequence selected from SEQ ID NOS: 1526-2789. At least one of the one or more gene modulatory reagents may comprise a sequence selected from SEQ ID NOS: 1526-2959. At least one of the one or more of gene modulatory reagents may comprise a sequence at least about 90% homologous or identical to a sequence selected from SEQ ID NOS: 1526-2959. At least one of the one or more gene modulatory reagents may comprise a sequence selected from SEQ ID NOS: 2790-2959. At least one of the one or more of gene modulatory reagents may comprise a sequence at least about 90% homologous or identical to a sequence selected from SEQ ID NOS: 2790-2959.
The modified cancer cells may have been modified by gene editing using a CRISPR-based method. As such, the gene modulatory reagents harbored by the modified cancer cells may comprise a gRNA sequence comprising homology to at least a portion of the gene whose function is knocked down or knocked out in the modified cancer cell. In some cases, the gRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene. The homology may be at least about 90% sequence homology or identity. The shRNA may be positioned within a vector, e.g., for viral delivery as discussed herein.
The modified cancer cells may also comprise an endonuclease, for instance, where the cells are modified using a gene editing system such as CRISPR. The endonuclease may comprise a Cas9 or Cas12a endonuclease. Non-limiting examples of Cas9 or Cas12a endonuclease include S. pyogenes Cas9 (SpCas9), SpCas9 D1135E variant, SpCas9 VRER variant, SpCas9 EQR variant, xCas9, SpCas9-NG, S. aureus Cas9 (SaCas9), Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LbCpfl), AsCpfl RR variant, LbCpfl RR variant, AsCpfl RVR variant, C. jejuni Cas9 (CjCas9), N. meningitidis (NmCas9), S. thermophilus (StCas9), T. denticola (TdCas9), and Mad7. The endonuclease may not comprise a Cas9 or Cas12a endonuclease.
The modified cancer cells may have been modified by gene silencing using shRNA gene modulatory reagents. Therefore, one or more of the gene modulatory reagents may comprise an shRNA sequence comprising homology to at least a portion of the gene whose function is knocked down in the modified cancer cell. The shRNA may comprise homology to about 10 to about 50 contiguous nucleotides of the gene. The homology may be at least about 90% sequence homology or identity. The shRNA may be positioned within a vector, e.g., for viral delivery as discussed herein.
Cell Propagation
In one aspect, provided herein are methods of propagating the plurality of genetically modified cells. For example, the genetically modified cells are modified using a CRISPR gene editing system or RNAi as described herein. The cells may be modified from primary cancer cells. In some embodiments, the plurality of modified cells is propagated in 2D format in vitro, 3D format in vitro, or in vivo. Non-limiting examples of the 3D in vitro format could include propagating cells embedded in sponge matrices (e.g., collagen-based), scaffolds, extracellular matrix (ECM) conditions such as basement membrane extract or Matrigel, in suspension, in organoid culture, or in microfluidic platforms. Exemplary materials constituting 3D in vitro format for cell propagation include collagen, gelatin, elastin, fibronectin, laminin, vitronectin, poly-lysine, poly-L-ornithine, silicone, polysaccharide polymers such as alginate, agar, dextran, carrageenan, chitosan, pectin, cellulose, gellan gum, xanthan gum, pullulan, glycosaminoglycan and any fragmented or derivative forms, hyaluronic acid, heparan, heparin, dermatan, chondroitin, or any hydrogel or biocompatible polymer. For in vitro approaches with cancer cells, the cancer cells are maintained under conditions that both support bulk cell survival while allowing selective pressure from induced mutations. For in vivo approaches, a propagation technique is selected which maximizes engraftment efficiency and survival. In some embodiments, in vivo cell propagation can include patient derived xenograft via either heterotopic implantation or orthotopic implantation. Additionally, for in vivo approaches, modified cancer cells may be implanted orthotopically (e.g., within the pancreas, for a pancreatic-origin tumor) or ectopically (e.g., subcutaneously, for a pancreatic origin tumor).
In one aspect, provided are methods of evaluating a sample of cells for the presence, absence, and/or quantity of a nucleic acid sequence from the genetic pharmacopeia. The power of the genetic pharmacopeia becomes evident in the ability to read out effects on cell growth directly via ‘barcode’ counting of modified cells (e.g., transduced cancer cells). Cells harboring a gRNA or shRNA impairing cell viability will be less represented in the overall population (i.e. will ‘dropout’); this manifests as less frequent appearance of the gRNA/shRNA sequence itself within the overall population of guide/shRNA sequences. The method may employ next-generation sequencing (NGS), which is well-established, cost effective, commercial scale, robust, highly quantitative, and highly amenable to multiplexed analysis.
Sequencing can be performed with any appropriate sequencing technology, including but not limited to, single-molecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis. Sequencing methods also include next-generation sequencing, e.g., modern sequencing technologies such as Illumina sequencing (e.g., Solexa), Roche 454 sequencing, Ion torrent sequencing, and SOLiD sequencing. In some cases, next-generation sequencing involves high-throughput sequencing methods. Additional sequencing methods available to one of skill in the art may also be employed.
The resulting barcode distributions are interpreted to determine the effect of individual perturbations on the viability of a subject's cells. In some implementations, raw sequencing read counts are interpreted and remapped back into ‘drug space’. For instance, in the hypothetical case described above, if a particular gRNA was found to be less prevalent than expected within the population, this would suggest that the protein encoded by the gene target of the gRNA is required for the survival or proliferation of the patient's cancer cells. As such, the drug targeting that protein (identified in step 1 above) is suggested to be a potentially higher value therapeutic for the patient.
An exemplary method of evaluating the functional effect of genetically modifying cancer cells from a subject comprises: sequencing a plurality of modified cancer cells, wherein each modified cancer cell harbors one or more gene modulatory reagents, each gene modulatory reagent capable of knocking down or knocking out the function of a gene that encodes a protein target in a library of protein targets; and wherein a gene modulatory reagent that impairs cell viability will have fewer sequence reads than a gene modulatory reagent that does not impair cell viability. The method may further comprise determining which gene modulatory regents have fewer than a threshold number of sequence reads. The threshold number of sequence reads may be an expected number of sequence reads if the gene modulatory reagent did not impair cell viability. In some cases the threshold number of sequence reads is an average number of sequence reads for each gene modulatory reagent in the plurality of modified cancer cells. In some embodiments, the method further comprises correlating each gene modulatory reagent that has fewer than the threshold number of sequence reads to its corresponding protein target in the library of protein targets. The method may then also comprise correlating the corresponding protein target to a therapeutic molecule. The library of protein targets may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5B. The library of protein targets may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5C. The library of protein targets may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5D. The library of protein targets may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 5A. The library of protein targets may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 3. The library of protein targets may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Table 4. At least one of the one or more of the gene modulatory reagents may comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. At least one of the one or more of the gene modulatory reagents may comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 2980-3071. At least one of the one or more of the gene modulatory reagents may comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 1526-2789.
Further provided herein are methods of treating a subject having a disease or condition, wherein the subject has been determined to be susceptible to a therapeutic agent using a method described herein. In some cases, the disease or condition is cancer. Non-limiting examples of cancer include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, central nervous system cancers, basal cell carcinoma of the skin, bile duct cancer, bladder cancer, bone cancer, brain tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, cardiac tumors, central nervous system cancers, embryonal tumors, germ cell tumor, primary CNS lymphoma, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumors, central nervous system cancer, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumors, hepatocellular cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (non-small cell and small cell), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous neck cancer with occult primary, midline tract carcinoma with NUT gene changes, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, myelogenous leukemia, chronic (CML), myeloid leukemia, acute (AML), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, rhabdomyosarcoma, vascular tumors, osteosarcoma, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer, small intestine cancer, squamous cell carcinoma of the skin, squamous neck cancer with occult primary, stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, ureter and renal pelvis, urethral cancer, uterine cancer, endometrial, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, and Wilms tumor and other childhood kidney tumors. In some embodiments, the therapeutic agent is selected from Table 2A. In some embodiments, the therapeutic agent is selected from Table 2B. In some embodiments, the therapeutic agent is selected from Table 3.
A non-limiting example of a method for treating cancer in a subject comprises: administering to the subject a therapeutic molecule selected from a library of therapeutic molecules, wherein the therapeutic molecule has been selected by a method comprising: modifying cancer cells from the subject to knock down or knock out the function of a plurality of genes, each gene in the plurality of genes encoding for a protein target of a therapeutic molecule in the library of therapeutic molecules, whereby the therapeutic molecule has been selected if knocking down or knocking out the function of the gene that encodes for the protein target of the selected therapeutic molecule impairs cancer cell viability. The library of therapeutic molecules may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 therapeutic agents of Table 2. The library of therapeutic molecules may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 therapeutic agents of Table 3. The one or more of the plurality of genes may encode for a protein of Table 5B. The one or more of the plurality of genes may encode for a protein of Table 5A. The one or more of the plurality of genes may encode for a protein of Table 5C. The one or more of the plurality of genes may encode for a protein of Table 5D. The one or more of the plurality of genes may encode for a protein of Table 3. The one or more of the plurality of genes may encode for a protein of Table 4.
Another exemplary method for treating cancer comprises: administering to the subject a therapeutic molecule selected from a library of therapeutic molecules; wherein the cancer of the subject has been determined to be susceptible to the selected therapeutic molecule by a method comprising: (a) contacting a sample of cancer cells from the subject with a library of gene modulatory reagents to generate a plurality of modified cancer cells, wherein each modified cancer cell harbors one or more of the gene modulatory reagents, and each gene modulatory reagent is capable of knocking down or knocking out the function of a gene that encodes a protein target of a therapeutic molecule in the library of therapeutic molecules, and (b) sequencing the plurality of modified cancer cells, wherein a gene modulatory reagent that impairs cell viability will have fewer sequence reads than a gene modulatory reagent that does not impair cell viability, and the gene that is knocked down or knocked out by the gene modulatory reagent that impairs cell viability encodes for the protein targeted by the selected therapeutic molecule. In some cases, prior to sequencing, the plurality of modified cancer cells has been propagated. Propagation may comprise maintenance of the modified cancer cells in a 2D in vitro culture. Propagation may comprise maintenance of the modified cancer cells in a 3D in vitro culture. Propagation may comprise maintenance of the modified cancer cells in vivo. Propagation may occur within an animal model, e.g., where the animal is a rodent.
In some embodiments, the cancer cells contacted with the library of gene modulatory reagents are primary cancer cells. Contacting may comprise introducing the one or more gene modulatory reagents into each cancer cell by a viral or non-viral delivery method. Each of the gene modulatory reagents in the library may be encoded on a viral vector. In non-limiting embodiments, the viral vector comprises a lentiviral vector, adenoviral vector, or adeno-associated viral vector. An exemplary non-viral delivery method comprises transposase-mediated transposition.
In some embodiments, the library of therapeutic molecules comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 therapeutic agents of Table 2. In some embodiments, the library of therapeutic molecules comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 therapeutic agents of Table 3. In some embodiments, the library of gene modulatory reagents comprises from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different gene modulatory reagents. In some embodiments, one or more gene modulatory reagents from the library of gene modulatory reagents comprises a nucleic acid sequence homologous to at least about 15 contiguous nucleotides of a gene encoding a protein of Table 5B. In some embodiments, one or more gene modulatory reagents from the library of gene modulatory reagents comprises a nucleic acid sequence homologous to at least about 15 contiguous nucleotides of a gene encoding a protein of Table 5C. In some embodiments, one or more gene modulatory reagents from the library of gene modulatory reagents comprises a nucleic acid sequence homologous to at least about 15 contiguous nucleotides of a gene encoding a protein of Table 5A. In some embodiments, one or more gene modulatory reagents from the library of gene modulatory reagents comprises a nucleic acid sequence homologous to at least about 15 contiguous nucleotides of a gene encoding a protein of Table 5D. In some embodiments, one or more gene modulatory reagents from the library of gene modulatory reagents comprises a nucleic acid sequence homologous to at least about 15 contiguous nucleotides of a gene encoding a protein of Table 3. In some embodiments, one or more gene modulatory reagents from the library of gene modulatory reagents comprises a nucleic acid sequence homologous to at least about 15 contiguous nucleotides of a gene encoding a protein of Table 4. The homology may be least about 90% sequence homology or identity.
In some cases, one or more of the gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 1-1525. In some cases, one or more of the gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 1-2789, 2980-3071. In some cases, one or more of the gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 2980-3071. In some cases, one or more of the gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 1526-2789. In some cases, one or more of the gene modulatory reagents each comprise a gRNA sequence comprising at least about 90% homology or identity to a sequence selected from SEQ ID NOS: 2790-2959. In some embodiments, each gene modulatory reagent comprises a gRNA sequence comprising homology to at least a portion of the gene that encodes a protein target of a therapeutic molecule in the library of therapeutic molecules. The gRNA may comprise homology to about 10 to about 50 contiguous nucleotides of the gene. The homology may be at least about 90% sequence homology or identity. The gRNA may be positioned within a vector, e.g., for viral delivery as discussed herein.
The method of determining susceptibility to the selected therapeutic molecule may further comprise contacting the cells with an endonuclease. In some embodiments, the endonuclease comprises a Cas9 or Cas12a endonuclease. Non-limiting examples of Cas9 or Cas12a endonucleases include S. pyogenes Cas9 (SpCas9), SpCas9 D1135E variant, SpCas9 VRER variant, SpCas9 EQR variant, xCas9, SpCas9-NG, S. aureus Cas9 (SaCas9), Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LbCpfl), AsCpfl RR variant, LbCpfl RR variant, AsCpfl RVR variant, C. jejuni Cas9 (CjCas9), N. meningitidis (NmCas9), S. thermophilus (StCas9), T. denticola (TdCas9), and Mad7. In some embodiments, the endonuclease does not comprise a Cas9 or Cas12a endonuclease.
In some embodiments, the gene modulatory reagents comprise a shRNA sequence comprising homology to at least a portion of the gene that encodes a protein target of a therapeutic molecule in the library of therapeutic molecules. The shRNA may comprise homology to about 10 to about 50 contiguous nucleotides of the gene. The homology may be at least about 90% sequence homology or identity. The shRNA may be positioned within a vector, e.g., for viral delivery as discussed herein.
(1) A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutic molecule selected from a library of therapeutic molecules; wherein the therapeutic molecule has been selected by a method comprising: modifying cancer cells from the subject to knock down or knock out the function of a plurality of genes, each gene in the plurality of genes encoding for a protein target of a therapeutic molecule in the library of therapeutic molecules, whereby the therapeutic molecule has been selected if knocking down or knocking out the function of the gene that encodes for the protein target of the selected therapeutic molecule impairs cancer cell viability.
(2) The method of embodiment 1, wherein the library of therapeutic molecules comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 therapeutic agents of Tables 2-3.
(3) The method of embodiment 1 or embodiment 2, wherein one or more of the plurality of genes encode for a protein of Table 3-5D.
(4) A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutic molecule selected from a library of therapeutic molecules; wherein the cancer of the subject has been determined to be susceptible to the selected therapeutic molecule by a method comprising:
(5) The method of embodiment 4, wherein prior to sequencing, one or more of the plurality of modified cancer cells have been propagated.
(6) The method of embodiment 5, wherein propagation comprises maintenance of the modified cancer cells in a 2D in vitro culture.
(7) The method of embodiment 5, wherein propagation comprises maintenance of the modified cancer cells in a 3D in vitro culture.
(8) The method of embodiment 5, wherein propagation comprises maintenance of the modified cancer cells in vivo.
(9) The method of embodiment 8, wherein propagation occurs within an animal model.
(10) The method of embodiment 9, wherein the animal is a rodent.
(11) The method of any one of embodiments 4-10, wherein the cancer cells are primary cancer cells.
(12) The method of any one of embodiments 4-11, wherein contacting comprises introducing the one or more gene modulatory reagents into each cancer cell by a viral or non-viral delivery method.
(13) The method of embodiment 12, wherein one or more of the gene modulatory reagents in the library are encoded on a viral vector.
(14) The method of embodiment 13, wherein the viral vector comprises a lentiviral vector, adenoviral vector, or adeno-associated viral vector.
(15) The method of embodiment 12, wherein the non-viral delivery method comprises transposase-mediated transposition.
(16) The method of any one of embodiments 4-15, wherein the library comprises from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different gene modulatory reagents.
(17) The method of any one of embodiments 4-16, wherein one or more gene modulatory reagents from the library of gene modulatory reagents comprises a nucleic acid sequence homologous to at least about 15 contiguous nucleotides of a gene encoding a protein of Tables 3-5D.
(18) The method of embodiment 17, wherein the homology is at least about 90% sequence homology.
(19) The method of embodiment 18, wherein the homology is at least about 90% sequence identity.
(20) The method of any one of embodiments 4-19, wherein the library of therapeutic molecules comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 therapeutic agents of Tables 2-3.
(21) The method of any one of embodiments 4-20, wherein the cancer comprises at least one cancer chosen from the group comprising acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, central nervous system cancers, basal cell carcinoma of the skin, bile duct cancer, bladder cancer, bone cancer, brain tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, cardiac tumors, central nervous system cancers, embryonal tumors, germ cell tumor, primary CNS lymphoma, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumors, central nervous system cancer, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumors, hepatocellular cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (non-small cell and small cell), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous neck cancer with occult primary, midline tract carcinoma with NUT gene changes, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, myelogenous leukemia, chronic (CML), myeloid leukemia, acute (AML), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, rhabdomyosarcoma, vascular tumors, osteosarcoma, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer, small intestine cancer, squamous cell carcinoma of the skin, squamous neck cancer with occult primary, stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, ureter and renal pelvis, urethral cancer, uterine cancer, endometrial, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, Wilms tumor, and other childhood kidney tumors.
(22) The method of any one of embodiments 4-21, wherein one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1-1525, SEQ ID NOS: 1-2789, SEQ ID NOS: 1526-2789, and/or SEQ ID NOS: 2980-3071.
(23) The method of embodiment 22, wherein the at least about 90% homology is at least about 90% identity.
(24) The method of any one of embodiments 4-23, wherein one or more of the gene modulatory reagents comprise a guide RNA (gRNA) sequence comprising homology to at least a portion of the gene that encodes a protein target of a therapeutic molecule in the library of therapeutic molecules.
(25) The method of embodiment 24, wherein the gRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene.
(26) The method of embodiment 24 or embodiment 25, wherein the homology is at least about 90% sequence homology.
(27) The method of embodiment 26, wherein the homology is at least about 90% sequence identity.
(28) The method of any one of embodiments 4-27, wherein the sample of cancer cells is contacted with an endonuclease.
(29) The method of embodiment 28, wherein the endonuclease comprises a Cas9 or Cas12a endonuclease.
(30) The method of embodiment 29, wherein the Cas9 or Cas12a endonuclease is selected from S. pyogenes Cas9 (SpCas9), SpCas9 D1135E variant, SpCas9 VRER variant, SpCas9 EQR variant, xCas9, SpCas9-NG, S. aureus Cas9 (SaCas9), Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LbCpfl), AsCpfl RR variant, LbCpfl RR variant, AsCpfl RVR variant, C. jejuni Cas9 (CjCas9), N. meningitidis (NmCas9), S. thermophilus (StCas9), T. denticola (TdCas9), and Mad7.
(31) The method of embodiment 28, wherein the endonuclease does not comprise a Cas9 or Cas12a endonuclease.
(32) The method of any one of embodiments 24-31, wherein the gRNA is positioned within a vector.
(33) The method of embodiment 32, wherein the vector further comprises genetic elements of a virus.
(34) The method of embodiment 33, wherein the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof.
(35) The method of any one of embodiments 32-34, wherein the vector further comprises an auxiliary nucleic acid sequence.
(36) The method of embodiment 35, wherein the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, and surface epitope expression cassette.
(37) The method of embodiment 36, wherein the marker is a fluorescent marker.
(38) The method of any one of embodiments 35-37, wherein the auxiliary nucleic acid allows for the selection of cancer cells that have been modified to harbor the one or more gene modulatory reagents.
(39) The method of any one of embodiments 4-23, wherein one or more of the gene modulatory reagents comprise a short hairpin RNA (shRNA) sequence comprising homology to at least a portion of the gene that encodes a protein target of a therapeutic molecule in the library of therapeutic molecules.
(40) The method of embodiment 39, wherein the shRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene.
(41) The method of embodiment 39 or embodiment 40, wherein the homology is at least about 90% sequence homology.
(42) The method of embodiment 41, wherein the homology is at least about 90% sequence identity.
(43) The method of any one of embodiments 39-42, wherein the shRNA is positioned within a vector.
(44) The method of embodiment 43, wherein the vector further comprises genetic elements of a virus.
(45) The method of embodiment 44, wherein the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof.
(46) The method of any one of embodiments 43-45, wherein the vector further comprises an auxiliary nucleic acid sequence.
(47) The method of embodiment 46, wherein the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette.
(48) The method of embodiment 47, wherein the marker is a fluorescent marker.
(49) The method of any one of embodiments 46-48, wherein the auxiliary nucleic acid allows for the selection of cancer cells that have been modified to harbor the one or more gene modulatory reagents.
(50) A method of generating a plurality of modified cancer cells from a subject having cancer, the method comprising delivering a library of gene modulatory reagents to a sample of cancer cells from the subject to generate the plurality of modified cancer cells; wherein each modified cancer cell harbors one or more of the gene modulatory reagents, and each gene modulatory reagent is capable of knocking down or knocking out the function of a gene that encodes a protein target from a library of protein targets.
(51) The method of embodiment 50, wherein one or more of the gene modulatory reagents comprises a guide RNA (gRNA) sequence comprising homology to at least a portion of the gene whose function is knocked down or knocked out in the modified cancer cell.
(52) The method of embodiment 51, wherein the gRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene.
(53) The method of embodiment 51 or embodiment 52, wherein the homology is at least about 90% sequence homology.
(54) The method of embodiment 53, wherein the homology is at least about 90% sequence identity.
(55) The method of embodiment 50, wherein one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1-1525, SEQ ID NOS: 1-2789, SEQ ID NOS: 1526-2789, and/or SEQ ID NOS: 2980-3071.
(56) The method of embodiment 55, wherein the homology is at least about 90% identity.
(57) The method of any one of embodiments 50-56, wherein the sample of cancer cells is contacted with an endonuclease.
(58) The method of embodiment 57, wherein the endonuclease comprises a Cas9 or Cas12a endonuclease.
(59) The method of embodiment 58, wherein the Cas9 or Cas12a endonuclease is selected from S. pyogenes Cas9 (SpCas9), SpCas9 D1135E variant, SpCas9 VRER variant, SpCas9 EQR variant, xCas9, SpCas9-NG, S. aureus Cas9 (SaCas9), Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LbCpfl), AsCpfl RR variant, LbCpfl RR variant, AsCpfl RVR variant, C. jejuni Cas9 (CjCas9), N. meningitidis (NmCas9), S. thermophilus (StCas9), T. denticola (TdCas9), and Mad7.
(60) The method of embodiment 57, wherein the endonuclease does not comprise a Cas9 or Cas12a endonuclease.
(61) The method of any one of embodiments 51-56, wherein the gRNA is positioned within a vector.
(62) The method of embodiment 61, wherein the vector further comprises genetic elements of a virus.
(63) The method of embodiment 62, wherein the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof.
(64) The method of any one of embodiments 61-63, wherein the vector further comprises an auxiliary nucleic acid sequence.
(65) The method of embodiment 64, wherein the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette.
(66) The method of embodiment 65, wherein the marker is a fluorescent marker.
(67) The method of any one of embodiments 64-66, wherein the auxiliary nucleic acid allows for the selection of cancer cells that have been modified to harbor the one or more gene modulatory reagents.
(68) The method of embodiment 50, wherein one or more of the gene modulatory reagents comprise a short hairpin RNA (shRNA) sequence comprising homology to at least a portion of the gene whose function is knocked down or knocked out in the modified cancer cell.
(69) The method of embodiment 68, wherein the shRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene.
(70) The method of embodiment 68 or embodiment 69, wherein the homology is at least about 90% sequence homology.
(71) The method of embodiment 70, wherein the homology is at least about 90% sequence identity.
(72) The method of any one of embodiments 68-71, wherein the shRNA is positioned within a vector.
(73) The method of embodiment 72, wherein the vector further comprises genetic elements of a virus.
(74) The method of embodiment 73, wherein the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof.
(75) The method of any one of embodiments 72-74, wherein the vector further comprises an auxiliary nucleic acid sequence.
(76) The method of embodiment 75, wherein the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette.
(77) The method of embodiment 76, wherein the marker is a fluorescent marker.
(78) The method of any one of embodiments 75-77, wherein the auxiliary nucleic acid allows for the selection of cancer cells that have been modified to harbor the one or more gene modulatory reagents.
(79) The method of embodiment 50, wherein delivering comprising transposase-mediated transposition.
(80) The method of any one of embodiments 50-79, wherein the sample of cancer cells comprises primary cancer cells.
(81) The method of any one of embodiments 50-80, wherein the sample of cancer cells comprises about 105 to about 108 cells.
(82) The method of any one of embodiments 50-81, wherein the library comprises from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different gene modulatory reagents.
(83) The method of any one of embodiments 50-82, wherein at least about 90% of the gene modulatory reagents are present in the library in a quantity within about 10% of the average gene modulatory reagent quantity.
(84) The method of any one of embodiments 50-83, wherein the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Tables 3-5D.
(85) The method of any one of embodiments 50-84, wherein the sample of cancer cells has been processed to preserve cell viability.
(86) The method of any one of embodiments 50-85, further comprising preparing the sample of cancer cells to preserve cell viability prior to and/or after delivery of the library of gene modulatory reagents.
(87) The method of any one of embodiments 50-86, further comprising propagating the modified cancer cells.
(88) The method of embodiment 87, wherein propagation comprises maintenance of the modified cancer cells in a 2D in vitro culture.
(89) The method of embodiment 87, wherein propagation comprises maintenance of the modified cancer cells in a 3D in vitro culture.
(90) The method of embodiment 87, wherein propagation comprises maintenance of the modified cancer cells in vivo.
(91) The method of embodiment 90, wherein propagation occurs within an animal model.
(92) The method of embodiment 91, wherein the animal model is a rodent.
(93) A compilation comprising a plurality of modified cancer cells, wherein each modified cancer cell harbors one or more gene modulatory reagents, and each gene modulatory reagent is capable of knocking down or knocking out the function of a gene that encodes a protein target from a library of protein targets.
(94) The compilation of embodiment 93, wherein at least one of the one or more gene modulatory reagents comprises a sequence selected from SEQ ID NOS: 1-1525, SEQ ID NOS: 1-2789, SEQ ID NOS: 1526-2789, and/or SEQ ID NOS: 2980-3071.
(95) The compilation of embodiment 93, wherein at least one of the one or more of gene modulatory reagents comprises a sequence at least about 90% homologous to a sequence selected from SEQ ID NOS: 1-1525, SEQ ID NOS: 1-2789, SEQ ID NOS: 1526-2789, and/or SEQ ID NOS: 2980-3071.
(96) The compilation of embodiment 95, wherein the homology is 90% identity.
(97) The compilation of any of embodiments 93-96, wherein the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Tables 3-5D.
(98) The compilation of any of embodiments 93-97, wherein one of more of the gene modulatory reagents comprise a guide RNA (gRNA) sequence comprising homology to at least a portion of the gene whose function is knocked down or knocked out in the modified cancer cell.
(99) The compilation of embodiment 98, wherein the gRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene.
(100) The compilation of embodiment 98 or embodiment 99, wherein the homology is at least about 90% sequence homology.
(101) The compilation of embodiment 100, wherein the homology is at least about 90% sequence identity.
(102) The compilation of any one of embodiments 93-101, further comprising an endonuclease.
(103) The compilation of embodiment 102, wherein the endonuclease comprises a Cas9 or Cas12a endonuclease.
(104) The method of embodiment 103, wherein the Cas9 or Cas12a endonuclease is selected from S. pyogenes Cas9 (SpCas9), SpCas9 D1135E variant, SpCas9 VRER variant, SpCas9 EQR variant, xCas9, SpCas9-NG, S. aureus Cas9 (SaCas9), Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LbCpfl), AsCpfl RR variant, LbCpfl RR variant, AsCpfl RVR variant, C. jejuni Cas9 (CjCas9), N. meningitidis (NmCas9), S. thermophilus (StCas9), T. denticola (TdCas9), and Mad7.
(105) The compilation of embodiment 102, wherein the endonuclease does not comprise a Cas9 or Cas12a endonuclease.
(106) The compilation of any one of embodiments 98-105, wherein the gRNA is positioned within a vector.
(107) The compilation of embodiment 106, wherein the vector further comprises genetic elements of a virus.
(108) The compilation of embodiment 107, wherein the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof.
(109) The compilation of any one of embodiments 106-108, wherein the vector further comprises an auxiliary nucleic acid sequence.
(110) The compilation of embodiment 109, wherein the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette.
(111) The compilation of embodiment 110, wherein the marker is a fluorescent marker.
(112) The compilation of any one of embodiments 109-111, wherein the auxiliary nucleic acid allows for the selection of the modified cancer cells.
(113) The compilation of embodiment 93, wherein one or more of the gene modulatory reagents comprise a short hairpin RNA (shRNA) sequence comprising homology to at least a portion of the gene whose function is knocked down or knocked out in the modified cancer cell.
(114) The compilation of embodiment 113, wherein the shRNA comprises homology to about 10 to about 50 contiguous nucleotides of the gene.
(115) The compilation of embodiment 113 or embodiment 114, wherein the homology is at least about 90% sequence homology.
(116) The compilation of embodiment 115, wherein the homology is at least about 90% sequence identity.
(117) The compilation of any one of embodiments 113-116, wherein the shRNA is positioned within a vector.
(118) The compilation of embodiment 117, wherein the vector further comprises genetic elements of a virus.
(119) The compilation of embodiment 118, wherein the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof.
(120) The compilation of any one of embodiments 117-119, wherein the vector further comprises an auxiliary nucleic acid sequence.
(121) The compilation of embodiment 120, wherein the auxiliary nucleic acid sequence comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette.
(122) The compilation of embodiment 121, wherein the marker is a fluorescent marker.
(123) The compilation of any one of embodiments 120-122, wherein the auxiliary nucleic acid allows for the selection of the modified cancer cells.
(124) The compilation of any one of embodiments 93-105, wherein delivering comprising transposase-mediated transposition.
(125) The compilation of any one of embodiments 93-124, wherein the modified cancer cells are modified primary cancer cells.
(126) The compilation of any one of embodiments 93-125, comprising from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different gene modulatory reagents.
(127) The compilation of any one of embodiments 93-126, comprising from about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, from about 50 to about 2,000, from about 100 to about 2,000, or from about 500 to about 2,000 different populations of modified cancer cells.
(128) A method of evaluating the functional effect of genetically modifying cancer cells from a subject, the method comprising: sequencing a plurality of modified cancer cells, wherein each modified cancer cell harbors one or more gene modulatory reagents, each gene modulatory reagent capable of knocking down or knocking out the function of a gene that encodes a protein target in a library of protein targets; and wherein a gene modulatory reagent that impairs cell viability will have fewer sequence reads than a gene modulatory reagent that does not impair cell viability.
(129) The method of embodiment 128, further comprising determining which gene modulatory regents have fewer than a threshold number of sequence reads.
(130) The method of embodiment 129, wherein the threshold number of sequence reads is an expected number of sequence reads if the gene modulatory reagent did not impair cell viability.
(131) The method of embodiment 129, wherein the threshold number of sequence reads is an average number of sequence reads for each gene modulatory reagent in the plurality of modified cancer cells.
(132) The method of any one of embodiments 128-131, further comprising correlating each gene modulatory reagent that has fewer than the threshold number of sequence reads to its corresponding protein target in the library of protein targets.
(133) The method of embodiment 132, further comprising correlating the corresponding protein target to a therapeutic molecule.
(134) The method of any one of embodiments 128-133, wherein the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Tables 3-5D.
(135) The method of any one of embodiments 128-134, wherein one or more of the gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1-1525, SEQ ID NOS: 1-2789, SEQ ID NOS: 1526-2789, and/or SEQ ID NOS: 2980-3071.
(136) The method of embodiment 135, wherein the at least about 90% homology is at least about 90% identity.
(137) A library comprising a plurality of gene modulatory reagents, each gene modulatory reagent capable of knocking down or knocking out the function of a gene that encodes a protein target from a library of protein targets.
(138) The library of embodiment 137, wherein the plurality of gene modulatory reagents is capable of knocking down or knocking out the function of at least about 50% of the genes that encode for the protein targets in the library.
(139) The library of embodiment 138, wherein the at least about 50% is at least about 60%.
(140) The library of embodiment 139, wherein the at least about 60% is at least about 70%.
(141) The library of embodiment 140, wherein the at least about 70% is at least about 80%.
(142) The library of embodiment 141, wherein the at least about 80% is at least about 90%.
(143) The library of any one of embodiments 137-142, wherein the library of protein targets comprises all known proteins targeted by known drugs capable of treating a particular disease or condition.
(144) The library of embodiment 143, wherein the disease or condition is cancer.
(145) The library of embodiment 144, wherein the cancer comprises at least one cancer from the group comprising acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytomas, atypical teratoid/rhabdoid tumor, central nervous system cancers, basal cell carcinoma of the skin, bile duct cancer, bladder cancer, bone cancer, brain tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, cardiac tumors, central nervous system cancers, embryonal tumors, germ cell tumor, primary CNS lymphoma, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumors, central nervous system cancer, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumors, hepatocellular cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (non-small cell and small cell), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous neck cancer with occult primary, midline tract carcinoma with NUT gene changes, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, myelogenous leukemia, chronic (CML), myeloid leukemia, acute (AML), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, rhabdomyosarcoma, vascular tumors, osteosarcoma, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer, small intestine cancer, squamous cell carcinoma of the skin, squamous neck cancer with occult primary, stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, ureter and renal pelvis, urethral cancer, uterine cancer, endometrial, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, Wilms tumor, and other childhood kidney tumors.
(146) The library of any one of embodiments 143-145, wherein the known drugs comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 drugs of Tables 2-3.
(147) The library of any one of embodiments 137-146, wherein the library of protein targets comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 protein targets of Tables 3-5D.
(148) The library of any one of embodiments 137-147, wherein one or more of the plurality of gene modulatory reagents each comprise a guide RNA (gRNA) sequence comprising at least about 90% homology to a sequence selected from SEQ ID NOS: 1-1525, SEQ ID NOS: 1-2789, SEQ ID NOS: 1526-2789, or SEQ ID NOS: 2980-3071.
(149) The library of embodiment 148, wherein the at least about 90% homology is at least about 90% identity.
(150) The library of any one of embodiments 137-149, wherein the plurality of gene modulatory reagents is capable of knocking down or knocking out the function of about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, about 50 to about 2,000, or about 100 to about 2,000 genes.
(151) The library of any one of embodiments 137-150, wherein the library comprises about 10 to about 2,000, from about 10 to about 500, from about 10 to about 200, from about 10 to about 150, from about 50 to about 500, from about 50 to about 200, about 50 to about 2,000, or about 100 to about 2,000 gene modulatory reagents.
(152) The library of any one of embodiments 137-151, wherein at least one of the gene modulatory reagents is capable of knocking out the function of a gene.
(153) The library of embodiment 152, wherein at least one of the gene modulatory reagents comprise a gRNA sequence having homology to at least a portion of the gene whose function is knocked out by the gene modulatory reagent.
(154) The library of any one of embodiments 137-147, wherein at least one of the gene modulatory reagents is capable of knocking down the function of a gene.
(155) The library of embodiment 154, wherein at least one of the gene modulatory reagents comprise a shRNA sequence having homology to at least a portion of the gene whose function is knocked down by the gene modulatory reagent.
(156) The library of embodiment 153 or embodiment 155, wherein the homology is at least about 90% sequence homology.
(157) The library of embodiment 156, wherein the homology is at least about 90% sequence identity.
(158) The library of embodiment 155 or embodiment 156, wherein the at least a portion is at least about 15 contiguous nucleotides.
(159) The library of any one of embodiments 137-158, wherein at least one of the gene modulatory reagents is positioned within a vector.
(160) The library of embodiment 159, wherein the vector comprises an adapter sequence.
(161) The library of embodiment 160, wherein the adapter sequence comprises a type IIS restriction enzyme cleavage sites.
(162) The library of any one of embodiments 159-161, wherein the vector further comprises genetic elements of a virus.
(163) The library of embodiment 162, wherein the virus comprises an adenovirus, retrovirus, adeno-associated virus (AAV), pox virus, parvovirus, baculovirus, measles virus, herpes simplex virus (HSV), Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV), Murine Stem cell Virus (MSCV), or human immunodeficiency virus (HIV), or a combination thereof.
(164) The library of any one of embodiments 159-163, wherein the vector further comprises a sequence encoding a marker, an antibiotic resistance cassette, or surface epitope expression cassette.
(165) The library of embodiment 164, wherein the marker is a fluorescent marker.
Tables 2-3 provide exemplary therapeutic agents, one or more of which may be a member of a drug library described herein.
Tables 4-5C provide exemplary protein targets of known therapeutic agents, one or more of which may be useful in a target library described herein.
Tables 6A-6C provide lists of gene modulatory reagents, one or more of which may be used in a method of cell editing described herein.
A drug library comprising molecularly targeted oncology drugs of Table 2B was generated. The drug library is updated periodically to include additional targeted oncology drugs as they are identified. A genetic pharmacopeia was generated to represent the genetic targets of the drug library (Table 5B).
A library of gene modulatory reagents comprising guide RNA (gRNA) sequences associated with each gene target was designed. As shown in Table 6A, five potential gRNA sequences were designed for each oncology drug target to generate gRNA sequences having SEQ ID NOS: 1-1525. The library of gene modulatory reagents is constructed to comprise at least one gRNA sequence selected from SEQ ID NOS: 1-1525. The library is constructed in a format compatible with use in primary cancer cells using a viral delivery method (adenovirus for Cas nuclease delivery, lentivirus for gRNA delivery).
A method is performed to determine the functional susceptibility of a patient's cancer cells to one or more perturbagens which model the action of the targeted oncology drugs identified in Example 1. The library comprising at least one gRNA sequence selected from SEQ ID NOS: 1-1525 and associated gene editing agent(s) (e.g., RNA-guided nuclease) are delivered to primary cancer cells derived from the patient in order to genetically modify the cancer cells. The Cas nuclease and gRNA are delivered by lentivirus. In this example, genetic modification occurs via gene editing using a CRISPR-based method. The modified cancer cells are propagated in vivo, however, the method may be employed in in vitro environments that mimic the in vivo context. The effect of each gene edit is evaluated by screening the modified cancer cells in a pooled or array format. Next-generation sequencing is performed to determine the effect of the individual perturbations on the viability of the patient's cancer cells. Oncology drug(s) associated with the perturbagens that reduce viability of the cancer cells are selected as a putative therapeutic for the patient.
Methods for the identification of patient-specific tumor therapeutic vulnerabilities were performed utilizing function genomics as outlined in
Guide-RNA Library Cloning
A library of guide RNAs (gRNA) with 1685 elements having 1585 gRNAs directed against drug target genes and 100 control gRNAs was designed (
Another library of gRNAs directed against drug target genes was prepared comprising the gRNAs of Table 6D. The library also includes gRNAs having SEQ ID NOS: 2972-2979 directed to B2M, and control gRNAs having SEQ ID NOS: 2890-2905 and 2960-2971.
Virus Production
Lentiviral particles containing viral genome encoding expression units for the gRNA library and Cas9 were generated by transfecting 293FT cells with transfer vector and 2nd generation lentiviral packaging plasmids (DR8.9 and pCMV-VSVG) in a ratio of 4:3:1 using Lipofectamine-3000 (Thermo) according to the manufacturer's instructions. Six hours after transfection, medium was changed to DMEM harvest medium containing 10% FCS. Virus containing supernatant was harvested at 30 and 54 hours after transfection, centrifuged for 5 minutes at 2500 rpm to remove debris and filtered through a 45 μm filter before pooling. Virus was precipitated from culture supernatants by incubation with PEG-8000 at 10% final concentration for >4 hours. PEG-precipitate was centrifuged for 1 hour at 4000 rpm and the pellet resuspended in ˜1/100 the original volume. Aliquots were stored at −80° C. until use.
Tumor Processing
Tumor pieces were finely chopped using sterile razor blades in 0.5 ml digestion mix (DMEM/F12 with 1 mg/ml collagenase IV, 10 uM Y27632 and 20 ug/ml DNase). These were digested for 30 min at 37C, triturated with a 10 ml pipette, then digested for an additional 15 min at 37 C. The mixture was strained through a 100 uM strainer. Cells were washed once with FACS buffer (PBS with 0.% BSA, 1 mM EDTA) and resuspended in organoid medium (Advanced DMEM/F12 with 10 uM SB202190, 1× HEPES, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1X Glutamax, 1X Primocin, 5% Knockout Serum Replacement, 1× B27 supplement, 0.1 nM cholera toxin, 0.5 uM A83-01, 10 uM Y27632, 1 uM PGE2, 10 nM [Leu15]-Gastrin I, 10 ng/ml rhFGF10, 10 ng/ml rhFGF2, 50 ng/ml EGF, 0.3 ug/ml hydrocortisone). For FACS analysis, 10 ul of cell sample was diluted with 190u1 FACS buffer containing 5 nM ToPro-3.
Tumor Cell Infection and Culture
Cells were mixed in organoid medium with lentivirus at a target MOI of <1 in the presence 4 ug/ml polybrene, and incubated for 1 hour at room temperature. The suspension was then spun, the pellet resuspended in a minimal volume of organoid medium, and then plated onto collagen sponges (Ethicon) for 3D culture (
Sponge Harvest
Sponges were digested for 15 min at 37C with lmg/ml Collagenase IV in DMEM-F12. Analysis of the re-isolated cells demonstrated the outgrowth of the small tumor-derived tumoroids/organoids (
Cancer Cell Line Culture
A549 lung carcinoma cells (American Type Culture Collection) were grown in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% (v/v) fetal bovine serum, 1× Glutamax, and 1× antibiotic/antimycotic.
DNA Preparation, PCR, and Next-Generation Sequencing (NGS)
Genomic DNA was isolated using the Zymo Quick-DNA Miniprep Plus kit. 5 ug of purified genomic DNA was used as input for first round PCR amplification using the Q5 2X Master Mix and primers specific to the lentiviral vector. 10% of the resulting first round reaction products was then used as input for the second round of PCR amplification, utilizing barcoding primers to allow multiplex NGS readout. Samples were analyzed on the Illumina MiSeq using standard Illumina sequencing primers (Admera).
Sequence Analysis
Read1 sequences corresponding to the PCR barcodes were used for de-multiplexing, generating single-sample FASTA files containing gRNA readcounts. Sequencing data was analyzed using the CRISPRCloud2 platform, generating both CPM-normalized readcounts as well as statistical analysis of gRNA abundance based beta-binomial modeling. Data were visualized as ‘volcano’ plots (DataGraph), describing the relationship between statistical significance and fold-change in gRNA abundance. Typically, comparison was made between gRNA abundance immediately following lentiviral transduction and at the end of the in vitro culture period.
Analysis of dropout frequency using library screening in the A549 lung cancer cell line demonstrated clear loss of gRNAs corresponding to known essential genes (e.g. TOP2A, TUBG1 and others), while non-targeting control gRNAs demonstrated no corresponding decrease in abundance (
Analysis of dropout frequency using library screening in primary human melanoma tumor sample demonstrated clear loss of gRNAs corresponding to a known melanoma therapeutic vulnerability (e.g. BRAF), while non-targeting control gRNAs demonstrated no corresponding decrease in abundance (
Oncology drugs targeting presumptive cancer therapeutic vulnerabilities identified in Example 3, are tested in an in vivo animal model of the patient's cancer. Drugs that show efficacy for treating the cancer in the animal model are selected for treating the patient's cancer.
While preferred embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the scope of this application. Various alternatives to the embodiments described herein may be employed in practicing the scope of this application.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/865,047, filed Jun. 21, 2019, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/38503 | 6/18/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62865047 | Jun 2019 | US |
