Abstract: The present invention includes dead-guide RNA (dgRNA) libraries and methods of use in immunology and immunotherapy thereof. Also provided are engineered primary or chimeric antigen receptor (CAR) T cells that overexpress Prodh2, Ccnblip1, Sreklip1, or Wdr37 or a fragment thereof, and methods of use in immunology and immunotherapy thereof.

Abstract: Compositions and methods for efficient cellular genomic engineering that transduce diverse cell types with minimal toxicity, leading to efficient and stable genomic modifications are described. The compositions and methods are applicable to development of chimeric antigen receptor engineered T cell therapy (CAR-T). An exemplary method introduces a gene of interest into cells by introducing to the cell a viral vector including a transposon encoding the gene of interest and mRNA including a sequence that encodes transposase enzymes configured to mediate targeted integration of the transposon into the cellular genome, whereby the mRNA is introduced to the cell via electroporation. Also disclosed are genetically modified cells and pharmaceutical compositions and methods of use thereof for treating subjects having diseases or disorders.

Abstract: The present disclosure provides in one aspect a method of enhancing an immune response in a subject in need thereof. In certain embodiments, the method comprises administering to the subject an effective amount of a gene silencing system comprising a plurality of gRNAs, wherein the gene silencing system decreases expression of at least one endogenous immunosuppressive gene in a target cell, thereby enhancing the immune response. Also included are methods and compositions for enhancing anti-tumor immune responses in subject in need thereof.

Abstract: Compositions and methods for improved ACT are provided. Compositions of CAR-T cells including CAR modified by fusion with one or more copies of a polypeptide including from 20 to 44 amino acids from the transmembrane and/or cytosolic domain of CTLA-4 (CT) are described. CAR-CT-T cells have reduced trogocytosis, reduced T-cell fratricide, enhanced tumor antigen presentation and overall enhanced anti-tumor efficacy as compared with CAR-T cells lacking the CT domain(s). In preferred embodiments, CAR-TC fusion peptides include a CT domain having 2 copies of the YVKM motif. The compositions and methods provide enhanced CAR-T cell therapy for cancer, auto-immune disease as well as other disease and disorders. Also disclosed are genetically modified cells and pharmaceutical compositions and methods of use thereof for treating subjects having diseases or disorders.

Abstract: The present invention includes compositions and methods for identification of membrane targets for enhancement of T cell activity against a disease, disorder or condition, and/or enhancing T cell anti-tumor activity in a subject in need thereof. In some embodiments, the disease is cancer. In further embodiments, the cancer is glioblastoma (GBM) or breast cancer.

Abstract: The current disclosure includes coronavirus vaccines that protect against pathogenic coronavirus species, as well as their variants. In certain embodiments, SARS-CoV-2 variant specific and multivalent coronavirus vaccines are described. The vaccines typically include a modified mRNA which encodes at least one coronavirus derived immunogen, such as a spike protein or a fragment thereof. The mRNA can be encapsulated into lipid nanoparticles or other carriers and formulated as pharmaceutical compositions which can be used to generate an immune response to coronavirus in a subject. The vaccines can be used to elicit potent B and T cell responses against SARS-CoV-2 variants and to confer protective immunity against SARS-CoV-2, as well as other pathogenic coronavirus species such as SARS-CoV and/or MERS-CoV.

Abstract: The present invention relates to in vivo methods for modeling tumor formation and/or tumor evolution comprising the use of eukaryotic cells in which one or more genetic target locus has been altered by the CRISPR/Cas system, and which cells are transplanted in non-human eukaryote as a model system for tumor formation and tumor evolution. In particular in vivo genetic screening methods for identifying genes involved in tumorigenesis and metastasis are disclosed. The invention further relates to kits and components for practicing the methods, as well as materials obtainable by the methods, in particular tumor and metastasis samples and cells or cell lines derived therefrom. The invention also relates to diagnostic and therapeutic methods derived from the information obtained in the modeling methods.

Abstract: The present disclosure provides a set of high-throughput methods for rapid and efficient identification and generation of monoclonal antibodies. Also provided are highly potent human and humanized monoclonal antibodies and antigen-binding fragments and bispecific antibodies which are capable of binding to a wild-type or variant SARS-COV-2 spike protein receptor-binding domain (RBD), isolated nucleic acids and expression vectors encoding the antibodies and antigen-binding fragments and bispecific antibodies, cells comprising the nucleic acids and/or expression vectors, and methods for detecting, diagnosing, and/or neutralizing SARS-COV-2 and for treating at least one sign or symptom of a condition, disorder, or disease caused by SARS-COV-2 infection, including COVID-19.

Abstract: The present invention includes compositions and methods for T cell genome editing and screening in vivo. In certain aspects, the invention includes an sgRNA library for genome-scale mutagenesis.

Abstract: Compositions and methods for cellular genome engineering that permit simple and efficient targeted knock-in of a CAR and simultaneous knockout of individual genes are described. The compositions and methods are especially applicable to massively parallel engineering, selection, and identification of CAR T cell variants exhibiting a desired phenotype. AAV vectors containing crRNA and CAR expression cassettes and homology arms for targeted genomic integration thereof are provided. Also provided are libraries containing a plurality of AAV vectors and methods of use thereof in screens for identifying desirable CAR T cell variants. Methods of treatment using CAR T cell variants exhibiting improvements in one or more phenotypes are also provided.

Abstract: The present invention includes compositions and methods comprising viral vector systems for multiplexed activation of endogenous genes as immunotherapy and viral-based immune-gene therapy.

Abstract: The present invention includes compositions and methods for identification of membrane targets for enhancement of T cell activity against a disease, disorder or condition, and/or enhancing T cell anti-tumor activity in a subject in need thereof. In some embodiments, the disease is cancer. In further embodiments, the cancer is glioblastoma (GBM) or breast cancer.

Abstract: The present invention includes compositions and methods for T cell genome editing and screening in vivo. In certain aspects, the invention includes an sgRNA library for genome-scale mutagenesis.

Abstract: The present invention includes compositions and methods for enhancing T cell based immunotherapy. In certain aspects, the invention includes modified T cells and inhibitors of Dhx37 for use in enhancing T cell based immunotherapy and treating cancer.

Abstract: Disclosed are compositions and methods for cellular genome engineering that permit simple, efficient, and versatile permutations of combinatorial or simultaneous knockout and knock-in genomic modifications. An exemplary method includes modifying the genome of a cell by introducing to the cell a Cpf1 endonuclease and one or more AAV vectors encoding one or more crRNAs that direct the endonuclease to one or more target genes. The AAV vectors further contain one or more HDR templates that provide a sequence that encodes a reporter gene, a chimeric antigen receptor (CAR), or combinations thereof, and sequences homologous to one or more target sites. Also disclosed are pharmaceutical compositions containing genetically modified cells and methods of use thereof in treating a subject having a disease or disorder, such as cancer. The disclosed compositions and methods are especially applicable to development of enhanced chimeric antigen receptor engineered T cell therapy (CAR-T).

Abstract: The invention involves inducing 3-50 or more mutations (e.g., any whole number between 3 and 50 of mutations, with it noted that in some embodiments there can be up to 16 different RNA(s), e.g., sgRNAs each having its own a promoter, in a vector, such as AAV, and that when each sgRNA does not have its own promoter, there can be twice to thrice that amount of different RNA(s), e.g., sgRNAs, e.g., 32 or even 48 different guides delivered by one vector) in transgenic Cas9 eukaryotes to model genetic disease, e.g. cancer. The invention comprehends testing putative treatments with such models, e.g., testing putative chemical compounds that may be pharmaceutically relevant for treatment or gene therapy that may be relevant for treatment, or combinations thereof. The invention allows for the study of genetic diseases and putative treatments to better understand and alleviate a genetic disease or a condition, e.g., cancer.

Abstract: The present invention includes dead-guide RNA (dgRNA) libraries and methods of use in immunology and immunotherapy thereof. Also provided are engineered primary or chimeric antigen receptor (CAR) T cells that overexpress Prodh2, Ccnblip1, Sreklip1, or Wdr37or a fragment thereof, and methods of use in immunology and immunothereapy thereof.

Abstract: The present invention includes compositions and methods for multiplexed genome editing and screening in vivo. In certain aspects, the invention includes an CCAS library for multiplexed genome-scale mutagenesis.

Abstract: The present invention includes compositions and methods for enhancing homology directed repair (HDR) and/or reducing non-homologous end joining (NHEJ) in a cell following CRISPR-mediated editing.

Abstract: The present invention includes compositions and methods for treating or preventing cancer. Embodiments include cell-based and viral vector-based vaccines that utilize gene expression activation systems to augment the product of endogenous genes to treat or prevent cancer.