Compositions and methods comprising viral vector systems for multiplexed activation of endogenous genes as immunotherapy and viral-based immune-gene therapy

1 . A method of developing a cancer immunotherapy, the method comprising:

a) administering a clustered regularly interspaced short palindromic repeats activation (CRISPRa) system comprising a single guide RNA (sgRNA) library targeting a plurality of genes to a plurality of cancer cells obtained from a human subject, thereby generating a plurality of modified cancer cells in which the expression of the plurality of targeted genes is increased as compared to an unmodified cancer cell from the subject,

b) administering the plurality of modified cancer cells to an immunocompetent non-human mammal whereby the non-human mammal develops tumors from the plurality of modified cancer cells,

c) isolating tumor tissue from the non-human mammal, wherein modified cancer cells comprising increased expression of targeted genes that enhance immune recognition by the non-human mammal's immune system are depleted in the isolated tumor tissue,

d) determining the sgRNAs and thereby the plurality of targeted genes that are decreased in cells of the isolated tumor tissue as compared to an unmodified cancer cell from the subject, wherein determining the plurality of targeted genes in the cells of the isolated tumor tissue comprises nucleotide sequencing and analysis, and

e) designing a cancer immunotherapy that increases the expression of targeted genes determined in step d) comprising packaging open reading frames (ORFs) encoding the targeted genes into a vector;

wherein the sgRNA library comprises every nucleic acid sequence of SEQ ID NOs. 86-192 or SEQ ID NOs. 193-411; and

wherein the cancer immunotherapy is selected from the group consisting of Multiplexed Activation of Endogenous Genes as an Immunotherapy (MAEGI) and direct ORF-based viral immune gene therapy.

2 . The method of claim 1 , wherein the sgRNA library comprises every nucleic acid sequence of SEQ ID NOs. 193-411.

3 . The method of claim 1 , wherein the CRISPRa system comprises a vector comprising the nucleotide sequence set forth in SEQ ID NO: 1.

4 . The method of claim 1 , wherein the vector is an adeno-associated viral (AAV) vector or an adenoviral vector.