CN-112823011-B - Gene targets for T cell-based immunotherapy

Abstract

Provided herein are genetically modified T cells that exhibit increased proliferation when stimulated as compared to wild-type T cells, methods of producing such T cells, and methods of using the T cells to treat diseases such as cancer.

Inventors

• A. MASON
• E. Sylvester
• J. Canevali
• A. Ashworth

Assignees

• 加利福尼亚大学董事会

Dates

Publication Date

20260508

Application Date

20190709

Priority Date

20180709

Claims (18)

1. 1. A genetically modified primary hematopoietic cell, wherein the primary hematopoietic cell is a T cell comprising a genetic modification of a gene that inhibits expression or activity of a polypeptide product encoded by a RASA2 gene, wherein expression or activity of the polypeptide product is inhibited by at least 60% as compared to a control wild-type hematopoietic cell, and inhibition of expression exhibits increased cell proliferation, wherein expression or activity of the polypeptide product encoded by the RASA2 gene is inhibited using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a transcription activator-like effector nuclease (TALEN) system, a zinc finger protease system, or a meganuclease system.
2. 2. The genetically modified primary hematopoietic cell of claim 1, wherein the genetic modification of the gene inactivates the RASA2 gene.
3. 3. The genetically modified primary hematopoietic cell of claim 1, wherein the T cell is a cd8+ T cell or a cd4+ T cell.
4. 4. The genetically modified primary hematopoietic cell of claim 1, wherein inhibition of expression increases proliferation induced by T cell receptor signaling.
5. 5. The genetically modified primary hematopoietic cell of claim 1, wherein the cell is obtained from a subject having cancer.
6. 6. The genetically modified primary hematopoietic cell of claim 5, wherein the cancer is melanoma.
7. 7. The genetically modified primary hematopoietic cell of any one of claims 1 to 6, wherein Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is used to inhibit the expression or activity of a polypeptide product encoded by the RASA2 gene.
8. 8. The genetically modified primary hematopoietic cell of any one of claims 1-6, wherein the expression or activity of the polypeptide product encoded by the RASA2 gene is inhibited using a transcription activator-like effector nuclease (TALEN) system, a zinc finger protease system, or a meganuclease system.
9. 9. A population of cells comprising the genetically modified primary hematopoietic cell of any one of claims 1-6.
10. 10. Use of a population of genetically modified primary hematopoietic cells for the preparation of a medicament for treating melanoma in a subject, wherein the genetically modified primary hematopoietic cells are T cells comprising a genetic modification of a gene that inhibits expression or activity of a polypeptide product encoded by a RASA2 gene, wherein expression or activity of the polypeptide product is inhibited by at least 60% compared to a control wild-type hematopoietic cell, and inhibition of expression exhibits increased cell proliferation, wherein expression or activity of the polypeptide product encoded by the RASA2 gene is inhibited using a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, a transcription activator-like effector nuclease (TALEN) system, a zinc finger protease system, or a meganuclease system.
11. 11. The use of claim 10, wherein the genetic modification of the gene inactivates the RASA2 gene.
12. 12. The use of claim 10 or 11, wherein the T cells are cd8+ or cd4+ T cells.
13. 13. The use of claim 10 or 11, wherein expression of a polypeptide product encoded by the RASA2 gene in the genetically modified primary hematopoietic cell is inhibited using a CRISPR system, a TALEN system, a zinc finger nuclease system or a meganuclease system.
14. 14. The use of claim 10 or 11, wherein CRISPR cells are used to inhibit the expression of a polypeptide product encoded by the RASA2 gene.
15. 15. The use of claim 10 or 11, wherein the T cells are obtained from the subject.
16. 16. A method of producing the genetically modified primary hematopoietic cell of claim 1, the method comprising: Inhibiting expression or activity of a RASA2 gene in one or more cells of a population of hematopoietic cells obtained from a patient, wherein the hematopoietic cells are T cells, and wherein the expression or activity of a polypeptide product encoded by the RASA2 gene is inhibited using a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, a transcription activator-like effector nuclease (TALEN) system, a zinc finger protease system, or a meganuclease system, and Expanding the hematopoietic cell population ex vivo.
17. 17. The method of claim 16, wherein the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is used to inhibit the expression or activity of a polypeptide product encoded by the RASA2 gene.
18. 18. The method of claim 16 or 17, further comprising selecting hematopoietic cells in which RASA2 gene expression is inhibited prior to expanding the hematopoietic cell population ex vivo.

Description

Gene targets for T cell-based immunotherapy Cross Reference to Related Applications This application claims priority from U.S. provisional patent application No. 62/695,672 filed on 7.7.9, 2018, which is incorporated herein by reference. Background Cytotoxic T cells play an important role in immune-mediated tumor control and autoimmunity. Immunotherapy, such as checkpoint blockade or engineered cell-based therapies, is radically altering cancer treatment, achieving a sustained response in some patients with other refractory malignancies. However, although significant effects are achieved in some patients, most patients do not respond to available immunotherapy. Next generation adoptive cell therapies are being developed using CRISPR-Cas9 genome engineering. Cas9 ribonucleoprotein can be delivered to primary human T cells to knock out checkpoint genes efficiently (Ren et al, 2017; rupp et al, 2017; schumann et al, 2015) or even to overwrite endogenous genomic sequences (Roth et al). While deletion of a typical checkpoint gene encoding PD-1 may enhance the response to some cancers (Ren et al, 2017; rupp et al, 2017), an expanded set of targets would provide additional therapeutic opportunities. Progress in immunotherapy depends on further insight into the genetic program that determines how T cells react when they encounter their target antigens. Promising gene targets may enhance cell proliferation and effective effector responses upon stimulation. In addition, immunosuppressive cells and soluble molecules such as cytokines and metabolites can accumulate within tumors and block effective anti-tumor T cell responses. Gene targets that affect T cells' ability to overcome the immunosuppressive tumor microenvironment can extend adoptive cell therapy to solid organ cancers. Animal models and cell line studies for decades have identified modulators of T cell inhibition and activation, but there is still a lack of systematic strategies to fully analyze the function of genes that regulate human T cell responses. Gene knockdown using a selected RNA interference library is directed to a target that enhances proliferation of antigen-reactive T cells in vivo in a mouse model (Zhou et al, 2014). Recently, CRISPR-Cas9 began a new era of functional genetics research (Doench, 2018). Large libraries of single guide RNAs (sgrnas) are easily designed to target genomic sequences. Transduction of cells with lentiviruses encoding these sgrnas produced pools with different genomic modifications that could be tracked by the sgRNA sequences in the integrated provirus. This approach has been used in a cell line engineered for stable Cas9 expression and in a Cas9 transgenic mouse model (Parnas et al, 2015; shang et al, 2018). Pooled CRISPR screens have revealed gene targets in human cancer cells that regulate T cell immunotherapy responses (Manguso et al, 2017; pan et al, 2018; patel et al, 2017). Disclosure of Invention The present disclosure is based (in part) on a novel method, namely, sgRNA lentiviral transfection (SLICE) with Cas9 protein electroporation, to identify modulators of stimulatory responses in primary human T cells. Whole genome loss of function screening identified important T cell receptor signaling components and genes that negatively regulate proliferation upon stimulation. Targeted ablation of individual candidate genes validated hits (hit) and identified perturbations (perturbations) that enhanced cancer cell killing. SLICE coupled to single cell RNA-seq reveals a characteristic stimulus-response gene program altered by critical genetic perturbation. SLICE whole genome screening is also useful for identifying mediators of immunosuppression, revealing genes that control the adenosine signaling response. Accordingly, provided herein are hematopoietic cells, such as stem cells and T cells, modified to inhibit expression of a target gene. Thus, for example, the genetic modifications described in the present disclosure can be used to modulate cd8+ T cell proliferation and function. In one aspect, provided herein is a genetically modified hematopoietic cell comprising a genetic modification to a T cell inhibitory gene that inhibits the expression or activity of a polypeptide product encoded by the T cell inhibitory gene, wherein the expression or activity of the polypeptide product is inhibited by at least 60% as compared to a control wild-type hematopoietic cell. In some embodiments, genetic modification to a T cell inhibitory gene inactivates the gene. In some embodiments, the genetically modified hematopoietic cell is a hematopoietic stem cell. In some embodiments, the genetically modified hematopoietic cell is a hematopoietic T cell. In some embodiments, the T cell is a cd8+ T cell or a cd4+ T cell. In some embodiments, clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems are used to suppress T cell inhibitory genes. Alternatively, a transcription activator-like effector nuclease (TALEN) syst