Intracellular genomic transplant and methods of therapy
Inventors
Moriarity, Branden • Webber, Beau • Largaespada, David • Choudhry, Modassir • Rosenberg, Steven A.
Assignees
Intima Bioscience Inc • University of Minnesota System • US Department of Health and Human Services
Publication Number
US-11925664-B2
Publication Date
2024-03-12
Expiration Date
2036-07-29
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Abstract
Genetically modified compositions, such as non-viral vectors and T cells, for treating cancer are disclosed. Also disclosed are the methods of making and using the genetically modified compositions in treating cancer.
Core Innovation
Despite significant advances in cancer therapeutics, many tumor types remain resistant to chemotherapy, radiotherapy, or biotherapy, particularly in advanced stages where surgical options are not feasible. Genetic engineering of lymphocytes to recognize molecular tumor targets has achieved remarkable tumor remissions but has been limited mainly to hematologic cancers. The broader application to solid tumors is limited by the absence of identifiable molecules uniquely expressed by tumor cells and the lack of molecules that can specifically bind tumor targets to mediate destruction.
The invention discloses genetically modified compositions including non-viral vectors and T cells for treating cancer. The methods involve identifying cancer-specific T Cell Receptors (TCRs) targeting unique immunogenic mutations in a patient's cancer, and inserting transgenes encoding these TCRs into T cells using non-viral gene integration methods such as CRISPR, TALEN, transposon-based, ZEN, meganuclease, or Mega-TAL systems. This approach enables precise genomic modification by integrating TCR transgenes into gene loci, including immune checkpoint genes like PD-1, disrupting their function while introducing cancer-specific receptors.
The engineered cells, which can be primary immune cells such as T cells, stem cells, or progenitor cells, may comprise single or multiple TCR chains forming functional receptors recognizing antigens, including neoantigens identified by whole-exome sequencing. The process can involve homologous recombination facilitated by recombination arms complementary to genomic loci. Furthermore, homology-directed repair efficiency can be enhanced using homologous recombination enhancers such as viral proteins (E1B55K, E4orf6), ligase inhibitors (SCR7), or small molecule enhancers. Additionally, methods to reduce cellular toxicity from exogenous polynucleic acids include use of modifying compounds targeting innate immune sensing pathways to improve viability and function of modified cells.
Claims Coverage
The patent contains multiple claims focusing on methods for non-viral genomic engineering of human immune cells by integrating exogenous immune receptor sequences and disrupting endogenous genes using RNA-guided nuclease systems.
Integration of exogenous TCR or CAR with immune checkpoint gene disruption
A method of ex vivo or in vitro modifying human immune cells comprising contacting the cells with a polynucleic acid encoding an exogenous T cell receptor or chimeric antigen receptor that binds a cancer antigen, a ribonuclear protein complex (including a Cas protein and guide RNA) targeting a gene locus selected from SEQ ID NOS: 75-86 to perform genomic disruption reducing protein production from that locus, and a homologous recombination enhancer that suppresses non-homologous end joining.
Multiplexed gene disruption using multiple ribonuclear protein complexes
The method can comprise contacting cells with a second or third ribonuclear protein complex comprising Cas protein and guide RNA targeting additional gene loci, including T Cell Receptor Alpha Constant and Beta loci, for multiplex gene editing to disrupt multiple target genes.
Use of viral vector delivery system for exogenous receptor sequence
Delivery of the exogenous TCR or CAR encoding polynucleic acid via a viral vector.
Electroporation method for introducing ribonuclear protein complex
Employing electroporation to deliver the Cas protein and guide RNA ribonuclear protein complex to the human immune cells, with electroporation optionally preceding polynucleic acid delivery.
Use of specific Cas protein variant and cell culture conditions
Use of Cas9 as the Cas protein, culturing genomically modified cells in IL-2, IL-7, IL-15 or combinations thereof, and expanding the modified cells.
Targeting of immune cells including T cells and NK cells
Modifying human immune cells comprising T cells including CD3+, CD4+, CD8+ subsets or natural killer cells.
Binding specificity of exogenous receptors
Engineering of cell receptors to bind cancer antigens such as NY-ESO-1 or LAGE-1, targeting solid cancer cells like melanomas or liquid cancers such as multiple myeloma.
Composition of homologous recombination enhancers
Employing homologous recombination enhancers that inhibit KU70, KU80, DNA Ligase IV, or comprising adenoviral proteins E1B55K or E4orf6 to facilitate gene integration.
The claims cover methods for non-viral genomic engineering of human immune cells to integrate exogenous antigen receptors targeting cancer, combined with disruption of immune checkpoint or T cell receptor genes using RNA-guided Cas nucleases, enhanced by homologous recombination enhancers, with provisions for multiplex gene editing, electroporation delivery, and expanded culture conditions.
Stated Advantages
The disclosed compositions and methods provide high efficiency gene transfer and expression of transgenes in human immune cells, including improved cell survival rates.
The approach favors homology directed repair mechanisms over non-homologous end joining, enhancing precision in genomic modification.
Use of homologous recombination enhancers increases the efficiency of targeted integration of therapeutic receptors into immune cell genomes.
Introduction of modifying compounds reduces cytotoxicity from exogenous polynucleic acids, improving viability and expansion of engineered cells.
Non-viral delivery methods such as CRISPR and electroporation facilitate precise genetic modification without viral vector-related risks.
Documented Applications
Therapeutic treatment of cancer in humans by adoptive cellular transfer of engineered immune cells expressing tumor-specific T cell receptors or chimeric antigen receptors.
Application for treating solid tumors including melanoma and hematologic cancers such as multiple myeloma.
Engineering of T cells or tumor infiltrating lymphocytes (TILs) for improved immunotherapy, including configuration for autologous or allogeneic transplantation.
Potential use in reducing or eliminating immune checkpoint proteins like PD-1 and CTLA-4 to enhance anti-tumor immune responses.
Use of engineered cells in combination with cytokines (e.g., IL-2, IL-7, IL-15) during culture for improved expansion and function prior to patient administration.
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