Generation of CD19-Targeted CAR-NK Cells Derived from Primary Sources Using CRISPR/AAV

• Grant number: 24/22467-2
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree
• Start date: May 01, 2025
• End date: October 31, 2025
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Virginia Picanço e Castro
• Grantee: Camilly Melo Garcia Ferreira
• Supervisor: Dean Anthony Lee
• Host Institution: Hemocentro de Ribeirão Preto. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil
• Institution abroad: Nationwide Children's Hospital, United States
• Associated to the scholarship: 24/01305-4 - Development of integrated strategies for lentiviral particles purification, BP.MS

Abstract

Natural Killer (NK) cells are a promising alternative for chimeric antigen receptor (CAR) therapy over CAR-T cells. Unlike CAR-T cells, CAR-NK cells retain their intrinsic ability to recognize tumor cells through their receptors. Additionally, NK cells have less restrictive HLA compatibility requirements and do not have the potential to cause graft-versus-host disease (GVHD). This makes CAR-NK cells ready for allogeneic therapeutic use (off-the-shelf). Allogeneic CAR therapies represent a solution to limitations inherent in autologous processes. Given the significance of this therapeutic approach and the enhanced safety linked to primary NK cell sources compared to cell lines such as NK-92, it is necessary to improve the applicability of primary-sourced NK cells, particularly achieving higher levels of transduction and expansion. Primary NK cells require activation signals to promote robust growth and expansion, which can be challenging to achieve ex vivo. To address this, we opted to use feeder cells, which have been shown to provide essential co-stimulatory signals and cytokine support, leading to effective and sustained ex vivo proliferation of NK cells. The CSTX002 cell line, developed by Professor Lee's lab, is an engineered version of K562 feeder cells designed to express co-stimulatory molecules 4-1BBL and membrane-bound IL-21 (mbIL-21). These enhancements provide essential activation signals, making CSTX002 highly effective in promoting robust and scalable expansion of primary NK cells, positioning it as a critical tool for advancing CAR-NK therapies. Despite this, a significant challenge persists: primary NK cells exhibit resistance to lentiviral transduction, which is the method currently used in our laboratory for introducing CAR constructs. To address this limitation, our collaborative project aims to test an innovative gene modification platform developed by Prof. Dean Lee's group. This new approach combines Cas9/RNP electroporation with AAV transduction, a method proven to enable precise, site-specific insertion of CAR genes in primary human NK cells. We will compare this technique with our current lentiviral vector (LV) method to determine which strategy yields superior transduction efficiency and CAR expression, ultimately optimizing the genetic modification process for primary NK cells.