Horizontal Gene Transfer in Multiple Myeloma and Development of a Non-Viral Gene Delivery System
Authors: Martinez-Medina Lourdes , Cinar Munevver , El-Rayes Bassel , Nagaraju Ganji , Gries Jean-Michel , Bernal-Mizrachi Leon 
Published: November 2021

Abstract
Multiple myeloma (MM) is a heterogeneous malignant plasma cell disorder with complex molecular and genetic abnormalities. While current therapies have improved outcomes in MM, many patients relapse. Part of the development of the resistance to treatment relates to the genetic heterogeneity of tumors that allows for the development of resistant clones. Our laboratory demonstrated that MM circulating tumor DNA (ctDNA) serves as a mechanism for horizontal gene transfer between cancer cells. Using novel 3-D imaging analysis methodologies, we demonstrated that ctDNA target specifically cancer cells of similar tumor phenotype. Furthermore, using novel in vitro models and whole genome sequencing, we identify certain transposable elements (TE) responsible of allowing DNA insertion into cancer cells. Additionally, our analysis discovered a particular retro-transposon sequence unique of multiple myeloma (MM zip-code, MMZC). After chemically synthesizing the MMZC and using flow cytometry methods, we demonstrated that a MMZC was capable of targeting and internalizing within MM cells after 30 minutes of incubation when compared to a control non-carrying TE sequence. Moreover, after culturing for 24 hours, we observed an increase on the number of cells that have internalized the MMZC. To evaluate genome integration, we ligated the MMZC to a linearized CMV-GFP and a linearized CMV-mCherry fragment prior to adding to MM cell culture. Within 12 hours, live imaging showed expression of GFP and mCherry in MM cells. This results are being validated in single cell sequencing. To evaluate a potential therapeutic potential of MMZC cargo delivery, we ligated MMZC to HSV-Tk-GFP linearized vector and tested the cell specificity targeting and killing effect of MMZC-HSV-Tk-GFP in MM, Pancreatic cancer and colon cancer cell lines. MMZC-HSV-Tk-GFP did not elicit a reduction in cell viability. However, MMZC-HSV-Tk-GFP reduce MM cell viability when ganciclovir was added to the cell culture. The capacity of gene delivery has been validated in-vivo using a MM xenograft mice. PCR evaluating for MMZC-HSV-Tk-GFP after 48 hours of tail injection demonstrated that tumor, but not other organs, amplified HSV-Tk. Furthermore, tumor tissue expressed GFP. To our knowledge this is the first description of the use of humanized non-viral synthetic technology capable of delivering therapeutic cargo material into MM cell.

Disclosures
Martinez-Medina: Kodikaz Therapeutic Solutions: Current Employment. El-Rayes: Bayer: Research Funding; Pfizer: Research Funding; Merck: Research Funding; Novartis: Research Funding; Boston biomedical: Research Funding; Bristol Myers Squibb: Research Funding; Astra Zeneca: Consultancy, Research Funding; exelixis: Consultancy; erytotech: Research Funding; dicephera pharmaceutical: Consultancy. Gries: Kodikaz Therapeutic Solutions: Current Employment; Feldan Therapeutics: Consultancy. Bernal-Mizrachi: Winship Cancer Institute of Emory University: Current Employment; Takeda Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions: Consultancy, Current holder of individual stocks in a privately-held company, Patents & Royalties; Bigene: Membership on an entity's Board of Directors or advisory committees.