WO2025010729A1

WO2025010729A1 - Methods of preparing chimeric antigen receptor engineered cells

Info

Publication number: WO2025010729A1
Application number: PCT/CN2023/107269
Authority: WO
Inventors: Ming Kuen Patrick TANG
Original assignee: Chinese University of Hong Kong CUHK
Current assignee: Chinese University of Hong Kong CUHK
Priority date: 2023-07-07
Filing date: 2023-07-13
Publication date: 2025-01-16
Anticipated expiration: 2026-01-07

Abstract

Provided is a method of preparing chimeric antigen receptor (CAR) engineered cells. The method comprises the steps of: (i) incubating a CRISPR plasmid comprising at least a portion of a CRISPR-mediated gene editing system and a CAR sequence with a cell culture comprising target cells; and (ii) delivering an ultrasound to the cell culture by an ultrasound delivery system comprising an ultrasound transducer, thereby delivering the CRISPR plasmid to the target cells. The method can achieve a high transfection efficiency, maintain genomic integrity and ensure stable and robust CAR expression in engineered cells.

Description

METHODS OF PREPARING CHIMERIC ANTIGEN RECEPTOR ENGINEERED CELLS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to, and the benefit of, U.S. Provisional Application having Serial No. 63/512, 287 filed on 7 July 2023. The entire contents of the foregoing application are hereby incorporated by reference in its entirety for all purposes.

REFERENCE TO SEQUENCE LISTING

This application contains one or more sequence listings in computer readable form, which are incorporated herein by reference in their entireties. This application contains a sequence listing which has been submitted electronically in ST. 26 (xml) format and is hereby incorporated by reference in its entirety. Said ST. 26 copy, created on 29 Jun, 2023, is named “C064.004. PROUS. xml” and is 91.5 kilobytes in size.

FIELD OF INVENTION

This application relates to methods of genetic cell engineering. In particular, this application relates to methods of preparing chimeric antigen receptor engineered cells and their uses.

BACKGROUND OF INVENTION

The advent of chimeric antigen receptor (CAR) engineering has revolutionized the field of immunotherapy, with significant advances in treating cancers such as leukemia and showing potential for addressing conditions beyond cancers including cardiac injury, atherosclerosis, diabetes, and osteoarthritis. However, the high costs and safety concerns of conventional virus-based cell engineering methods have limited the widespread adoption of CAR immunotherapy such as CAR T-cell (CAR-T) and CAR Natural killer cell (CAR-NK) immunotherapy.

In 2021, there were 186400 new cases of leukemia in the US, with about 20%of patients receiving second-line therapy, thus approximately 37280 potential candidates for CAR cell-based therapy. Improved gene editing methods and systems that are affordable, safe, and efficient are highly desired.

SUMMARY OF INVENTION

Disclosed herein are novel methods, systems, kits, and uses that are useful for engineering immune cells to express CAR such as the processes for delivering genetic materials into recipient cells, preparation and usage of the kits and systems thereof, and intermediates used in preparing/assembling/engineering/modifying the cells, kits and systems thereof. In certain embodiments, provided is a novel approach which combines the mechanical ultrasound-guided microbubble system (USMB) with the precision and accuracy of CRISPR/Cas9 guided Homology-directed repair knockin (KI) system, creating a novel solution USMB-KI for highly efficient CAR-T/NK cell engineering.

In some embodiments, provided is a method of preparing chimeric antigen receptor (CAR) engineered cells, comprising the steps of: (i) incubating a CRISPR plasmid comprising at least a portion of a CRISPR-mediated gene editing system and a CAR sequence with a cell culture comprising target cells; and (ii) delivering an ultrasound to the cell culture by an ultrasound delivery system comprising an ultrasound transducer, thereby delivering the CRISPR plasmid to the target cells.

In some embodiments, provided is a method of preparing chimeric antigen receptor (CAR) engineered cells, comprising the steps of: (i) incubating a CRISPR plasmid comprising at least a portion of a CRISPR-mediated gene editing system and a CAR sequence with a cell culture comprising target cells with about 1 x 10⁶ cells in about 5 ml harvested at log phase in a cell culture flask; (ii) positioning an ultrasound transducer below the cell culture flask containing the cell culture; (iii) applying an ultrasound gel between the ultrasound transducer and the bottom surface of the cell culture flask; (iv) delivering an ultrasound to the cell culture by an ultrasound delivery system comprising an ultrasound transducer, wherein the ultrasound transducer is configured to deliver an ultrasound having a frequency range of the frequency range is about 1 MHz, the energy output is about 1 W/cm², and the duration is about 60 seconds; thereby delivering the CRISPR plasmid to the target cells; and (v) refreshing medium of the cell culture with a complete growth medium and incubating the target cells for about two days.

There are many advantages of the invention. Certain embodiments provide a novel and highly efficient platform to achieve ex vivo andin vitro genetic cell engineering in a virus-free manner. In certain embodiments, the methods, systems, kits, and uses can generate engineered immune cells showing persistent CAR expression with very high cell viability (>80%) and yield (>60%) by conducting a clean and simple process in a closed system. In certain embodiments, the genetic modification is strategically placed at a defined genomic insertion site considered safe, therefore effectively preserving the genome integrity of the engineered cells compared to conventional viral methods.

In some embodiments, the provided methods, systems, kits, and uses can be used for producing CAR engineered cells with persistent CAR overexpression for immunotherapy in clinic as well as a convenient tool for conducting gene transfer on cells with low proliferation rate in the laboratory. In certain embodiments, the provided methods, systems, kits, and uses offer superior transfection rates, making them a highly versatile virus-free cell engineering platform for cancer therapy and beyond.

In some embodiments, the provided methods, systems, kits, and uses comprise a modified ultrasound-guided microbubble (USMB) platform and CRISPR/Cas9 knock-in system to persistently overexpress single or multiple genes in targeted cells. In certain embodiments, USMB capable to transfer the plasmids of CRISPR/Cas9 gene editing system into cells with very low sonication energy in vitro, allowing the engineered CAR sequence to be precisely incorporated into the host genome at a defined safe insertion site, the AAVS1 locus, resulting in permanent and high-level expression of the CAR protein on the engineered cells. This platform allows multi-CAR delivery in a single application, offering flexible and cost-effective CAR engineering for immunotherapy.

In some embodiments, the methods, systems, kits, and uses described herein can be applied to engineered cell-based immunotherapy for human diseases beyond blood cancers (e.g., leukemia) , such as solid tumors, tissue fibrosis, inflammation, autoimmune diseases, diabetes, aging-related conditions. etc. In certain embodiments, the simplicity of the provided methods and systems tackle the safety concerns and high costs of virus-based methods, providing a cost-effective, virus-free alternative that increases the accessibility of CAR-T/NK therapy, particularly in developing countries.

In certain embodiments, the methods, systems, kits, and uses described herein have superior transfection efficiency in contrast to current virus-free methods with low efficiency and high cytotoxicity, which is suitable for conducting gene transfer on cells with extremely low proliferation rate in vitro, e.g., stem cells, primary immune cells, senescent cells, etc.

In some embodiments, the methods, systems, kits, and uses described herein offer a superior transfection rate (>60%) which outperforms existing virus-free methods with transfection rates of 2-15%. The markedly enhanced production yield of engineered cells is particularly helpful for immunotherapy of patients in advanced disease stages, with few immune cells available for producing own CAR-engineered cells.

In some embodiments, the methods, systems, kits, and uses described herein provide a superior cell viability (>80%) and yield (>60%) which outperforms existing virus-free methods with cell viability below 15%. In certain embodiments, the low sonication energy applied is low and the cell recovery time is very short which largely shortens the production time needed to generate sufficient engineered cells for therapy.

In some embodiments, the methods, systems, kits, and uses described herein produce cells with high expression level of CAR by permanently incorporating the CAR sequence (s) into the host genome using CRISPR/Cas9 technology, markedly enhancing the persistence and stability of the engineered cells' anticancer capacity and activity.

In some embodiments, the methods, systems, kits, and uses described herein preserve the genome integrity of the engineered cells, as the gene insertion is precisely designed to incorporate into the host genome at a defined safe genomic harbor, the AAVS1 locus. This markedly improves the safety of the engineered cells compared to existing methods, which may introduce potential carcinogenesis due to random insertion of the CAR sequence into the engineered cells.

In some embodiments, the methods, systems, kits, and uses described herein enable multi-CAR engineering through a single transfection by simultaneously delivering multiple CARs or genes without size restrictions, providing greater flexibility in CAR engineering and cutting costs in producing multiple virus particles for each gene.

In some embodiments, it was surprisingly discovered that transfection efficiency is further improved when microbubble reagent is absent during the ultrasound mediated gene transfer. The absence of transfection agents during gene transfer markedly reduces the allergenic risk of the microbubble reagents to the end-users of the engineered cells. In certain embodiments, the methods, systems, kits, and uses described herein facilitate cost-effective large-scale manufacturing as no transfection agents are needed to achieve gene transfer.

In some embodiments, the provided methods, systems, kits, and uses described herein (e.g., the USMB-KI platform) address the safety concerns that have long plagued viral methods, such as insertional mutagenesis and transactivation of proto-oncogenes. In certain embodiments, methods, systems, kits, and uses not only achieve a high transfection efficiency comparable to viral methods but also maintain genomic integrity and ensure stable and robust CAR expression in engineered cells. As a result, the performance and persistence of these cells are significantly improved. The platform can represent a paradigm shift in immunotherapy, overcoming limitations of traditional virus-based methods and offering a safer, more effective alternative.

In some embodiments, the provided methods, systems, kits, and uses described herein provide a cost-effective solution for CAR-T/NK cell production, making the therapy more accessible for clinical use. The platform also can bypass numerous safety tests and tightly regulated standard operating procedures associated with virus-based CAR engineering in current good manufacturing practices (cGMP) laboratories, significantly reducing the cost of cell production and quality assurance. In certain embodiments, methods, systems, kits, and uses have the potential to transform CAR-T/NK immunotherapy into an affordable therapeutic option in the clinic.

In certain embodiments, the provided methods, systems, kits, and uses described herein have wide-ranging implications for the future of immunotherapy, with potential applications in cancer treatment and other human diseases globally. As a highly efficient, safe, and cost-effective virus-free platform for producing clinical-grade CAR-T/NK cells, provided methods and systems can mark a turning point in the field, overcoming the limitations of conventional protocols and paving the way for innovative and accessible therapies. In certain embodiments, the methods, systems, kits, and uses described herein can change the immunotherapy landscape, ushering in a new era of safer, more efficient, and affordable treatments for cancer and other life-threatening diseases.

In some embodiments, the provided methods, systems, kits, and uses described herein offer significant improvements over existing CAR engineering methods in terms of transfection efficiency, cell recovery period, persistence of CAR expression and overall safety.

In some embodiments, the provided methods, systems, kits, and uses described herein provide a superior transfection rate (>60%) and cell viability (>80%) compared to existing virus-free methods, ensuring higher yields of functional immune cells. In certain embodiments, the methods, systems, kits, and uses described herein achieve permanent and high-level CAR expression, enhancing the persistence and anticancer activity of the engineered cells. In certain embodiments, the methods, systems, kits, and uses described herein enable multi-CAR engineering with a single transfection, increasing flexibility in CAR engineering. In certain embodiments, the methods, systems, kits, and uses described herein precisely insert the CAR sequences into host genome at defined position, and achievable without any transfection reagent. Its simplicity and cost-effectiveness largely accelerate the development of engineered cell-based immunotherapy for human diseases.

In some embodiments, the provided methods, systems, kits, and uses described herein have additional advantages over lentiviral methods. In certain embodiments, methods, systems, kits, and uses are non-viral approaches, which eliminate the risk of insertional mutagenesis and potential complications associated with viral vector integration into the host genome and thus have improved safety.

In certain embodiments, the provided methods, systems, kits, and uses described herein are relatively simple and easy to perform compared to the complex process of producing viral vectors, thus requiring fewer steps and less stringent quality control measures.

In certain embodiments, the provided methods, systems, kits, and uses described herein have low immunogenicity, i.e. they do not induce a significant immune response, whereas viral vectors can trigger immune reactions that may lead to clearance of the transduced cells or even lethal side effects.

In certain embodiments, the provided methods, systems, kits, and uses described herein have broad applicability that they can be used to transfect a wide range of cell types, including primary cells and cell lines that may be resistant to viral transduction.

In certain embodiments, the provided methods, systems, kits, and uses described herein allows for the delivery of large plasmids or multiple plasmids simultaneously, whereas viral vectors often have size limitations for the genetic material they can carry.

In certain embodiments, the provided methods, systems, kits, and uses described herein are generally more cost-effective than viral methods, especially when working with large numbers of cells or performing multiple transfections.

In certain embodiments, the provided methods, systems, kits, and uses described herein can be completed within minutes, whereas the process of viral transduction may take longer time from a few hours to overnight.

In certain embodiments, with provided methods, systems, kits, and uses, researchers can easily control the amount of DNA introduced into the cells, allowing for fine-tuning of gene expression levels.

BRIEF DESCRIPTION OF FIGURES

Figure 1 is a vector map of plasmid #1 (CAR19 Donor DNA, see Table 1) according to an example embodiment.

Figure 2 is a vector map of plasmid #2 (pSUPER-EGFP-puro, see Table 1) , according to an example embodiment.

Figure 3 is a vector map of plasmid #3 (pSLCAR-CD19-CD3z, see Table 1) , according to an example embodiment.

Figure 4 is a vector map of plasmid #5 (USMB-KI EV Donor, see Table 1) , according to an example embodiment.

Figure 5 is a vector map of PX458-AAVS1 gRNA, according to an example embodiment.

Figure 6A shows a schematic diagram which illustrates an example system (i.e., ultrasound-guided microbubble (USMB) delivery system) , which actively delivers the capsuled plasmids into targeted cells within minutes via sonication-induced sonoporation in vitro, according to an example embodiment.

Figure 6B shows USMB effectively delivered plasmid #2 into the NK-92 cells compared to the free plasmid treated cells (control) , according to an example embodiment. Data represent 3 independent in vitro experiments.

Figures 6C-D show the gene transfer efficiency of USMB was much higher than conventional lipofectamine transfection by the dramatic increase in green fluorescent protein (GFP) expression at both mRNA (Figure 6C) and protein levels (Figure 6D) in independent in vitro experiments, according to an example embodiment. Data represent 3 independent in vitro experiments.

Figure 6E shows USMB resulted in a better and acceptable cell viability compared to electroporation.

Figure 7A is a schematic diagram of the molecular structure of a αCD19-CAR, according to an example embodiment.

Figure 7B shows USMB was far more efficient than lipofectamine for transferring plasmid #3 (αCD19-CAR overexpressing plasmids) into the NK-92 cells, evaluated by mRNA expression level of CAR19 with real-time polymerase chain reaction (PCR) , according to an example embodiment.

Figure 7C shows the production of anticancer effector interferon gamma (INF-γ) was markedly increased in the NK-92 cells treated with USMB-mediated plasmid #3 transfer (USMB-CAR19) under cocultivation with a CD19+ Pre-B leukemia cell line 697 cells, compared to the control transferred with plasmid #4 only (USMB-EV) , according to an example embodiment. Data represent 3 independent in vitro experiments.

Figures 7D, 7D’ and 7E show the USMB-CAR19 treated NK-92 cells demonstrated a better CD19-dependent anticancer activity, confirmed by their enhanced cytotoxicity against CD19-positive leukemia 697 (Figure 7D) and RS4; 11 cells (Figure 7D’) but not the CD19-negative A549 cells in vitro (Figure 7E) , according to an example embodiment. Data represent 3 independent in vitro experiments.

Figures 8A-B show USMB effectively transferred plasmid #3 into human T cell line Jurkat (Figure 8A) and results in >70%cell viability detecting by MTT assay (Figure 8B) , according to an example embodiment. Data represent 3 independent in vitro experiments.

Figures 8C-D show USMB successfully increased CAR19 expression (Figure 8C) and direct cancer-killing activity of Jurkat cells in vitro (n=3) (Figure 8D) , compared to the plasmid #4 control plasmid (pSUPER-puro) treated control, according to an example embodiment. Data represent 3 independent in vitro experiments.

Figures 8E-F show USMB demonstrated promising gene transfer efficiency on human peripheral blood derived primary T cells (Primary T) as conventional Lentivirus method (n=3) , evaluated by the expression levels of GFP mRNA (Figure 8E) and CAR19 mRNA (Figure 8F) , according to an example embodiment. Data represent 3 independent in vitro experiments.

Figure 8G shows the enhanced anticancer activity of human primary T cells engineered from USMB was comparable to the conventional virus method. Data represent 3 independent in vitro experiments.

Figure 9A is a graphical illustration of an example ultrasound-guided microbubble knockin (USMB-KI) system, according to an example embodiment. Said system specifically insert CAR19 into a safe genomic site at AAVS1 in a virus-free manner by CRISPR/Cas9 gene knock-in technology, improving the safety and efficiency for gene insertion.

Figures 9B, 9C and 9C’s how USMB-KI-CAR19 not only showed superior gene transfer efficiency (Figure 9B) and also produced Jurkat (Figure 9C) and primary human peripheral blood mononuclear cells (Primary PBMC) cells with better cancer-killing activity than the original method (USMB-CAR19) in vitro (n=3) (Figure 9C’) , according to an example embodiment.

Figures 9D and 9D’s how the USMB-KI produced CAR-T Jurkat cells (USMB-KI-CAR19) that markedly inhibits the cancer abundance in mice with xenografts of human CD19⁺ leukemia 697 cells, compared to the mice received plasmid #4 transferred Jurkat cells (USMB-KI-EV) and saline treated control group in vivo (n=3) , according to an example embodiment. Figure 9D is a bioluminescence image of mice having xenograft of human CD19+ leukemia 697 cells upon receipt treatment with USMB-KI-CAR-19, USMB-KI-EV or saline (control) . Figure 9D’ is a graph representing the measurement of the cancer abundance expressed in nominal radiance ratio based om the bioluminescence imaging.

Figures 10A-B show employing ultrasound alone (US) surprisingly increased plasmid delivery into Jurkat (Figure 10A) and NK-92 cells (Figure 10B) substantially compared to the original method (USMB) in vitro (n=5) , according to an example embodiment. This improvement was evidenced by the elevated GFP mRNA expression detected by quantitative polymerase chain reaction (qPCR) .

DETAILED DESCRIPTION

As used herein and in the claims, the terms “comprising” (or any related form such as “comprise” and “comprises” ) , “including” (or any related forms such as “include” or “includes” ) , “containing” (or any related forms such as “contain” or “contains” ) , means including the following elements but not excluding others. It shall be understood that for every embodiment in which the term “comprising” (or any related form such as “comprise” and “comprises” ) , “including” (or any related forms such as “include” or “includes” ) , or “containing” (or any related forms such as “contain” or “contains” ) is used, this disclosure/application also includes alternate embodiments where the term “comprising” , “including, ” or “containing, ” is replaced with “consisting essentially of” or “consisting of” . These alternate embodiments that use “consisting of” or “consisting essentially of” are understood to be narrower embodiments of the “comprising” , “including, ” or “containing, ” embodiments.

For example, alternate embodiments of “a system comprising A, B, and C” would be “a system consisting of A, B, and C” and “a system consisting essentially of A, B, and C.”Even if the latter two embodiments are not explicitly written out, this disclosure/application includes those embodiments. Furthermore, it shall be understood that the scopes of the three embodiments listed above are different.

For the sake of clarity, “comprising” , including, and “containing” , and any related forms are open-ended terms which allows for additional elements or features beyond the named essential elements, whereas “consisting of” is a closed end term that is limited to the elements recited in the claim and excludes any element, step, or ingredient not specified in the claim.

As used herein, the singular forms “a” , “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Where a range is referred in the specification, the range is understood to include each discrete point within the range. For example, 1-7 means 1, 2, 3, 4, 5, 6, and 7.

As used herein, the term “about” is understood as within a range of normal tolerance in the art and not more than ±10%of a stated value. By way of example only, about 50 means from 45 to 55 including all values in between. As used herein, the phrase “about” a specific value also includes the specific value, for example, about 50 includes 50.

As used herein and in the claims, an “effective amount” , is an amount that is effective to achieve at least a measurable amount of a desired effect. For example, the amount may be effective to elicit an immune response, and/or it may be effective to elicit a protective response.

As used herein and in the claims, a “subject” refers to animals such as mammals, including, but not limited to, primates (e.g., humans) , cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In certain examples, the subject is a human.

As used herein, the term “treat, ” “treating” or “treatment” refers to methods of alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition either prophylactically and/or therapeutically.

As used herein, the term “CRISPR” or “clustered regularly interspaced short palindromic repeat” refers to a segment of genetic material found in or derived from the genomes of prokaryotes (such as some bacteria and archaea) that consists of repeated short sequences of nucleotides interspersed at regular intervals between unique sequences of nucleotides derived from the DNA of pathogens (such as viruses) which had previously infected the bacteria and that functions to protect the bacteria against future infection by the same pathogens.

As used herein, the term “Cas9” or “CRISPR associated protein 9” refers to a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNAs with complementarity to the single-guide RNA (sgRNA) .

As used herein, the terms “CRISPR/Cas9 system” , “CRISPR/Cas9 technology” or “CRISPR/Cas9 gene editing system” are interchangeable and refers to a gene editing technology involving the use of CRISPR and Cas9.

As used herein, the terms “gRNA” or “guide RNA” refers to a piece of genetic material such as RNA that functions as a guide for RNA-or DNA-targeting enzymes, with which it forms complexes.

As used herein, the terms “sgRNA” or “single-guide RNA” refers to a single genetic material such as gRNA molecule chain that contains CRISPR RNA (crRNA) and a trans-acting CRISPR RNA (tracrRNA) .

As used herein, the term “CRISPR plasmid” refers to a plasmid containing at least a portion of a CRISPR (such as CRISPR/Cas9) mediated gene editing system or construct. In some examples, the CRISPR plasmid further comprises one or more gene of interest or target sequence.

As used herein, the term “ultrasound” refers to a sound with frequencies greater than 20 kilohertz.

As used herein, the term “microbubbles” refers to spherical vesicles which can be used as ultrasound contrast/transfection agents.

As used herein, the term “ultrasound-guided microbubble” or “USMB” are interchangeable to refer to an example in vitro gene transfer system or method as described in any one of the example embodiments herein, involving steps and systems of delivering a plasmid or a nucleic acid fragment to the target cells by an ultrasound delivery system. For clarity sake, the system or method may or may not use transfecting agent or microbubble.

As used herein, the term “knockin” or “KI” are interchangeable to refer to a genetic engineering technology that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found within the locus.

As used herein, the term “empty vector” or “EV” are interchangeable to refer to a vector or plasmid in which gene of interest (such as CAR19) is not present.

As used herein, the term “chimeric antigen receptor” or ‘CAR” refers to a receptor protein that has been engineered to give cells modified therewith the ability to target a specific antigen.

As used herein, the term “chimeric antigen receptor (CAR) engineered cells” , “chimeric antigen receptor engineered cells” , or “CAR engineered cells” refers to cells that contain chimeric antigen receptor (CAR) sequence (s) . Examples include, but not limited to, cells whose genome includes CAR sequence (s) , cells that express one or more CAR, cells that overexpress one or more CAR, cells that have been genetically modified to express CAR, and cells that have been genetically modified to express CAR on their surfaces.

As used herein, the term “CAR19” refers to an anti-CD19 chimeric antigen receptor. For example, a schematic diagram of CAR19 is shown in Figure 7A.

As used herein, the term “AAVS1” refers to a genetic locus adeno-associated virus integration site 1.

As used herein, the term “T cell” or “T lymphocyte” refers to a type of white blood cell, including, but not limited to, a helper T cell, a cytotoxic T cell, and any other subset of T cells.

As used herein, the term “Jurkat” refers to an immortalized line of human T lymphocyte cells.

As used herein, the term “primary T cell” refers to cell (s) directly expanded from the extracted T lymphocytes.

As used herein, the term “NK cell” or “natural killer cell” refers to a type of white blood cells cell that has granules with enzymes that can kill target cells.

As used herein, the term “NK-92” refers to an immortalized cell line that has the characteristics of natural killer cells.

As used herein, the term “primary peripheral blood mononuclear cell” or “PBMC” refers to a heterogeneous population of blood cells with a single round nucleus.

As used herein, the term “refresh” , “refreshing” , or “refreshment” when referring to medium or growth medium refers to a process of maintaining the growth medium of a cell culture in a certain condition for the health and viability of the cell population, including, but not limited to, replacing an old cell culture medium with a fresh growth medium, transferring cells from an old culture vessel to a new culture vessel, adding fresh growth medium into an existing cell culture medium. In some embodiments, the fresh growth medium that is added is a different type of growth medium, such as a complete growth medium.

As used herein, the term “complete medium” or “complete growth medium” refers to a medium for a cell culture that contains the supplemental nutrients as well as the basic nutrients sufficient to support the growth requirements of the cell culture.

As used herein, the term “cancer” refers to a proliferative disorder caused or characterized by the proliferation of cells which have lost susceptibility to normal growth control. A “cancer” may include tumors and any other proliferative disorders. Cancers of the same tissue type usually originate in the same tissue, and may be divided into different subtypes based on their biological characteristics. Examples include but not limited to leukemias, lymphomas, breast, stomach, uterine, ovarian, bladder cancer, and lung cancers.

Although the description referred to particular embodiments, the disclosure should not be construed as limited to the embodiments set forth herein.

NUMBERED EMBODIMENTS

Embodiment 1. A method of preparing chimeric antigen receptor (CAR) engineered cells, comprising the steps of: (i) incubating a CRISPR plasmid comprising at least a portion of a CRISPR-mediated gene editing system and a CAR sequence with a cell culture comprising target cells; and (ii) delivering an ultrasound to the cell culture by an ultrasound delivery system comprising an ultrasound transducer, thereby delivering the CRISPR plasmid to the target cells.

Embodiment 2. The method of embodiment 1, wherein the target cell is an immune cell.

Embodiment 3. The method of embodiment 2, wherein the immune cell is a Natural Killer (NK) cell or a T cell. In certain embodiments, the target cells are stem cells, primary immune cells (e.g. macrophage, dendritic cell, neutrophil) , senescent cells, etc.

Embodiment 4. The method of any one of the preceding embodiments, wherein a total energy applied by the ultrasound transducer is in the range of 10J/ml to 40J/ml to a cell culture housed in a container with a cell density in the range of 1x10⁵ to 5x10⁶ cells/ml.

Embodiment 5. The method of any one of the preceding embodiments, wherein the cell culture has about 1 x 10⁶ cells in about 5ml of cell culture, and the total energy applied to the cell culture by the ultrasound transducer is 50 to 200 J.

Embodiment 6. The method of any one of the preceding embodiments, wherein the ultrasound transducer is configured to deliver an ultrasound having a frequency range of about 0.5-5 MHz, an energy output of about 0.5-5 W/cm², and a duration of about 15-120 seconds.

Embodiment 7. The method of embodiment 6, wherein the frequency range is about 1 MHz, the energy output is about 1 W/cm², and the duration is about 60 seconds.

Embodiment 8. The method of any one of the preceding embodiments, wherein the CRISPR plasmid comprises AAVS1 targeting sequences.

Embodiment 9. The method of any one of the preceding embodiments, wherein the CRISPR plasmid comprises an αCD19-CAR (CAR19) sequence.

Embodiment 10. The method of embodiment 9, wherein the CRISPR plasmid comprises sequence ID NO. 1.

Embodiment 11. The method of any one of embodiments 1-10, further comprising a step prior to step (i) of mixing the CRISPR plasmid with a transfection agent and incubating for around 15 minutes at room temperature.

Embodiment 12. The method of embodiment 11, wherein the transfection agent comprises microbubble. In certain embodiments, the microbubble solution comprises sulfur hexafluoride microbubbles. In certain embodiments, the microbubble solution used is Sonovue (Bracco Imaging) .

Embodiment 13. The method of any one of Embodiments 1-10, further comprising a step prior to step (i) of preparing the CRISPR plasmid in saline without a transfection agent.

Embodiment 14. The method of any one of the preceding embodiments, wherein the cell culture is harvested at the log growth phase.

Embodiment 15. The method of any one of the preceding embodiments, wherein prior to step (ii) , further comprises a step of: positioning the ultrasound transducer below a cell culture flask containing the cell culture.

Embodiment 16. The method of embodiment 15, wherein prior to step (ii) , further comprises a step of: applying ultrasound gel between the ultrasound transducer and the bottom surface of the cell culture flask.

Embodiment 17. The method of any one of the preceding embodiments, after step (ii) , further comprises the step of: refreshing medium of the cell culture and incubating the target cells for about two days.

Embodiment 18. The method of any one of the preceding embodiments, wherein the target cell culture is a mammalian cell culture.

Embodiment 19. The method of embodiment 18, wherein the mammalian cell culture is a human cell culture.

Embodiment 20. A method of preparing chimeric antigen receptor (CAR) engineered cells, comprising the steps of: (i) incubating a CRISPR plasmid comprising at least a portion of a CRISPR-mediated gene editing system and a CAR sequence with a cell culture comprising target cells with about 1 x 10⁶ cells in about 5 ml harvested at log phase in a cell culture flask; (ii) positioning an ultrasound transducer below the cell culture flask containing the cell culture; (iii) applying an ultrasound gel between the ultrasound transducer and the bottom surface of the cell culture flask; (iv) delivering an ultrasound to the cell culture by an ultrasound delivery system comprising an ultrasound transducer, wherein the ultrasound transducer is configured to deliver an ultrasound having a frequency range of the frequency range is about 1 MHz, the energy output is about 1 W/cm², and the duration is about 60 seconds; thereby delivering the CRISPR plasmid to the target cells; and (iv) refreshing medium of the cell culture with a complete growth medium and incubating the target cells for about two days.

Embodiment 21. The method of embodiment 20, further comprising the step of preparing the CRISPR plasmid in saline.

Embodiment 22. The method of embodiment 20, further comprising the step of mixing the CRISPR plasmid with about 9mg/ml microbubble solution in saline and incubating for around 15 minutes at room temperature. In certain embodiments, the microbubble solution comprises sulfur hexafluoride microbubbles. In certain embodiments, the microbubble solution used is Sonovue (Bracco Imaging) .

Embodiment 23. The method of any one of the preceding embodiments, wherein the human cell culture is selected from the group consisting of NK-92, Jurkat, Primary T cells, and primary peripheral blood mononuclear (PBMC) cells. In certain embodiments, the target cells are stem cells, primary immune cells (e.g., T cell or NK cell) , etc.

Embodiment 24. The method of any one of the preceding Embodiments, wherein the method is virus-free and achieves a transfection rate of >60%and/or a cell viability rate >80%.

Embodiment 25. A method of treating a disease, health condition or disorder in a subject in need of treatment, comprising the step of: administrating a therapeutically effective amount of CAR engineered cells to the subject, wherein the CAR engineered cells made according to a method of any one of embodiments 1-24.

Embodiment 26. The method of Embodiment 25, wherein the disease, health condition or disorder is selected from the group consisting of leukemia, blood cancer, solid tumors, tissue fibrosis, inflammation, autoimmune disease, diabetes and aging-related condition.

EXAMPLES

Provided herein are examples that describe in more detail certain embodiments of the present disclosure. The examples provided herein are merely for illustrative purposes and are not meant to limit the scope of the invention in any way. All references given below and elsewhere in the present application are hereby included by reference.

Example 1: USMB mediated gene transfer in vitro

1.1 General procedures of USMB mediated gene transferin vitro

Materials:

- SonoVue microbubbles (Bracco Imaging)

- Ultrasound equipment with an ultrasound transducer for cell culture vessels

- In vitro cell culture system (including cell culture flasks)

- Cell culture media and supplements for respective target cell culture:

a. NK-92: MEM Alpha Medium (Gibco, #12571063) , supplemented with 12.5%fetal bovine serum (Gibco, #10500064) , 12.5%horse serum (Gibco, #26050088) , 20ng/ml IL-2 (Biolegend, #791906)

b. Jurkat: RPMI 1640 Medium (Gibco, #11875119) supplemented with 10%fetal bovine serum (Gibco, #10500064)

c. Primary T: ImmunoCult^TM-XF T Cell Expansion Medium (Stemcell Technologies, #10981) supplemented with 10ug/mL IL-2 (Biolegend, #791906)

d. Primary peripheral blood mononuclear cells (PBMC) : RPMI 1640 Medium (Gibco, #11875119) supplemented with 10%fetal bovine serum (Gibco, #10500064) and 20ng/ml IL-2 (Biolegend, #791906)

- Plasmids of interest as indicated in Table 1

Table 1. Summary of plasmids of interest used in USMB mediated gene transfer in vitro

Methods:

i) Preparation of loaded microbubbles as a transfection agent:

The lyophilized powder of SonoVue microbubbles was reconstituted by mixing 5ml 0.9%saline with 45mg microbubble with shaking for 20 seconds to prepare a homogeneous solution. 20 μg plasmids were combined with 1ml microbubbles for each transfection by mixing and incubating for 15 minutes at room temperature. The mixture was vortexed briefly before adding it to the cells to be transfected (NK-92, Jurkat, Primary T and PBMC 1x10⁶ cells/5ml) .

ii) Cell culture preparation:

Selected target cells of interest were cultured according to the respective methods described in Examples 2-6 below. Target cell culture obtained as a result was harvested at the log growth phase (5-7x10⁶/20ml growth medium) . 1 x 10⁶ cells/5ml per flask was seeded for each transfection.

iii) Ultrasound-guided delivery (USMB) :

1ml loaded microbubbles from step (i) was added to the target cell culture with 1 x 10⁶ cells from step (ii) . The cell culture flask was placed in a suitable holder, ensuring that the ultrasound transducer was positioned below the flask with complete contact between the bottom surface of the flask and the transducer. A thin layer of ultrasound gel was applied between the transducer and cell culture flask surface. The ultrasound settings were adjusted to the optimized range, depending on the cells of interest, usually ranging between 0.5-5 MHz (frequency) , 0.5-5 W/cm² (energy) , and 15-120 seconds (time) , to ensure the release of the payload with minimal effects on cell viability. For the cell strains mentioned above, 1 MHz, 1W/cm², 60 seconds conditions were applied. The culture medium was replaced with complete medium 3 hours after the delivery process. Figure 6A shows a schematic diagram of an example USMB gene transfer delivery method and system, which actively delivers the capsuled plasmids into targeted cells within minutes via sonication-induced sonoporation in vitro.

1.2 General approach to evaluate USMB mediated gene transferin vitro

The target cells generated based on methods of Example 1.1 were collected two days after the delivery process. The uptake of the plasmids was evaluated in terms of GFP or CAR19 expression using appropriate techniques, including fluorescence microscopy, real-time quantitative polymerase chain reaction (qPCR) , and flow cytometry as described in the following Examples 2-6. The function of CAR-engineered cells was assessed in terms of cell viability, gene expression changes, or functional assays.

Table 2 is a summary of the primer sequences used during real-time polymerase chain reaction (PCR) in Examples 2-6.

Table 2. Primer sequences for real-time qPCR in Examples 2-6

2.1 Quantification of GFP+ NK-92 cells between control and USMB-transferred groups under fluorescent microscopy

Materials:

- In vitro cell culture system (cell culture flasks)

- Cell culture media and supplements for NK-92 cells: MEM Alpha Medium (Gibco, #12571063) , supplemented with 12.5%fetal bovine serum (Gibco, #10500064) , 12.5%horse serum (Gibco, #26050088) , 20ng/ml IL-2 (Biolegend, #791906)

- Fluorescent microscope

- Microscope slides and cover slips

- 4%paraformaldehyde solution

- Plasmid (s) of interest: plasmid #2 (pSUPER-EGFP-puro; see Figure 2) and USMB delivery system (according to the method/system of Example 1.1)

Methods:

i) Cells preparation:

NK-92 cells were cultured and received USMB-based delivery of plasmid #2 according to the methods of Example 1.1 (USMB-GFP) . A control group (control) was prepared with cells cultured under the same conditions which received the addition of plasmid #2, yet without USMB-mediated delivery as depicted in Example 1.1.

ii) Fixation and mounting:

48 hours after delivery, cells were gently washed with phosphate-buffered saline (PBS) to remove any residual culture media. Cells were fixed by adding a 4%paraformaldehyde solution to each flask and incubating for 15 minutes at room temperature. Fixed cells were washed twice with PBS. Cells were centrifuged at 300 x g for 5 minutes to obtain a cell pellet. The supernatant was removed, and the cell pellet was resuspended in a small volume of PBS. A small drop of the cell suspension was placed on a microscope slide and covered with a cover slip.

iii) Fluorescent microscopy:

Cells were observed under a fluorescent microscope using excitation and emission filters for GFP. Images of representative fields of view for both the control and USMB-GFP groups were captured. The number of GFP+ cells in each field of view for both groups was counted. The percentage of GFP+ cells was calculated by dividing the number of GFP+cells by the total number of cells in each field of view.

Results:

The USMB-GFP group observed an outstanding increase (～75%) in gene transfer efficiency of plasmid #2 into NK-92 cells compared to the control group (<10%) (see Figure 6B) . Results showed that USMB a superior transfection rate compared to the control cells treated with free plasmid.

2.2 Quantification of GFP mRNA expression between control, lipofectamine, and USMB-transferred groups of NK-92 cells with qPCR

Materials:

- In vitro cell culture system (cell culture flasks)

- Plasmid (s) of interest: Plasmid #2 (pSUPER-EGFP-puro) and USMB delivery system (according to the method/system of Example 1.1)

- Lipofectamine 2000 (Thermo Fisher Scientific, #11668019)

- RNA extraction kit (Qiagen, QRNeasy Mini Kit, #74104)

- cDNA synthesis kit (Promega, GoScript^TM Reverse Transcription System, #A5001)

- qPCR master mix (Bio-rad, iTaq^TM UniversalGreen Supermix, #1725122)

- Primer pair for Green Fluorescent Protein (GFP) (SEQ ID NO: 5-6) and housekeeping gene (GAPDH) (SEQ ID NO: 9-10)

- Real-time PCR instrument (Applied Biosystems QuantStudio)

- Microcentrifuge tubes and pipettes

Methods:

i) Cell culture preparation and transfection:

NK-92 cells were cultured and received USMB-based delivery of plasmid #2 according to the method of Examples 1.1 (USMB-GFP) . A control group (control) was prepared with cells cultured under the same conditions and received the addition of plasmid #2, yet without USMB-mediated delivery as depicted in Example 1.1. Another group of cells (lipo-GFP) was transfected with plasmid #2 using Lipofectamine reagent according to the manufacturer’s instructions.

ii) RNA extraction:

After 48 hours post-transfection, cells from each group (control, lipo-GFP, and USMB-GFP) were harvested and transferred to microcentrifuge tubes. Total RNA was extracted from the cells using an RNA extraction kit (QRNeasy Mini Kit, #74104) , according to the manufacturer’s instructions. Briefly, samples were first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate was then loaded onto the RNeasy silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. Pure, concentrated RNA of each sample was eluted in water.

iii) cDNA synthesis:

The concentration and purity of the extracted RNA were determined using a spectrophotometer (NanoDrop^TM 2000, Thermo-fisher) with default setting detecting wavelengths of 230/260/280nm with blank of pure water. cDNA was synthesized from an equal amount of total RNA (1 μg) for each sample using a cDNA synthesis kit following the manufacturer’s protocol. Briefly 1μg RNA, 0.5μg oligo (dt) primer, were mixed in 1X reaction buffer with 1.5mM MgCl2, PCR Nucleotide Mix (0.5mM each dNTP) , 20 units RecombinantRibonuclease Inhibitor, 1μl GoScript^TM Reverse Transcriptase, and ran with program 25℃ (5 mins) , 42℃ (40 mins) , 70℃ (15 mins) .

iv) qPCR analysis:

A qPCR reaction mix containing cDNA, qPCR master mix, and primers for GFP and GAPDH genes (Table 2) were prepared. The qPCR reactions (triplicate for each sample) were run in a real-time PCR instrument with program: 95℃ (30s) and 50 cycles of 95℃ (5s) to 60℃ (30s) .

v) Data analysis:

The relative expression of GFP mRNA in each group was calculated using the 2^(-ΔΔCT) method, normalizing to the housekeeping gene expression. GFP mRNA expression levels between the control, lipo-GFP, and USMB-GFP groups were compared using one-way ANOVA followed by post-hoc tests.

Results:

Results showed that gene transfer efficiency of USMB-GFP group (about 400000 fold change) was surprisingly much higher than the conventional lipofectamine transfection (lipo-GFP) group (<50000 fold change) , showing by the dramatic increase in GFP expression at mRNA level (see Figure 6C) in NK-92 cells of USMB group over Lipo or control groups. Results showed that the gene transfer efficiency of USMB is surprisingly higher than that of conventional lipofectamine transfection methods at mRNA level.

2.3 Flow cytometric quantification of GFP+ NK92 cells between control, lipofectamine, and USMB-transferred groups

Materials:

- In vitro cell culture system (cell culture flasks)

- Cell culture media and supplements

- Lipofectamine 2000 (Thermo Fisher Scientific)

- Phosphate-buffered saline (PBS)

- Flow cytometry buffer (Thermo Fisher Scientific)

- Flow cytometer (BD LSRFortessa)

- Flow cytometry analysis software (Cytobank)

Methods:

i) Cell culture preparation and transfection:

NK-92 cells were cultured and received USMB-based delivery of plasmid #2 according to methods of Example 1.1 (USMB-GFP) . A control group (control) was prepared with cells cultured under the same conditions and received the addition of plasmid #2, yet without USMB-mediated delivery as depicted in Example 1.1. Another group of cells (lipo-GFP) was transfected with plasmid #2 using Lipofectamine reagent according to the manufacturer’s instructions.

ii) Cell preparation for flow cytometry:

After 48 hours post-transfection, cells from each group (control, lipo-GFP, and USMB-GFP) were harvested and transferred to flow cytometry tubes. The cells were washed twice with PBS and resuspended in flow cytometry buffer to a final concentration of approximately 1 x 10⁶ cells/mL.

iii) Flow cytometry analysis:

Cell samples were acquired on a flow cytometer using the laser and filter settings for GFP detection (488 nm excitation, 530/30 nm emission) . A gating strategy selecting linear region in FSC-A vs. FSC-H plot was applied to exclude debris, doublets, and dead cells, and to identify the GFP+ cell population. The percentage of GFP+ cells for each sample (control, lipo-GFP, and USMB-GFP groups) was recorded. Flow cytometry data was analysed with flow cytometry analysis software (Cytobank, Beckman Coulter Life Sciences) in histogram mode of GFP-A channel to quantify the percentage of GFP+ cells in each group. The GFP+ cell populations between the control, lipo, and USMB groups were compared using one-way ANOVA followed by post-hoc tests.

Results:

Flow analysis results showed that USMB showed an excellent transfection rate of 63.12%, compared to 19.74%and 2.94%of lipofectamine and control delivery, respectively (see Figure 6D) . Results showed that the gene transfer efficiency of USMB is surprisingly higher than that of conventional lipofectamine transfection methods at protein level.

2.4 Evaluation of cell viability of transfected NK-92 cells between control, lipofectamine, electroporation, and USMB-transferred groups with MTT assay

Materials:

- In vitro cell culture system (cell culture plates, e.g., 96-well plates)

- Lipofectamine 2000 (Thermo Fisher Scientific)

- Electroporation system (Amaxa Nucleofector, Lonza bioscience)

- MTT reagent (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide)

- Dimethyl sulfoxide (DMSO)

- Plate reader with absorbance detection (BioTek 800TS)

Methods:

i) Cell culture preparation and transfection:

NK-92 cells were cultured, and the USMB-based delivery of plasmid #2 was performed according to Example 1.1 (USMB-GFP) . A control group (control) was prepared with cells cultured under the same conditions and received the addition of plasmid #2 yet without USMB-mediated delivery as depicted in Example 1.1. Another group of cells was transfected with the plasmids using Lipofectamine reagent according to the manufacturer’s instructions (lipo-GFP) . A third group of cells was transfected with the plasmids using an electroporation system according to the manufacturer’s instructions (electro-GFP) .

ii) MTT assay:

Cells were seeded in 96-well plates at a density of 1 x 10⁴ cells/well after delivery or transfection. 10 μL of MTT reagent (5 mg/mL in PBS) was added to each well, and the plate was incubated at 37℃ for 4 hours. After incubation, the medium was carefully removed by centrifugation (1500rpm, 5mins) , and 100 μL of DMSO was added to each well to dissolve the formazan crystals. The plate was gently shaken for 5-10 minutes to ensure complete dissolution of the formazan crystals.

iii) Absorbance measurement and data analysis:

The absorbance of each well was measured at 570 nm using a plate reader (BioTek 800TS) . The average absorbance for each group (control, lipo-GFP, electro-GFP, and USMB-GFP) was calculated, and the relative cell viability was determined by normalizing to the control group with formula:

Cell viability (%) = { [Absorbance (Control) –Absorbance (experiment group) ] / Absorbance (Control) } x 100.

The cell viability between the different groups was compared using one-way ANOVA followed by post-hoc tests.

Results:

USMB showed a better and acceptable cell viability (about 80%) compared to electroporation (electro-GFP) (< 10%) . The cell viability of USMB was comparable to the control and conventional lipofectamine group (See Figure 6E) . Results showed that USMB provide superior cell viability which outperforms the electroporation methods.

Example 3: Evaluating USMB-mediated CAR19 plasmid transfer (USMB- CAR19) into NK-92 cellsin vitro

3.1 qPCR quantification of CAR19 mRNA expression between control, lipofectamine, and USMB-transferred groups of NK-92 cells

Materials:

- In vitro cell culture system (cell culture flasks)

- Plasmid (s) of interest: plasmid #3 (pSLCAR-CD19-CD3z; see Figure 3) and USMB delivery system (according to the method/system of Example 1.1)

- Lipofectamine 2000 (Thermo Fisher Scientific)

- RNA extraction kit (Qiagen, QRNeasy Mini Kit, #74104)

- qPCR master mix (Bio-rad, iTaq^TM Universal Green Supermix, #1725122)

- Primers for GFP (SEQ ID NO: 5-6) and housekeeping gene (GAPDH) (SEQ ID NO: 9-10)

- Real-time PCR instrument (Applied Biosystems QuantStudio)

- Microcentrifuge tubes and pipettes

Methods:

i) Cell culture preparation and transfection:

NK-92 cells were cultured and received USMB-based delivery of plasmid #3 according to Example 1.1 (USMB-CAR19) . A control group (control) was prepared with cells cultured under the same conditions with the addition of plasmid #3 but without USMB-mediated delivery. Another group of cells was transfected with plasmid #3 using Lipofectamine reagent according to the manufacturer’s instructions (lipo-CAR19) .

ii) RNA extraction:

48 hours after delivery or transfection, cells from each group (control, lipo-CAR19, and USMB-CAR19) were harvested and transferred to microcentrifuge tubes. Total RNA was extracted from the cells using an RNA extraction kit according to the manufacturer’s instructions. Briefly, Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in water.

iii) cDNA synthesis:

iv) qPCR analysis:

A qPCR reaction mix containing cDNA, qPCR master mix, and primers for CAR19 and GAPDH genes were prepared. The qPCR reaction (triplicate/sample) was run in a real-time PCR instrument with program: 95℃ (30s) and 50 cycles of 95℃ (5s) to 60℃(30s) .

v) Data analysis:

The relative expression of CAR19 mRNA in each group was calculated using the 2^(-ΔΔCT) method, normalizing to the housekeeping gene expression. The CAR19 mRNA expression levels between the control, lipofectamine, and USMB-transferred groups were analysed by one-way ANOVA followed by post-hoc tests.

Results:

USMB-CAR19 group showed a >300000 fold increase in CAR19 mRNA expression, which is much greater than the lipofectamine group (<100000 fold) (see Figure 7B) , evaluated by mRNA expression level of CAR19 with real-time PCR. Results showed that USMB was surprisingly far more efficient than lipofectamine for transferring αCD19-CAR (CAR19) overexpressing plasmids into the NK-92 cell at mRNA level.

3.2 Quantification of IFN-γ mRNA expression ofCAR19 transfected NK92 cells in contact with 697 cancer cells compared to EV transfected group

Materials:

- In vitro cell culture system (cell culture flask, 12-well plate)

- Cell culture media and supplements for NK92 (MEM Alpha Medium (Gibco, #12571063) , supplemented with 12.5%fetal bovine serum (Gibco, #10500064) , a 12.5%horse serum (Gibco, #26050088) , 20ng/ml IL-2 (Biolegend, #791906) ) and 697 cancer cells (RPMI 1640 Medium (Gibco, #11875119) supplemented with 10%fetal bovine serum (Gibco, #10500064) )

- Plasmid (s) of interest: plasmid #3 (pSLCAR-CD19-CD3z) and plasmid #4 (pSUPER-puro; control plasmid)

- USMB delivery system (according to the method/system of Example 1.1)

- RNA extraction kit (Qiagen, QRNeasy Mini Kit, #74104)

- qPCR master mix (Bio-rad, iTaq^TM UniversalGreen Supermix, #1725122)

- qPCR primer pairs for IFN-γ (SEQ ID NOs.: 11-12) : and reference gene (GAPDH) (SEQ ID NOs.: 9-10)

- Real-time PCR system (Applied Biosystems QuantStudio)

Methods:

i) Cell culture and transfection:

NK92 cells were cultured at 37℃ and 5%carbon dioxide (CO₂) and received plasmid #3 (USMB-CAR19) or plasmid #4 (USMB-EV) according to Example 1.1.697 cancer cells were cultured in RPMI1640 medium supplemented with 10%FBS in 37℃incubator with 5%CO₂.

ii) Co-culture of NK92 and 697 cancer cells:

48 hours after USMB delivery, transfected NK92 cells (USMB-CAR19 and USMB-EV control groups) were harvested and cell densities were adjusted to a 10: 1 effector-to-target (E: T) ratio. NK-92 (10⁵ cells/well) and 697 cancer cells (10⁴ cells/well) were seeded in 12-well plates and incubated for 24 hours.

iii) RNA extraction and cDNA synthesis:

After the co-culture incubation, cells were harvested, and total RNA was isolated using RNA extraction kit (QRNeasy Mini Kit, #74104) , according to the manufacturer’s instructions. Briefly, Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in water.

cDNA was synthesized from the extracted RNA with cDNA synthesis kit according to the manufacturer's instructions.

iv) Quantitative PCR:

qPCR reactions were prepared using the synthesized cDNA, qPCR master mix, and primer pairs specific for IFN-γ and GAPDH. qPCR reactions (triplicate/sample) were run in a real-time PCR system following the program: 95℃ (30s) and 50 cycles of 95℃(5s) to 60℃ (30s) .

v) Data analysis:

qPCR data were analyzed using the comparative Ct (ΔΔCt) method to determine the relative IFN-γ mRNA expression levels in CAR19-transfected NK92 cells compared to the EV-transfected control group. IFN-γ mRNA expression levels between CAR19-and EV-transfected NK92 cells with or without co-culture with 697 cancer cells were compared.

Results:

As shown in Figure 7C, production of anticancer effector interferon gamma (INF-γ) was markedly increased in the NK-92 cells treated with USMB-mediated CAR19 plasmid transfer (USMB-CAR19, >6 fold increase) under cocultivation with a CD19+ Pre-B leukemia cell line 697 cells, compared to the control transferred with plasmid #4 only (USMB-EV, <2 fold increase) . Results showed that USMB-mediated CAR19 plasmid transfer can produce CAR engineered cells with outstanding performance in expression of anticancer effector interferon gamma in NK-92 cells in mRNA level.

3.3 Quantification ofcancer cells killing activity of EV-and CAR19-transfected NK-92 cells against 697, RS4; 11 cells and A549 cells

Materials:

- In vitro cell culture system (96-well plates)

- Cell culture media and supplements for NK-92 (MEM Alpha Medium (Gibco, #12571063) , supplemented with 12.5%fetal bovine serum (Gibco, #10500064) , 12.5%horse serum (Gibco, #26050088) , 20ng/ml IL-2 (Biolegend, #791906) ) , 697, RS4; 11, and A549 cells (RPMI 1640 Medium (Gibco, #11875119) supplemented with 10%fetal bovine serum (Gibco, #10500064) )

- USMB delivery system (according to the method/system of Example 1.1)

- CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, #G1780)

Methods:

i) Cell culture and transfection:

NK92 cells were cultured and received USMB delivery of plasmid #3 (USMB-CAR19) or plasmid #4 (USMB-EV) control according to Example 1.697, RS4; 11, and A549 target cells were continuously cultured at log phase (5-8 x 10⁶ cells per 20ml) in RPMI1640 medium supplemented with 10%FBS at in 37℃ and 5%CO2.

ii) Co-culture of NK-92 and target cells:

48 hours after USMB delivery, transfected NK92 cells (USMB-CAR19 and USMB-EV control groups) were harvested and cell densities were adjusted to achieve the desired effector-to-target (E: T) ratios of 5: 1, 10: 1, and 20: 1.697, RS4; 11, and A549 target cells were seeded in 96-well plates. Transfected NK-92 cells (USMB-CAR19 and USMB-EV groups) were added to the target cells and the co-culture was incubated for 12 hours.

iii) Cytotoxicity assay:

After the co-culture incubation, the co-culture was centrifuged (2000rpm, 5mins) to obtain cell-free supernatant for cytotoxic assay. The Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay (#G1780) was performed according to the manufacturer’s instructions to measure the level of target cell death caused by NK-92 cells. Briefly, a reaction solution containing LDH substrate, cofactor, and catalyst was prepared, and added to the supernatant collected in a new 96-well plate. The plate was then incubated in the dark at room temperature for 20 minutes to develop colour for absorbance measurement at 490 nm with microplate reader.

iv) Data analysis:

The percentage of cancer cell-killing activity for each group (USMB-EV and USMB-CAR19 transfected NK-92 cells) at each E: T ratio (5: 1, 10: 1, and 20: 1) against 697, RS4; 11, and A549 target cells were calculated with below formula:

Statistical analysis (one-way ANOVA) was performed to compare USMB EV-and CAR19-transfected groups.

Results:

The USMB-CAR19 treated NK-92 cells showed a better CD19-dependent anticancer activity than USMB-EV treated cells, confirming by their enhanced cytotoxicity against CD19-positive leukemia 697 (Figure 7D) and RS4; 11 cells (Figure 7D’) but not the CD19-negative A549 cells in vitro (Figure 7E) . Results showed that the USMB-CAR19 has better CD19-dependent anticancer activity with NK-92 cells compared to control.

Example 4: Evaluating the performance of USMB in producing human CAR-T cells against conventional virus method

4.1 Quantification of GFP+ Jurkat T cells between control and USMB-transferred groups under fluorescent microscopy

Materials:

- In vitro cell culture system (cell culture flasks)

- Cell culture media and supplements for Jurkat: RPMI 1640 Medium (Gibco, #11875119) supplemented with 10%fetal bovine serum (Gibco, #10500064)

- Fluorescent microscope

- Microscope slides and cover slips

- 4%paraformaldehyde solution

- Plasmid (s) of interest) : plasmid #3 (pSLCAR-CD19-CD3z) and USMB delivery system (according to the method/system of Example 1)

Methods:

i) Cell preparation:

Jurkat cells were cultured and received USMB-based delivery of plasmid #3 according to Example 1.1 (USMB-CAR19) . A control group (control) was prepared with cells cultured under the same conditions and received the addition of plasmid #3 yet without USMB-mediated delivery.

ii) Fixation and mounting:

48 hours after delivery, cells were gently washed with phosphate-buffered saline (PBS) to remove any residual culture media. Cells were fixed by adding 4%paraformaldehyde solution to each flask and incubating for 15 minutes at room temperature. Fixed cells were washed twice with PBS. Cells were centrifuged at 300 x g for 5 minutes to obtain a cell pellet. The supernatant was removed, and the cell pellet was resuspended in a 10-50 μl of PBS. A small drop of the cell suspension was placed on a microscope slide and covered with a cover slip.

iii) Fluorescent microscopy:

Cells were observed under a fluorescent microscope using excitation and emission filters for GFP. Images of representative fields of view were captured for both the control and USMB-transferred groups. The number of GFP+ cells in each field of view for both groups was counted. The percentage of GFP+ cells was calculated by dividing the number of GFP+ cells by the total number of cells in each field of view.

Results:

USMB also effectively transferred GFP-expressing plasmids into human T cell line Jurkat, demonstrating a transfection rate of >60% (see Figure 8A) . Results showed that USMB has a superior transfection rate with human Jurkat T cells compared to the control cells treated with free plasmid.

4.2 Evaluation of cell viability ofJurkat T cell received USMB delivery with MTT assay

Materials:

- In vitro cell culture system (cell culture flasks, 96-well plates)

- Cell culture media and supplements for Jurkat cells: RPMI 1640 Medium (Gibco, #11875119) supplemented with 10%fetal bovine serum (Gibco, #10500064)

- Plasmid (s) of interest: plasmid #3 (pSLCAR-CD19-CD3z) and USMB delivery system according to the method/system of Example 1.1

- Dimethyl sulfoxide (DMSO)

- Plate reader with absorbance detection (BioTek, #800TS)

Methods:

i) Cell culture preparation and transfection:

Jurkat cells were cultured, and the USMB-based delivery of plasmid #3 was performed according to the method of Example 1.1 (USMB-CAR19) . A control group (control) was prepared with cells cultured under the same conditions and received the addition of plasmid #3 but without USMB-mediated delivery.

ii) MTT assay:

Cells were seeded in 96-well plates at a density of 1 x 10⁴ cells/well after USMB-delivery. 10 μL of MTT reagent (5 mg/mL in PBS) was added to each well, and the plate was incubated at 37℃ for 4 hours. After incubation, the medium was carefully removed by centrifugation, and 100 μL of DMSO was added to each well to dissolve the formazan crystals. The plate was gently shaken for 5-10 minutes to ensure complete dissolution of the formazan crystals.

iii) Absorbance measurement and data analysis:

The absorbance of each well was measured at 570 nm using a plate reader. The average absorbance for each group (control and USMB-CAR19) was calculated and normalized to the control group to determine the relative cell viability. The cell viability between groups was compared using statistical analysis (Student's t-test) .

Results:

>70%cell viability was achieved via USMB, as detected by MTT assay (Figure 8B) . Results showed that USMB produced a superior cell viability with human Jurkat T cells compared to the control cells treated with free plasmid.

4.3 Quantification of CAR19 mRNA expression and 697 cells killing activity of CAR19 transfected Jurkat T cells compared to EV transfected group

Materials:

- In vitro cell culture system (cell culture flasks, 96-well plates)

- Cell culture media for Jurkat T cells and 697 cells (see Example 1.1)

- Plasmid (s) of interest: plasmid #3 (pSLCAR-CD19-CD3z) and plasmid #4 (pSUPER- puro; control plasmid)

- USMB delivery according to the method/system of Example 1.1)

- qPCR master mix (Bio-rad, iTaq^TM UniversalGreen Supermix, #1725122)

- RNA extraction kit (Qiagen, QRNeasy Mini Kit, #74104)

- Real-time PCR instrument (Applied Biosystems, QuantStudio 7)

- Primers for CAR19 (SEQ ID NO: 57-8) and GAPDH (SEQ ID NO: 9-10)

- CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega)

Methods:

i) Cell culture and transfection:

Jurkat T cells were cultured and transfected with plasmid #3 (USMB-CAR19) or plasmid #4 (USMB-EV) using USMB delivery, according to the methods of Example 1.1. 697 target cells were cultured under RPMI1640 supplemented with 10%FBS.

ii) Quantification of CAR19 mRNA expression:

After 48 hours of USMB delivery, the transfected Jurkat T cells (USMB-CAR19 and USMB-EV groups) were harvested. Total RNA was isolated from the harvested cells using an RNA extraction kit, following the manufacturer's instructions. Briefly, Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in water. cDNA synthesis was performed using a reverse transcription kit, following the manufacturer's instructions. Briefly 1μg RNA, 0.5μg oligo (dt) primer, were mixed in 1X reaction buffer with 1.5mM MgCl2, PCR Nucleotide Mix (0.5mM each dNTP) , 20 units RecombinantRibonuclease Inhibitor, 1μl GoScript^TM Reverse Transcriptase, and ran with program 25℃ (5 mins) , 42℃ (40 mins) , 70℃ (15 mins) . qPCR was carried out using specific primers for CAR19 and GAPDH with program: 95℃ (30s) and 50 cycles of 95℃ (5s) to 60℃ (30s) to assess relative mRNA expression levels, and the data were analyzed using the comparative Ct (ΔΔCt) method as formula below:

ΔCt = Ct (CAR19) –Ct (GAPDH)

ΔΔCt = ΔCt (Sample) –ΔCt (Control average)

iii) Co-culture of Jurkat T cells and target cells:

The transfected Jurkat T cells (USMB-CAR19 and USMB-EV groups) were harvested and cell densities were adjusted to achieve the desired effector-to-target (E: T) ratios (5: 1, 10: 1, and 20: 1) . 697 target cells were seeded in 96-well plates. The transfected Jurkat T cells (USMB-CAR19 and USMB-EV groups) were added to the target cells, and the co-culture was incubated for 12 hours at 37℃ and 5%CO2.

iv) Cytotoxicity assay:

After the co-culture incubation, the co-culture was centrifuged to obtain cell-free supernatant for the cytotoxic assay. The Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay (#G1780) was performed according to the manufacturer’s instructions to measure the level of 697 cancer cell death caused by Jurkat T cells. Briefly, a reaction solution containing LDH substrate, cofactor, and catalyst was prepared, and added to the supernatant collected in a new 96-well plate. The plate was then incubated in the dark at room temperature for 20 minutes to develop colour for absorbance measurement at 490 nm with microplate reader.

v) Data analysis:

Statistical analysis of CAR19 mRNA expression and 697 cell-killing activity for each group (USMB-EV and USMB-CAR19 transfected Jurkat T cells) at each E: T ratio was performed using Student's t-test to compare the groups.

Results:

USMB-CAR19 surprisingly increased CAR19 mRNA expression (～800000 fold increase) (Figure 8C) and direct cancer-killing activity of Jurkat cells in vitro at all 3 E: T ratios (n=3) (Figure 8D) , compared to the plasmid #4 treated control (USMB-EV) . Results showed that USMB-mediated CAR19 plasmid transfer can produce CAR engineered cells with outstanding performance in expression of CAR19 in mRNA level, and also outstanding performance in cancer-killing activity with Jurkat cells.

4.4 Quantification of CAR19 mRNA expression and 697 cells killing activity of USMB-CAR19 transfected primary T cells (derived from human peripheral blood of healthy donor) compared to EV-and lentivirus-CAR19 transfected group

Materials:

- In vitro cell culture system (cell culture flasks, 96-well plate)

- Cell culture media and supplements for 697 cells (RPMI 1640 Medium (Gibco, #11875119) supplemented with 10%fetal bovine serum (Gibco, #10500064) ) and primary T cells (ImmunoCult^TM-XF T Cell Expansion Medium (Stemcell Technologies, #10981) supplemented with 10ug/mL IL-2 (Biolegend, #791906) )

- Plasmid (s) of interest: plasmid #3 (pSLCAR-CD19-CD3z) and USMB delivery system (according to the method/system of Example 1.1)

- Lentiviral particles containing CAR19 plasmid (prepared based on Chan et al. “R4 RGS proteins suppress engraftment of human hematopoietic stem/progenitor cells by modulating SDF-1/CXCR4 signaling” . Blood Adv. 2021 Nov 9; 5 (21) : 4380-4392, hereinafter “Chan” )

- Real-time quantitative PCR (qPCR) reagents and equipment

- Primers for CAR19 (SEQ ID NOs: 7-8) , GFP (SEQ ID NOs: 5-6) , and housekeeping gene GAPDH (SEQ ID NOs: 9-10)

- CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, #G1780)

Methods:

i) Cell culture and transfection:

Human peripheral blood derived primary T cells (derived from a human donor) were cultured and underwent USMB-based delivery of plasmid #3 (USMB-CAR19) . Alternatively, primary T cells were transfected with lentivirus containing plasmid #3 according to established protocols (lentivirus-CAR19) . Lentiviral particles containing the CAR19 plasmid were prepared based on the method as described in Chan using a standard calcium phosphate precipitation protocol, further referencing to an additional reference Meng et al. “Erythroid Promoter Confines FGF2 Expression to the Marrow after Hematopoietic Stem Cell Gene Therapy and Leads to Enhanced Endosteal Bone Formation” . PLoS One. 2012; 7 (5) : e37569) . The lentiviral vectors were packaged in 293T cells (CRL-3216; ATCC, Manassas, VA) and subsequently concentrated 100-fold by high-speed centrifugation. The functional viral titers were determined by transducing HT1080 cells (CCL-121, ATCC) and analyzing the transduction efficiency using flow cytometry. The titers obtained were around 4x10⁷/mL. A control group (control) was prepared with cells cultured under the same conditions and received the addition of plasmid #3 but without USMB-mediated delivery. 697 target cells were cultured under RPMI1640 supplemented with 10%FBS.

ii) Quantification of CAR19 mRNA expression:

48 hours after transfection, the control and transfected primary T cells (USMB-CAR19, lentivirus-CAR19 groups) were harvested. Total RNA was isolated from the harvested cells using an RNA isolation kit (QRNeasy Mini Kit, #74104) , according to the manufacturer's instructions. Briefly, Samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in water. cDNA synthesis was performed using a reverse transcription kit, following the manufacturer's instructions. qPCR was carried out using specific primers for CAR19, GFP, and housekeeping gene GAPDH to assess relative mRNA expression levels. The data were analyzed using the comparative Ct (ΔΔCt) method as below:

ΔCt = Ct (CAR19 or GFP) –Ct (GAPDH)

ΔΔCt = ΔCt (Sample) –ΔCt (Control average)

iii) Co-culture of primary T cells and target cells:

The control and transfected primary T cells (USMB-CAR19, lentivirus-CAR19 groups) were harvested, and cell densities were adjusted to achieve the desired effector-to-target (E: T) ratios (20: 1) . 697 target cells were seeded in 96-well plates. The control and transfected primary T cells (USMB-CAR19, lentivirus-CAR19 groups) were added to the 697 target cells, and the co-culture was incubated for 12 hours at 37℃ and 5%CO2.

iv) Cytotoxicity assay:

After the co-culture incubation, the co-culture was centrifuged at 2000rpm for 5 mins to obtain cell-free supernatant for the cytotoxic assay. The Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay (#G1780) was performed according to the manufacturer’s instructions to measure the level of target cell death caused by primary T cells. Briefly, a reaction solution containing LDH substrate, cofactor, and catalyst was prepared, and added to the supernatant collected in a new 96-well plate. The plate was then incubated in the dark at room temperature for 20 minutes to develop colour for absorbance measurement at 490 nm with microplate reader.

v) Data analysis:

Statistical analysis (one-way ANOVA) was performed to compare CAR19 and GFP mRNA expression as well as 697 cell-killing activity between the control and transfected primary T cells (USMB-CAR19, lentivirus-CAR19) groups.

Results:

USMB-CAR19 showed a high gene transfer efficiency on human peripheral blood derived primary T cells (Primary T) . Transfection with USMB-CAR19 resulted in a >400000 fold increase in both mRNA expression of GFP (Figure 8E) and of CAR19 (Figure 8F) , which are comparable to the conventional Lentivirus method (lentivirus-CAR19) (n=3) . As shown in Figure 8G, the enhanced anticancer activity of human primary T cells engineered from USMB-CAR19 (～50%cell cytotoxicity) was also comparable to the conventional virus method. Results showed that the USMB-CAR19 which is a safe, simple virus-free platform which has an outstanding expression level in mRNA level and an enhanced anticancer activity with human primary T cells over control group, and the performance is comparable to that of conventional Lentivirus method.

Example 5: Evaluating USMB-KI virus-free gene knock-in platform for CAR-T/NK engineering

5.1 Quantification of CAR19 mRNA expression and cancer cell killing activity of Jurkat T cells and primary PBMC received USMB or USMB-KI CAR19 engineering

Materials:

- In vitro cell culture system (cell culture plates)

- Cell culture media and supplements:

a. Primary PBMC: RPMI 1640 Medium (Gibco, #11875119) supplemented with 10%fetal bovine serum (Gibco, #10500064) and 20ng/ml IL-2 (Biolegend, #791906)

b. Jurkat T cells, and 697 cancer cells: RPMI 1640 Medium (Gibco, #11875119) supplemented with 10%fetal bovine serum (Gibco, #10500064)

- Plasmid (s) of interest: plasmid #3 (pSLCAR-CD19-CD3z) , plasmid #1 (CAR19 Donor DNA; see Figure 1) , plasmid #4 (pSUPER-puro; control plasmid) , plasmid #5 (USMB-KI EV donor; control plasmid; see Figure 4)

- Cas9/gRNA plasmid: PX458-AAVS1 gRNA for USMB-KI. Plasmid detailed information is available in Addgene (Plasmid #48138) . PX458-AAVS1 gRNA comprises the sequence of SEQ ID NO: 4. See vector map in Figure 5.

- USMB delivery system (according to the method/system of Example 1.1)

- RNA extraction kit (Qiagen, QRNeasy Mini Kit, #74104)

- qPCR master mix (Bio-rad, iTaq^TM UniversalGreen Supermix, #1725122)

- Primers for CAR19 (SEQ ID NO: 7-8) and GAPDH (SEQ ID NO: 9-10)

- Real-time PCR system (Applied Biosystems, QuantStudio 7)

- CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, #G1780)

Methods:

i) Cell culture and transfection:

Jurkat T cells and primary PBMCs were cultured and seeded in cell density of 1x10⁶/5ml to receive USMB-KI delivery of plasmid #1 (USMB-KI CAR19) or plasmid #5 (USMB-KI EV) together with PX458-AAVS1 gRNA plasmid, or USMB delivery of plasmid #3 (USMB-CAR19) , or plasmid #4 (USMB-EV) . 697 cancer cells were cultured under RPMI1640 medium supplemented with 10%FBS in 37℃ and 5%CO₂. The graphical illustration of the example ultrasound-guided microbubble knockin (USMB-KI) system is showed in Figure 9A.

ii) Quantification of CAR19 mRNA expression:

48 hours after USMB delivery, EV controls and transfected Jurkat T cells were harvested. Total RNA was isolated from the harvested cells using an RNA extraction kit (QRNeasy Mini Kit, #74104) following the manufacturer’s instructions. Briefly, samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in water. cDNA synthesis was performed using reverse transcription kit (Promega, GoScript^TM Reverse Transcription System, #A5001) , following the manufacturer’s instructions. Briefly 1μg RNA, 0.5μg oligo (dt) primer, were mixed in 1X reaction buffer with 1.5mM MgCl₂, PCR Nucleotide Mix (0.5mM each dNTP) , 20 units RecombinantRibonuclease Inhibitor, 1μl GoScript^TM Reverse Transcriptase, and ran with program 25℃ (5 mins) , 42℃ (40 mins) , 70℃ (15 mins) . qPCR was carried out using specific primers as table1 for CAR19 and GAPDH to assess relative mRNA expression levels, and the data were analyzed using the comparative Ct (ΔΔCt) method.

iii) Co-culture of Jurkat T cells, primary PBMCs, and 697 cancer cells:

48 hours after USMB delivery, transfected Jurkat T cells and primary PBMCs (USMB-CAR19, USMB-KI CAR19, USMB-KI EV and USMB-EV control groups) were harvested and cell densities were adjusted to the effector-to-target E: T ratios (5: 1, 10: 1, 20:1) . 2.5x10⁴, 5x10⁴ and 1x10⁵ Jurkat T cells and primary PBMCs were seeded against 5x10³ 697 cancer cells corresponding to E: T ratios (5: 1, 10: 1, 20: 1) in 96-well plates and incubated at 37℃ with 5%CO2 for 12 hours.

iv) Cytotoxicity assay:

After the co-culture incubation, the co-culture was centrifuged to obtain cell-free supernatant for the cytotoxic assay. The Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay (#G1780) was performed according to the manufacturer’s instructions to measure the level of 697 cancer cell death caused by Jurkat T cells or primary PBMC. Briefly, a reaction solution containing LDH substrate, cofactor, and catalyst was prepared, and added to the supernatant collected in a new 96-well plate. The plate was then incubated in the dark at room temperature for 20 minutes to develop colour for absorbance measurement at 490 nm with microplate reader.

v) Data analysis:

Statistical analysis of CAR19 mRNA expression and 697 cell-killing activity for each group at each E: T ratio was performed using one-way ANOVA.

Results:

USMB-KI CAR19 did not only show superior gene transfer efficiency (>500000 hold mRNA expression change) (Figure 9B) , but also produced Jurkat and primary human peripheral blood mononuclear cells (Primary PBMC) cells with nearly 2x higher cell cytotoxity than the original USMB method at all 3 E: T ratios (USMB-CAR19) (Figures 9C and 9C’) in vitro (n=3) . Results showed that USMB-KI showed superior gene transfer efficiency, higher cell cytotoxicity and an excellent cancer-killing capability with Jurkat and primary PBMC cells.

5.2 Photon metric quantification of the of luciferase-expressing 697 cells in NOD- SCID mice receiving saline, USMB-KI-EV, and USMB-KI CAR19-engineered Jurkat T cells

Materials:

- 697 cells with stably expression of luciferase (cell strain as Leung KT, et al. “CD9 blockade suppresses disease progression of high-risk pediatric B-cell precursor acute lymphoblastic leukemia and enhances chemosensitivity “, Leukemia, Mar 2020, 34 (3) : 709-720) , Jurkat T cells

- Cell supplements and media: 697 cells and Jurkat T cells: Jurkat: RPMI 1640 Medium (Gibco, #11875119) supplemented with 10%fetal bovine serum (Gibco, #10500064)

- Plasmid (s) of interest: plasmid #5 (USMB-KI EV Donor; control plasmid) and plasmid #1 (CAR19 Donor DNA)

- NOD-SCID mice (Laboratory Animal Services Centre, CUHK)

- D-luciferin (Thermo Fisher Scientific)

- In vivo imaging system (IVIS Spectrum, Perkin Elmer) and software (Living Image Software, #128113, Perkin Elmer)

- Cas9/gRNA plasmid: PX458-AAVS1 gRNA for USMB-KI (Plasmid detailed information available in Addgene (Plasmid #48138) ) . PX458-AAVS1 gRNA comprises the sequence of SEQ ID NO: 4. See vector map in Figure 5.

Methods:

i) Preparation of cells for injections:

697 cells stably expressing luciferase were cultured and harvested during the log-growth phase. Jurkat T cells were transfected with either plasmid #5 (USMB-KI EV) or plasmid #1 (USMB-KI CAR19) together with PX458-AAVS1 gRNA plasmid using the optimized USMB method as described in Example 1.1. Cell suspensions were prepared in 0.9%sodium chloride saline for injection.

ii) Mouse model and injections:

NOD-SCID mice were used as the animal model for this study. All experimental procedures were approved by the Animal Experimentation Ethics Committee (AEEC) and carried out in accordance with institutional guidelines. Mice were randomized into three groups (n=5/group) and inoculated intravenously with 1 x 10⁶ luciferase-expressing 697 cells. After the establishment of leukemia (～5 days after inoculation) , mice were intravenously injected with either saline, 1 x 10⁶ USMB-KI-EV, or USMB-KI CAR19-engineered Jurkat T cells, according to their assigned group.

iii) Bioluminescence imaging:

Mice were anesthetized, and D-luciferin (3mg per 20g mouse) was injected intraperitoneally 10-15 minutes prior to imaging. Bioluminescence imaging was performed using an in vivo imaging system (IVIS Spectrum) with 15-30 seconds exposure. Photon emission from each mouse was quantified using the imaging system software. Data were expressed as photons per second per square centimeter per steradian (p/s/cm2/sr) .

iv) Statistical analysis:

The data were analyzed using one-way ANOVA; results were considered statistically significant at p < 0.05.

Results:

USMB-KI produced CAR-T Jurkat cells (USMB-KI-CAR19) that markedly reduced the leukemia abundance (in terms of nominal radiance ratio) in mice with xenografts of human CD19+ leukemia 697 cells, compared to the mice received plasmid #4 transferred Jurkat cells (USMB-KI-EV) and saline treated control group in vivo (n=3) (see Figures 9D and 9D’) . Results indicated that USMB-KI can be a novel, efficient and safe virus-free platform for CAR-T/NK engineering.

Example 6: Comparing gene transfer efficiency using ultrasound with or without microbubble

6.1 Quantification of GFP mRNA expression of Jurkat T cells and NK-92 cells received ultrasound delivery with or without microbubble addition:

Materials

- SonoVue microbubbles (Bracco Imaging)

- Plasmid #2 (pSUPER-EGFP-puro) and USMB delivery system (according to the method/system of Example 1.1)

- Ultrasound equipment with a suitable transducer for cell culture vessels

- In vitro cell culture system (cell culture flasks)

- Cell culture media and supplements: NK-92 and Jurkat: RPMI 1640 Medium (Gibco, #11875119) supplemented with 10%fetal bovine serum (Gibco, #10500064)

- Primers for GFP (SEQ ID NOs: 5-6) amd GAPDH (SEQ ID NOs: 9-10)

- RNA extraction kit (Qiagen, QRNeasy Mini Kit, #74104)

- qPCR master mix (Bio-rad, iTaq^TM UniversalGreen Supermix, #1725122)

- Real-time PCR system (Applied Biosystems, QuantStudio 7)

- CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, #G1780)

Methods:

i) Cell culture and preparation:

Jurkat and NK-92 cells were cultured in RPMI1640 media supplemented with 10%FBS. Cells were harvested during the log-growth phase (5-8x10⁶ cells/20 ml medium) for the transfection experiment.

ii) Plasmid preparation:

Plasmid #2 was used as a reporter for transfection efficiency in both Jurkat and NK-92 cells.

iii) Ultrasound-based transfection:

Two groups were prepared for each cell type: one with ultrasound alone (US-GFP) and the other with the original ultrasound-microbubble method (USMB-GFP) for comparison.

For the USMB-GFP group, the original ultrasound-microbubble method was followed as described in Example 1.1.

For the US-GFP group, the optimized ultrasound parameters were followed as described in Example 1.1 without using microbubbles. In particular, in step i) of Example 1.1, 20 μg plasmid #2 were combined with saline, instead of microbubble. In other words, no tranfecting agent was added.

iv) RNA isolation and qPCR analysis:

Transfected Jurkat and NK-92 cells were harvested 48 hours post-transfection. Total RNA was isolated from the harvested cells using an RNA extraction kit (QRNeasy Mini Kit, #74104) following the manufacturer's instructions. Briefly, samples are first lysed and then homogenized. Ethanol is added to the lysate to provide ideal binding conditions. The lysate is then loaded onto the RNeasy silica membrane and RNA binds to the silica membrane, and all contaminants are efficiently washed away. Pure, concentrated RNA is eluted in water.

cDNA synthesis was performed using a reverse transcription kit (Promega, GoScript^TM Reverse Transcription System, #A5001) , following the manufacturer's instructions. Briefly 1μg RNA, 0.5μg oligo (dt) primer, were mixed in 1X reaction buffer with 1.5mM MgCl2, PCR Nucleotide Mix (0.5mM each dNTP) , 20 units Recombinant Ribonuclease Inhibitor, 1μl GoScript^TM Reverse Transcriptase, and ran with program 25℃ (5 mins) , 42℃ (40 mins) , 70℃ (15 mins) .

qPCR was carried out using specific primers for GFP and GAPDH to assess relative mRNA expression levels. The data were analyzed using the comparative Ct (ΔΔCt) method as formula below:

ΔCt = Ct (CAR19) –Ct (GAPDH)

ΔΔCt = ΔCt (Sample) –ΔCt (Control average)

v) Data analysis:

a. Statistical analysis (One way ANOVA) was performed to compare GFP mRNA expression levels between the US and USMB groups for both Jurkat and NK-92 cells.

Results:

Employing ultrasound alone (US-GFP) substantially increased plasmid delivery into Jurkat cells (Figure 10A) and NK-92 cells (Figure 10B) compared to the original method (USMB-GFP) in vitro (n=5) . The results showed a 40-50%improvement of target gene expression in Jurkat and NK-92 received ultrasound alone compared to USMB. Results showed that example method and system without using transfecting agent such as microbubble demonstrated even surprising improvements of target gene expression in Jurkat and NK-92 cells when compared to USMB method and system.

The exemplary embodiments of the present invention are thus fully described. Although the description referred to particular embodiments, it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific details. Hence this invention should not be construed as limited to the embodiments set forth herein.

For example, in certain embodiments, the cell culture having about 1 x 10⁶ cells in about 5ml medium were used for the step of delivering ultrasound, but other amount of cells and other volumes of medium can be used, and the parameters such as the total energy applied to the cell culture, the energy output and the duration can be adjusted accordingly.

Claims

A method of preparing chimeric antigen receptor (CAR) engineered cells, comprising the steps of:

(i) incubating a CRISPR plasmid comprising at least a portion of a CRISPR-mediated gene editing system and a CAR sequence with a cell culture comprising target cells; and

(ii) delivering an ultrasound to the cell culture by an ultrasound delivery system comprising an ultrasound transducer, thereby delivering the CRISPR plasmid to the target cells.
The method of claim 1, wherein the target cell is an immune cell.
The method of claim 2, wherein the immune cell is a Natural Killer (NK) cell or a T cell.
The method of any one of the preceding claims, wherein a total energy applied by the ultrasound transducer is in the range of 10J/ml to 40J/ml to a cell culture housed in a container with cell density in the range of 1x10⁵ to 5x10⁶ cells/ml.
The method of any one of the preceding claims, wherein the cell culture has about 1 x 10⁶ cells in about 5ml of cell culture, and the total energy applied to the cell culture by the ultrasound transducer is 50 to 200 J.
The method of any one of the preceding claims, wherein the ultrasound transducer is configured to deliver an ultrasound having a frequency range of about 0.5-5 MHz, an energy output of about 0.5-5 W/cm², and a duration of about 15-120 seconds.
The method of claim 6, wherein the frequency range is about 1 MHz, the energy output is about 1 W/cm², and the duration is about 60 seconds.
The method of any one of the preceding claims, wherein the CRISPR plasmid comprises AAVS1 targeting sequences.
The method of any one of the preceding claims, wherein the CRISPR plasmid comprises an αCD19-CAR (CAR19) sequence.
The method of claim 9, wherein the CRISPR plasmid comprises sequence ID NO. 1.
The method of any one of claims 1-10, further comprising a step prior to step (i) of mixing the CRISPR plasmid with a transfection agent and incubating for around 15 minutes at room temperature.
The method of claim 11, wherein the transfection agent comprises microbubble.
The method of any one of claims 1-10, further comprising a step prior to step (i) of preparing the CRISPR plasmid in saline without a transfection agent.
The method of any one of the preceding claims, wherein the cell culture is harvested at the log growth phase.
The method of any one of the preceding claims, wherein prior to step (ii) , further comprises a step of:

positioning the ultrasound transducer below a cell culture flask containing the cell culture.
The method of claim 15, wherein prior to step (ii) , further comprises a step of: applying ultrasound gel between the ultrasound transducer and the bottom surface of the cell culture flask.
The method of any one of the preceding claims, after step (ii) , further comprises the step of:

refreshing medium of the cell culture and incubating the target cells for about two days.
The method of any one of the preceding claims, wherein the target cell culture is a mammalian cell culture.
The method of claim 18, wherein the mammalian cell culture is a human cell culture.
A method of preparing chimeric antigen receptor (CAR) engineered cells, comprising the steps of:

(i) incubating a CRISPR plasmid comprising at least a portion of a CRISPR-mediated gene editing system and a CAR sequence with a cell culture comprising target cells with about 1 x 10⁶ cellsin about 5 ml harvested at log phase in a cell culture flask;

(ii) positioning an ultrasound transducer below the cell culture flask containing the cell culture;

(iii) applying an ultrasound gel between the ultrasound transducer and the bottom surface of the cell culture flask;

(iv) delivering an ultrasound to the cell culture by an ultrasound delivery system comprising an ultrasound transducer, wherein the ultrasound transducer is configured to deliver an ultrasound having a frequency range of the frequency range is about 1 MHz, the energy output is about 1 W/cm², and the duration is about 60 seconds; thereby delivering the CRISPR plasmid to the target cells; and

(v) refreshing medium of the cell culture with a complete growth medium and incubating the target cells for about two days.
The method of claim 20, further comprising the step of preparing the CRISPR plasmid in saline.
The method of claim 20, further comprising the step of mixing the CRISPR plasmid with about 9mg/ml microbubble solution in saline and incubating for around 15 minutes at room temperature.
The method of any one of the preceding claims, wherein the human cell culture is selected from the group consisting of NK-92, Jurkat, Primary T cells, and primary peripheral blood mononuclear (PBMC) cells.
The method of any one of the preceding claims, wherein the method is virus-free and achieves a transfection rate of >60%and/or a cell viability rate >80%.
A method of treating a disease, health condition or disorder in a subject in need of treatment, comprising the step of:

administrating a therapeutically effective amount of CAR engineered cells to the subject, wherein the CAR engineered cells made according to a method of any one of claims 1-24.
The method of claim 25, wherein the disease, health condition or disorder is selected from the group consisting of leukemia, blood cancer, solid tumors, tissue fibrosis, inflammation, autoimmune disease, diabetes and aging-related condition.