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WO2025152036A1 - Système et procédé pour augmenter la capacité de transduction et l'efficacité de reprogrammation dans le ciblage de cellules et de tissus - Google Patents

Système et procédé pour augmenter la capacité de transduction et l'efficacité de reprogrammation dans le ciblage de cellules et de tissus

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Publication number
WO2025152036A1
WO2025152036A1 PCT/CN2024/072575 CN2024072575W WO2025152036A1 WO 2025152036 A1 WO2025152036 A1 WO 2025152036A1 CN 2024072575 W CN2024072575 W CN 2024072575W WO 2025152036 A1 WO2025152036 A1 WO 2025152036A1
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magnet
ipscs
cells
reprogramming
mscs
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Guang-Yuh Chiou
Chian-Shiu Chien
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    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
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    • C12N2740/10011—Retroviridae
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    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
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    • C12N2770/00011—Details
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention pertains to a system and method for increasing the transduction capability and reprogramming efficiency in targeting cells and tissues.
  • Gene therapy is an advanced intervention technology that can replace the defective genes or restore the deficient genes in the host cells. Gene delivery can be achieved predominantly via using either viral or non-viral vectors. After the removal of pathogenicity and self-replication capabilities, viral vectors can be used as ideal tool for gene delivery to meet the demands of gene therapy or laboratory experiments. Viral vectors can be further divided into integrating vectors and non-integrating. Integrating viral vectors, such as retroviruses and lentiviruses, can integrate their genetic information into the patient DNA. Non-integrating viral vectors, such as adenoviruses, adeno-associated viruses (AAV) , and herpes simplex viruses (HSV) , retain the episomal DNA in the cytoplasm without integration. Episomal vectors do not replicate themselves in the patient's DNA and disappear within a short time.
  • AAV adeno-associated viruses
  • HSV herpes simplex viruses
  • iPSCs The conventional methods for establishing iPSCs require transductions of foreign genetic materials.
  • plasmids which expresses iPSCs reprogramming genes consisting of OCT4, SOX3, Klf4 and c-Myc.
  • Various reprogramming protocols had established and the efficiency of reprogramming is validated and tested.
  • Our team previously had successfully established a iPSCs reprogramming method by replacing c-Myc with non-oncogenic PARP1 gene.
  • the replacement of c-Myc with PARP1 generates high genome stability and the pluripotency are comparable to original iPSCs induction protocols established by Takahashi K et al.
  • RNAs and nanoparticles can be fully synthesized and produced in GMP-competent laboratories or factories. The production sources are controllable, tunable as compared with conventional laboratories-prepared expression vectors or virus soups for transfections.
  • MSCs Mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • the MSCs are lineage committed stem cells with limited cell differentiation capacities.
  • the safety of MSCs in biomedical applications is high due to the limited pluripotency of MSCs.
  • MSCs hold promising cell-based therapeutic application in degenerative diseases and tissue repair.
  • most of the majorities of the MSCs are collected from adipose tissues or cord blood biopsies.
  • MSCs are collected from various sources and the safety and cell operation procedures are exposed to high uncertainties in the regards of GTP-competent cell products.
  • the sources of MSCs are the major safety concern of MSCs in biomedical applications.
  • Majority of the MSCs are collected from biopsies by flowcytometry based cell sorting and separation methods.
  • Pathogen-free MSCs are extremely difficult to obtain albeit pathogen detections form MSCs collected from various biopsies are conducted.
  • GTP competent MSCs production processes are major challenges in using MSCs as cell-based therapeutic reagents. Production of pathogen-free iPSCs are more feasible than MSCs collected from various uncontrollable sources.
  • iPSCs and differentiations of iPSCs to MSCs can be conducted in GTP competent laboratories or cell producing facilitated with controllable operations and parameters.
  • MSCs prepared from these approaches have promising potentials in personalized/autologous or allogeneic cell transplantation and cell-based therapeutics.
  • the purity of MSCs-derived from iPSCs is a major concern of production quality of MSCs derived from iPSCs.
  • iPSCs The conventional methods for establishing iPSCs require transductions of foreign genetic materials.
  • plasmids which expresses iPSCs reprogramming genes consisting of OCT4, SOX3, Klf4 and c-Myc.
  • Various reprogramming protocols had established and the efficiency of reprogramming is validated and tested.
  • Our team previously had successfully established a iPSCs reprogramming method by replacing c-Myc with non-oncogenic PARP1 gene.
  • the replacement of c-Myc with PARP1 generates high genome stability and the pluripotency are comparable to original iPSCs induction protocols established by Takahashi K et al.
  • RNAs and nanoparticles can be fully synthesized and produced in GMP-competent laboratories or factories. The production sources are controllable, tunable as compared with conventional laboratories-prepared expression vectors or virus soups for transfections.
  • the transduction of genetic materials from any given vector into the host cells relies on the intensive exposure of vectors to the target cells or tissues.
  • these host cells/tissues cannot effectively sink in the culture media, therefore reducing the probably of the vector-to-cell contact and suppressing the transduction efficiency of exogenous genes.
  • the unstable gene transduction can be observed in the reprogramming of suspension somatic cells.
  • tissue slices that tended to be floating failed to be infected by viruses in the culture. Based upon these drawbacks in conventional setup for vector-mediated gene transduction or viral infections, an efficiency method that can enhance the contact between given vectors and host cells/tissues should be required.
  • the present invention provides a system and method by combing a novel device design with novel biomaterials to accelerate the production and quality assurance of iPSCs, wherein the quality and easy-of-operations of isolating MSCs derived from iPSCs can be achieved from the novel devices and utilizations of the newly formulated biomaterials.
  • the invention provides a system for enhancing efficiency of gene delivery and/or reprogramming of target cells or tissues, comprising a magnet field-generating device, which is assembled in a 12-well culture plate with magnet components including ring-shaped magnet, a magnet holder and a passive aligner to generate a magnet field, and magnet beads, which are used to label targeting cells or tissues, or which are incorporated into agarose gel.
  • the magnet field-generating device comprises a ring-shaped magnet; a magnet holder; a culture plate; and a passive aligner; wherein the ring-shaped magnet and the magnet holder are assembled, and then assembled in the culture plate.
  • the magnet adhesion of the target cells or tissues under the magnet filed increases the contact probability of cell-to-cell, cell-to-virus or antibody-to-target in the bottom of the culture plates during the process of viral infection or gene transduction.
  • the magnet field-generating system and method are able to enhance the transduction efficiency of the exogenous genes carried by any given bead-labeled vector.
  • a non-integrating self-amplifying RNA is prepared and encapsuled with nanoparticles based on modified iron oxide/PEI formulations, in combinations with customized 3D-printing prepared magnetic microfluidic device.
  • the magnetic device is modified with specialized hydrophobic surface modification for enhancing transfection efficiencies of transductions and iPSCs generations. All these improvements together in iPSCs production significantly increase the successful rate in iPSCs productions and due to high gene delivery efficiency, the amounts of cells required in iPSCs production is significantly reduced and therefore decreasing the technical challenges in establishing iPSCs.
  • the time-and cost-requirements for generating personalized and customized made iPSCs are cost feasible by these methods.
  • a magnet field-generating device is constructed by using 3D-printing technologies, which can be assembled in a 12-well culture plate and constitutively generate the magnet field.
  • the target cells/tissues were labeled by magnet beads or incorporated into a magnet bead-containing agarose gel.
  • the magnet adhesion of the target cells or tissues under the magnet filed will increased the contact probability of cell-to-virus in the bottom of culture plates during the process of viral infection and gene transduction.
  • the device and the 3D-printing-based magnet field-generating method enhance the transduction efficiency of exogenous genes carried by any given bead-labeled vector.
  • the present invention provides an easy-to-operate, scalable cell separation and production method based on magnetic facilitated antibodies for purifying MSCs with specific MSCs surface marker sets.
  • the method provides an affinity based MSCs separation and purification method with the customizable fluidic device by 3D-printing to maximize the in-well MSCs purifications and cost reduction.
  • Figure 4 (C) shows that the formulated iron oxide/PEI/saRNAs nanoparticles were incubated with PBMCs and the transfection efficiencies were direct compared with conventional transfection methods including liposome-, retrovirus-, and iron oxide/PEIs.
  • Figure 4 (D) shows that 5 individual colonies were isolated and the expressions of selective stemness genes including Oct4, Sox2, Nanog, Rex1 were analyzed by semi-quantitative PCRs. These results shown that formulation of iron oxide/PEI was capable to conduct gene deliveries in PBMCs and induce reprogramming of PBMCs as evidenced by inductions of Nanog expression.
  • Figure 5 shows that the magnet field-generating device increased the efficiency of iPSC reprogramming from PBMCs.
  • Figure 5 (A) shows that the endogenous Oct4 expression was higher in reprogramming cells transduced with the magnet field-generating device than that without the device.
  • Figure 5 (B) shows that the stemness gene Nanog presented a similar pattern as using the magnet field-generating device.
  • Figure 5 (C) shows that three days after the delivery of OSKM (Oct4, Nanog, Sox2, and c-Myc) genes, the expression of Oct4 and Nanog was increased in reprogramming cells transduced with the magnetic devices group.
  • Figure 5 (D) shows that the relative reprogramming efficiency was estimated by alkaline phosphatase staining and showed higher efficiency in reprogramming cells with the magnet field-generating devices than that without the device.
  • the magnet adhesion of the target cells or tissues under the magnet filed will increased the contact probability of cell-to-virus in the bottom of culture plates during the process of viral infection and gene transduction.
  • our device and the novel 3D-printing-based magnet field-generating method is able to enhance the transduction efficiency of exogenous genes carried by any given bead-labeled vector.
  • the 3D-printing-based magnet field-generating device is able to be assembled for most of any given commercial 12-well culture plates. After the assembly, the device is able to constitutive generate strong magnet field.
  • the components and the assembly process according to the invention include (1) representative ring-shaped magnets and magnet holders, (2) A commercial 12-well culture dish, an unassembled ring-shaped magnet, an unassembled magnet holder, and a passive aligner with two representative assembled magnet holders with ring-shaped magnets; (3) assemble the ring-shaped magnet into the magnet holder; (4) assemble the magnet holder with a ring-shaped magnet into the passive aligner; (5) assemble the passive aligner into the commercial 12-well culture dish.
  • the magnet field will promote the landing of bead-labeled culture cells which facilitate the viral infection of cells and enhance the transfection efficiency and resultant expression of exogenous gene.
  • compositions including particle sizes of iron oxides, types and concentrations of PEIs, and contents of RNAs of the nanoparticles for gene delivery device (cell reprogramming device) is tailorable.
  • the transfection and reprogramming edffection efficiencies were determined by counting eGFP-positive colonies and positive colonies of AP staining.
  • PBMC were transfected with retrovirus factors without or with magnetic devices.
  • the magnetic device enhanced retrovirus delivery were determined in Figure 3 (C) .
  • the reprogramming efficiencies of PBMC to iPSCs were determined by AP staining. These colonies were counted and quantitated as shown in Figure 3 (D) .
  • the regrogramming data showed significant among 2.5 folds increase in reprogramming effciencies with magnetic device.
  • PBMCs were transfected with formulated iron oxide/PEI/saRNA nanoparticles with magnetic devices.
  • the scheme of iron oxide/PEI/saRNAs mediated transfections were shown in Figure 4 (A) .
  • formulated iron oxides were coated with PEI and then co-incubated with saRNAs containing open reading frames of Yamanaka factor, SOX2, OCT4, c-Myc, and Klf4.
  • the transfection abilities were validated with iron oxide/PEI/eGFP formulations as shown in Figure 4 (B) .
  • HE staining and immunostaining showed that the organotypic culture system maintained the structure and characteristics of lung tissue, and the expression of ACE2, the SARS-CoV-2 entry receptor, was detectable ( Figure 8 (B) ) .
  • Western blot results showed that the expression of S and N proteins were not detectable in the organotypic culture system infected in a culture plate without the magnet field-generating device. This could be due to the low exposure and contact of the slices to the viruses in the culture, leading to the low expression of viral proteins in the host cells.

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Abstract

L'invention concerne un système pour améliorer l'efficacité d'administration et/ou de reprogrammation de gènes de cellules ou de tissus cibles, comprenant un dispositif de génération de champ magnétique qui est assemblé dans une plaque de culture à 12 puits et des composants magnétiques pour générer un champ magnétique, et des billes magnétiques, qui sont utilisées pour marquer des cellules ou des tissus ciblant, ou qui sont incorporées dans un gel d'agarose. L'invention concerne également le procédé utilisant le système.
PCT/CN2024/072575 2024-01-16 2024-01-16 Système et procédé pour augmenter la capacité de transduction et l'efficacité de reprogrammation dans le ciblage de cellules et de tissus Pending WO2025152036A1 (fr)

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Citations (5)

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Publication number Priority date Publication date Assignee Title
US20120220030A1 (en) * 2009-08-21 2012-08-30 Reijo Pera Renee A Enhanced Efficiency of Induced Pluripotent Stem Cell Generation from Human Somatic Cells
US20140220681A1 (en) * 2010-12-22 2014-08-07 Fate Therapeutics, Inc. Cell culture platform for single cell sorting and enhanced reprogramming of ipscs
CN106414721A (zh) * 2014-03-04 2017-02-15 菲特治疗公司 改良的重编程方法和细胞培养平台
CN110577967A (zh) * 2018-05-22 2019-12-17 中国人民解放军军事科学院军事医学研究院 诱导性多能干细胞及其制备方法
CN116615530A (zh) * 2020-11-04 2023-08-18 菲特治疗公司 靶向实体瘤的多重工程改造的iPSC和免疫效应细胞

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120220030A1 (en) * 2009-08-21 2012-08-30 Reijo Pera Renee A Enhanced Efficiency of Induced Pluripotent Stem Cell Generation from Human Somatic Cells
US20140220681A1 (en) * 2010-12-22 2014-08-07 Fate Therapeutics, Inc. Cell culture platform for single cell sorting and enhanced reprogramming of ipscs
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