Transduction Enhancers Enable Efficient Human Adenovirus Type 5-Mediated Gene Transfer into Human Multipotent Mesenchymal Stromal Cells.
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ABSTRACT:
SUBMITTER: Nilson R
PROVIDER: S-EPMC8231506 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In adenovirus type 5 (HAdV-5)-derived viral vectors, the fiber protein has been the preferred locale for modifications to alter the natural viral tropism. Hexon, the most abundant capsid protein, has rarely been used for retargeting purposes, likely because the insertion of larger targeting peptides into Hexon often interferes with the assembly of the viral capsid. We previously observed that positively charged molecules enhance the transduction of human multipotent mesenchymal stromal cells (hMSCs)-a cell type of significant interest for clinical development but inefficiently transduced by unmodified HAdV-5-based vectors. As efficient HAdV-5-mediated gene transfer would greatly increase the therapeutic potential of hMSCs, we tested the hypothesis that introducing positively charged amino acids into Hexon might enhance the transduction of hMSCs, enabling efficient expression of selected transgenes. From the constructs that could be rescued as functional virions, one (HAdV-5-HexPos3) showed striking transduction of hMSCs with up to 500-fold increased efficiency. Evaluation of the underlying mechanism identified heparan sulfate proteoglycans (HSPGs) to be essential for virus uptake by the cells. The ease and efficiency of transduction of hMSCs with this vector will facilitate the development of genetically modified hMSCs as therapeutic vehicles in different disciplines, including oncology or regenerative medicine.
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Project description:Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well. It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.
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Project description:Secretome of multipotent mesenchymal stromal cells (MSCs) is actively used in biomedical applications such as alveolar bone regeneration, treatment of cardiovascular disease, and neurodegenerative disorders. Nevertheless, hMSCs have low proliferative potential and production of the industrial quantity of their secretome might be challenging. Human fetal multipotent mesenchymal stromal cells (FetMSCs) isolated from early human embryo bone marrow are easy to expand and might be a potential source for pharmaceutical substances production based on their secretome. However, the secretome of FetMSCs was not previously analyzed. Here, we describe the secretome of FetMSCs using LC-MALDI shotgun proteomics. We identified 236 proteins. Functional annotation of the identified proteins revealed their involvement in angiogenesis, ossification, regulation of apoptosis, and immune response processes, which made it promising for biomedical applications. The proteins identified in the FetMSCs secretome are involved in the same biological processes as proteins from previously described adult hMSCs secretomes. Nevertheless, many of the common hMSCs secretome components (such as VEGF, FGF, Wnt and TGF-?) have not been identified in the FetMSCs secretome.
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Project description:Multipotent mesenchymal stromal cells (MSCs) are rare cells resident in bone marrow and other organs capable of differentiating into mesodermal lineage tissues. MSCs possess immunomodulatory properties and have extensive capacity for ex-vivo expansion. Early clinical studies demonstrated safety and feasibility of infusing autologous MSCs and suggested a role in enhancing engraftment after hematopoietic cell transplant (HCT). Subsequent pilot studies using allogeneic MSCs showed safety but presented contradictory results regarding efficacy in treating graft-versus-host disease (GVHD).Larger, phase II allogeneic MSC infusion studies, including cells obtained from haploidentical and third-party donors, showed efficacy in GVHD treatment; however, recent randomized, placebo-controlled studies failed to corroborate these results. New investigations include MSC infusions in umbilical cord blood transplantation, MSC therapy for tissue regeneration/repair, harvest and use of MSCs from adipose tissue and cell-tracking/imaging studies using radionuclides, gene and fluorescent dye-labeled MSCs.MSCs remain the subject of intense investigation in HCT because of their differentiation potential and immunomodulatory properties. Whereas infusions of autologous, allogeneic and third-party donor MSCs are well tolerated, further research is needed to clarify the optimal methods for harvesting and expansion, optimal timing of administration and efficacy in the setting of HCT.
| S-EPMC3748957 | biostudies-literature
Project description:The objective of this study was to investigate whether human placental multipotent mesenchymal stromal cell (hPMSC)-derived Slit2 and endothelial cell Roundabout (Robo) receptors are involved in placental angiogenesis. The hPMSC-conditioned medium and human umbilical vein endothelial cells were studied for Slit2 and Robo receptor expression by immunoassay and RT-PCR. The effect of the conditioned medium of hPMSCs with or without Slit2 depletion on endothelial cells was investigated by in vitro angiogenesis using growth factor-reduced Matrigel. hPMSCs express Slit2 and both Robo1 and Robo4 are present in human umbilical vein endothelial cells. Human umbilical vein endothelial cells do not express Robo2 and Robo3. The hPMSC-conditioned medium and Slit2 recombinant protein significantly inhibit the endothelial cell migration, but not by the hPMSC-conditioned medium with Slit2 depletion. The hPMSC-conditioned medium and Slit2 significantly enhance endothelial tube formation with increased cumulated tube length, polygonal network number and vessel branching point number compared to endothelial cells alone. The tube formation is inhibited by the depletion of Slit2 from the conditioned medium, or following the expression of Robo1, Robo4, and both receptor knockdown using small interfering RNA. Furthermore, co-immunoprecipitation reveals Slit2 binds to Robo1 and Robo4. Robo1 interacts and forms a heterodimeric complex with Robo4. These results suggest the implication of both Robo receptors with Slit2 signaling, which is involved in endothelial cell angiogenesis. Slit2 in the conditioned medium of hPMSCs has functional effect on endothelial cells and may play a role in placental angiogenesis.
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Project description:Oncolytic adenovirus (oAd)-mediated gene therapy is a promising approach for cancer treatment because of its cancer cell-restricted replication and therapeutic gene expression. However, systemic administration of oAd is severely restricted by their immunogenic nature and poor tumor homing ability, thus oAd cannot be utilized to treat disseminated metastases. In this study, human bone marrow-derived mesenchymal stromal cell (hMSCs) was used as a viral replication-permissive carrier for oAd with an aim to improve the systemic delivery of the virus to tumor tissues. To overcome the poor delivery of oAd into hMSCs, a relaxin (RLX)-expressing oncolytic Ad (oAd/RLX), which degrades dense tumor extracellular matrix of highly desmoplastic pancreatic cancer, was complexed with biodegradable polymer (poly (ethyleneimine)-conjugated poly(CBA-DAH); PCDP), generating oAd/RLX-PCDP complex. oAd/RLX-PCDP complex enhanced the internalization of oAd into hMSC, leading to superior viral production and release from hMSCs, along with high RLX expression. Furthermore, systemic administration of oAd/RLX-PCDP-treated hMSCs elicited more potent antitumor effect compared to naked oAd/RLX or oAd/RLX-treated hMSC in pancreatic tumor model. This potent antitumor effect of systemically administered oAd/RLX-PCDP-treated hMSCs was achieved by superior viral replication in tumor tissues than any other treatment group. In conclusion, these results demonstrate that hMSCs are effective carriers for the systemic delivery of oAd to tumor sites and treatment of pancreatic cancer.
| S-EPMC6659406 | biostudies-literature
Project description:Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking immunotherapy for cancer. However, the intricate and costly manufacturing process remains a hurdle. Improving the transduction rate is a potential avenue to cut down costs and boost therapeutic efficiency. Peptide nanofibrils (PNFs) serve as one such class of transduction enhancers. PNFs bind to negatively charged virions, facilitating their active engagement by cellular protrusions, which enhances virion attachment to cells, leading to increased cellular entry and gene transfer rates. While first-generation PNFs had issues with aggregate formation and potential immunogenicity, our study utilized in silico screening to identify short, endogenous, and non-immunogenic peptides capable of enhancing transduction. This led to the discovery of an 8-mer peptide, RM-8, which forms PNFs that effectively boost T cell transduction rates by various retroviral vectors. A subsequent structure-activity relationship (SAR) analysis refined RM-8, resulting in the D4 derivative. D4 peptide is stable and assembles into smaller PNFs, avoiding large aggregate formation, and demonstrates superior transduction rates in primary T and NK cells. In essence, D4 PNFs present an economical and straightforward nanotechnological tool, ideal for refining ex vivo gene transfer in CAR-T cell production and potentially other advanced therapeutic applications.
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Project description:BackgroundThe gene transduction efficiency of adenovirus to hematopoietic cells, especially T lymphocytes, is needed to be improved. The purpose of this study is to improve the transduction efficiency of T lymphocytes by using fiber-modified human adenovirus 5 (HAdV-5) vectors.ResultsFour fiber-modified human adenovirus 5 (HAdV-5) vectors were investigated to transduce hematopoietic cells. F35-EG or F11p-EG were HAdV-35 or HAdV-11p fiber pseudotyped HAdV-5, and HR-EG or CR-EG vectors were generated by incorporating RGD motif to the HI loop or to the C-terminus of F11p-EG fiber. All vectors could transduce more than 90% of K562 or Jurkat cells at an multiplicity of infection (MOI) of 500 viral particle per cell (vp/cell). All vectors except HR-EG could transduce nearly 90% cord blood CD34+ cells or 80% primary human T cells at the MOI of 1000, and F11p-EG showed slight superiority to F35-EG and CR-EG. Adenoviral vectors transduced CD4+ T cells a little more efficiently than they did to CD8+ T cells. These vectors showed no cytotoxicity at an MOI as high as 1000 vp/cell because the infected and uninfected T cells retained the same CD4/CD8 ratio and cell growth rate.ConclusionsHAdV-11p fiber pseudotyped HAdV-5 could effectively transduce human T cells when human EF1a promoter was used to control the expression of transgene, suggesting its possible application in T cell immunocellular therapy.
| S-EPMC6480437 | biostudies-literature
Project description:Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and alpha(v) integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34(+) cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34(+) cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an alpha(v) integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34(+) cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34(+) cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34(+) cells expressing alpha(v) integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34(+) cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34(+) cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34(+) c-Kit(+) cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34(+) c-Kit(+) cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.
| S-EPMC111745 | biostudies-literature
Project description:We have previously identified and purified multipotent mesenchymal stromal cell (MSC)-like cells in the highly regenerative endometrial lining of the human uterus (eMSC) as CD140b?CD146? cells. Due to ease of accessibility with minimal morbidity via biopsy, we are proposing to use eMSC in cell-based therapies; however, culture conditions compliant with Good Manufacturing Practice have not been established for eMSC. The aim of this study was to optimize serum-free and xeno-free culture conditions for expansion of eMSC for potential clinical use. Real-time cell assessment (Xcelligence) and MTS viability assays were used to measure attachment and proliferation of freshly isolated, flow cytometry-sorted CD140b?CD146? eMSC cultured in several commercially available and in-house serum-free and xeno-free media in combination with five attachment matrices (fibronectin, collagen, gelatin, laminin, and Cell Start-XF®). Comparisons were made with a standard serum-containing medium, DMEM/F-12/10% fetal bovine serum. Under all conditions examined, eMSC attachment and proliferation was greatest using a fibronectin matrix, with Lonza TP-SF® and our in-house DMEM/SF/FGF2/EGF serum-free xeno-product-containing medium similar to serum-containing medium. Hypoxia increased eMSC proliferation in the DMEM/SF/FGF2/EGF serum-free medium. Culture of eMSC for 7 days on a fibronectin matrix in DMEM/SF/FGF2/EGF serum-free media in 5% O? maintained greater numbers of undifferentiated eMSC expressing CD140b, CD146, and W5C5 compared to culture under similar conditions in Lonza TP-SF medium. However, the percentage of cells expressing typical MSC phenotypic markers, CD29, CD44, CD73, and CD105, were similar for both media. EMSC showed greater expansion in 2D compared to 3D culture on fibronectin-coated microbeads using the optimized DMEM/SF/FGF2/EGF medium in 5% O?. In the optimized 2D culture conditions, eMSC retained CFU activity, multipotency, and MSC surface phenotype, representing the first steps in their preparation for potential clinical use.
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