Therapeutic correction of hemophilia A by transplantation of hPSC-derived liver sinusoidal endothelial cell progenitors

Gage, Blair K.; Merlin, Simone; Olgasi, Cristina; Follenzi, Antonia; Keller, Gordon

doi:10.1016/j.celrep.2022.110621

Cited by 10 publications

(9 citation statements)

References 69 publications

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“…phenotypic correction (74)(75)(76)(82)(83)(84)(85)(86)(87). The use of iPSC also has the added advantage of the ease with which these cells can be modified with genome-editing platforms, such as CRISPR/Cas9, that require induction of a doublestrand break (DSB) following by homologous recombination repair (HRR), a process that only occurs in actively dividing cells (88-92).…”

Section: Discussionmentioning

confidence: 99%

“…Although a good deal of progress has been made using CRISPR/Cas9-mediated gene editing to correct hemophilia B (HB), only a few studies have reported using this approach to attempt to correct HA ( 70 – 79 ), due in large part to the difficulty of achieving CRISPR/Cas9-mediated knock-in of a transgene with the increased length and complexity of FVIII ( 80 , 81 ). The studies published to-date using CRISPR/Cas9 to correct HA have employed iPSCs, which enables selection of clones that have been edited successfully, which can then be differentiated into the desired cell types, such as endothelial cells or mesenchymal stromal cells (MSC) that can then be transplanted to mediate phenotypic correction ( 74 – 76 , 82 – 87 ). The use of iPSC also has the added advantage of the ease with which these cells can be modified with genome-editing platforms, such as CRISPR/Cas9, that require induction of a doublestrand break (DSB) following by homologous recombination repair (HRR), a process that only occurs in actively dividing cells ( 88 – 92 ).…”

Section: Discussionmentioning

confidence: 99%

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Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII

et al. 2022

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IntroductionPlacenta-derived mesenchymal cells (PLCs) endogenously produce FVIII, which makes them ideally suited for cell-based fVIII gene delivery. We have previously reported that human PLCs can be efficiently modified with a lentiviral vector encoding a bioengineered, expression/secretion-optimized fVIII transgene (ET3) and durably produce clinically relevant levels of functionally active FVIII. The objective of the present study was to investigate whether CRISPR/Cas9 can be used to achieve location-specific insertion of a fVIII transgene into a genomic safe harbor, thereby eliminating the potential risks arising from the semi-random genomic integration inherent to lentiviral vectors. We hypothesized this approach would improve the safety of the PLC-based gene delivery platform and might also enhance the therapeutic effect by eliminating chromatin-related transgene silencing.MethodsWe used CRISPR/Cas9 to attempt to insert the bioengineered fVIII transgene “lcoET3” into the AAVS1 site of PLCs (CRISPR-lcoET3) and determined their subsequent levels of FVIII production, comparing results with this approach to those achieved using lentivector transduction (LV-lcoET3) and plasmid transfection (Plasmid-lcoET3). In addition, since liver-derived sinusoidal endothelial cells (LSECs) are the native site of FVIII production in the body, we also performed parallel studies in human (h)LSECs).ResultsPLCs and hLSECs can both be transduced (LV-lcoET3) with very high efficiency and produce high levels of biologically active FVIII. Surprisingly, both cell types were largely refractory to CRISPR/Cas9-mediated knockin of the lcoET3 fVIII transgene in the AAVS1 genome locus. However, successful insertion of an RFP reporter into this locus using an identical procedure suggests the failure to achieve knockin of the lcoET3 expression cassette at this site is likely a function of its large size. Importantly, using plasmids, alone or to introduce the CRISPR/Cas9 “machinery”, resulted in dramatic upregulation of TLR 3, TLR 7, and BiP in PLCs, compromising their unique immune-inertness.DiscussionAlthough we did not achieve our primary objective, our results validate the utility of both PLCs and hLSECs as cell-based delivery vehicles for a fVIII transgene, and they highlight the hurdles that remain to be overcome before primary human cells can be gene-edited with sufficient efficiency for use in cell-based gene therapy to treat HA.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII

et al. 2022

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“… 30 The resulting cells have been used to correct hemophilia induced by factor VIII deficiency in animal models. 31 Forward programming combined with directed differentiation has also been applied to generate endothelial cells. 32 For this cell type, the simple expression of ETV2 seems sufficient to induce endothelial programming.…”

Section: Generating the Different Hepatic Cells From Hipscsmentioning

confidence: 99%

Modeling Nonalcoholic Fatty Liver Disease in the Dish Using Human-Specific Platforms: Strategies and Limitations

Rezvani¹,

Vallier²,

Guillot³

2023

Cellular and Molecular Gastroenterology and Hepatology

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“…Despite promising pre-clinical results, the development of effective cell therapy approaches for HA treatment may benefit from providing insights into non-genetically modified cell populations able to produce FVIII and endowed with high engraftment potential. Indeed, pre-clinical studies have already demonstrated the therapeutic potential of non-transduced cell populations in correcting the Haemophilia A phenotype, resorting to the transplantation into animal HA models of tissue-specific cells such as Liver Endothelial Sinusoidal Cells (LESCs) [21] and human pluripotent stem cell (hPSC)-derived LSEC progenitors [22]. Based on the previous research, either hepatocytes or LSECs could be candidate cells for the therapeutic intervention of HA; nevertheless, the transplantation of stem cells rather than already specialized cells seems to be the most promising approach due to the greater proliferative and differentiation potential of cell populations with stem/progenitor characteristics [23].…”

Section: Introductionmentioning

confidence: 99%

In Vitro Conditioning of Adipose-Derived Mesenchymal Stem Cells by the Endothelial Microenvironment: Modeling Cell Responsiveness towards Non-Genetic Correction of Haemophilia A

Barbon

Stocco

Rajendran

et al. 2022

IJMS

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In recent decades, the use of adult multipotent stem cells has paved the way for the identification of new therapeutic approaches for the treatment of monogenic diseases such as Haemophilia A. Being already studied for regenerative purposes, adipose-derived mesenchymal stem cells (Ad-MSCs) are still poorly considered for Haemophilia A cell therapy and their capacity to produce coagulation factor VIII (FVIII) after proper stimulation and without resorting to gene transfection. In this work, Ad-MSCs were in vitro conditioned towards the endothelial lineage, considered to be responsible for coagulation factor production. The cells were cultured in an inductive medium enriched with endothelial growth factors for up to 21 days. In addition to significantly responding to the chemotactic endothelial stimuli, the cell populations started to form capillary-like structures and up-regulated the expression of specific endothelial markers (CD34, PDGFRα, VEGFR2, VE-cadherin, CD31, and vWF). A dot blot protein study detected the presence of FVIII in culture media collected from both unstimulated and stimulated Ad-MSCs. Remarkably, the activated partial thromboplastin time test demonstrated that the clot formation was accelerated, and FVIII activity was enhanced when FVIII deficient plasma was mixed with culture media from the untreated/stimulated Ad-MSCs. Overall, the collected evidence supported a possible Ad-MSC contribution to HA correction via specific stimulation by the endothelial microenvironment and without any need for gene transfection.

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Therapeutic correction of hemophilia A by transplantation of hPSC-derived liver sinusoidal endothelial cell progenitors

Cited by 10 publications

References 69 publications

Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII

Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII

Modeling Nonalcoholic Fatty Liver Disease in the Dish Using Human-Specific Platforms: Strategies and Limitations

In Vitro Conditioning of Adipose-Derived Mesenchymal Stem Cells by the Endothelial Microenvironment: Modeling Cell Responsiveness towards Non-Genetic Correction of Haemophilia A

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