Mechanistic Insights into Factor VIII Immune Tolerance Induction via Prenatal Cell Therapy in Hemophilia A

Rodriguez, Martin; Porada, Christopher D.; Almeida-Porada, Graça

doi:10.1007/s40778-019-00165-y

Cited by 3 publications

(2 citation statements)

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“…Furthermore, as the AAVS1 site is thought to be a transcripionally active site with open chromatic structure and native insulators that can resist transgene silencing, it stood to reason that the use of CRISPR/Cas9 to introduce the EF1α-lcoET3 expression cassette into this site might also enhance the levels of expression of the fVIII transgene and thus improve therapeutic efficacy ( 55 ). As there are thousands of different mutations spanning the whole FVIII gene locus that can cause HA ( 69 ), the use of this “knock-in” approach to insert a functional fVIII transgene into the genome, rather than trying to correct a specific HA-causing mutation, was deemed to be far more practical, as it would yield a universal treatment that could be administered to all HA patients. PLCs were simultaneosuly modified with two common modes of gene delivery, lentivector transduction and transfection with lcoET3-expressing plasmid, and side-by-side comparisons performed to results obtained with CRISPR/Cas9 gene-editing.…”

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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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%

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

et al. 2022

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“…In-utero fetal cell therapy provides the opportunity to replace or augment a dysfunctional cellular population during development, with the potential to avoid or ameliorate a state of disease. In vivo experience to date has included replacing osteoblasts for fetuses affected by osteogenesis imperfecta1 and replacement of hematopoietic lineages for the prenatal treatment of thalassemia2 and hemophilia A 3. Successful in utero cell therapy requires 4 components: the isolation of a replacement cell population, in utero stem cell transplantation, cell engraftment in an appropriate biological niche with fetal immune tolerance, and ongoing appropriate physiological cell function (Fig.…”

Section: In Utero Cell Therapymentioning

confidence: 99%

In Utero Fetal Therapy: Stem Cells, Cell Transplantation, Gene Therapy, and CRISPR-Cas9

Shear

Massa

2021

Clinical Obstetrics &Amp; Gynecology

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In utero fetal therapy offers the opportunity to prevent and treat diseases with a cellular or genetic basis. Components of successful fetal treatment include isolation of a replacement cell population, in utero stem cell transplantation, cell engraftment with fetal immune tolerance, and ongoing cell function. Fetal gene therapy with CRISPR-Cas9 represents an exciting potential therapy for genetic diseases not amenable to gene supplementation via adenoviral vector transduction. These fetal therapies have unique ethical and safety considerations. Clinical trials for in utero cell therapy are underway, as additional discoveries in stem cell biology and gene therapy move closer to clinical translation.

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In Utero Cell Treatment of Hemophilia A Mice via Human Amniotic Fluid Mesenchymal Stromal Cell Engraftment

Kao,

Yen,

Fan

et al. 2023

IJMS

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Hemophilia is a genetic disorder linked to the sex chromosomes, resulting in impaired blood clotting due to insufficient intrinsic coagulation factors. There are approximately one million individuals worldwide with hemophilia, with hemophilia A being the most prevalent form. The current treatment for hemophilia A involves the administration of clotting factor VIII (FVIII) through regular and costly injections, which only provide temporary relief and pose inconveniences to patients. In utero transplantation (IUT) is an innovative method for addressing genetic disorders, taking advantage of the underdeveloped immune system of the fetus. This allows mesenchymal stromal cells to play a role in fetal development and potentially correct genetic abnormalities. The objective of this study was to assess the potential recovery of coagulation disorders in FVIII knockout hemophilia A mice through the administration of human amniotic fluid mesenchymal stromal cells (hAFMSCs) via IUT at the D14.5 fetal stage. The findings revealed that the transplanted human cells exhibited fusion with the recipient liver, with a ratio of approximately one human cell per 10,000 mouse cells and produced human FVIII protein in the livers of IUT-treated mice. Hemophilia A pups born to IUT recipients demonstrated substantial improvement in their coagulation issues from birth throughout the growth period of up to 12 weeks of age. Moreover, FVIII activity reached its peak at 6 weeks of age, while the levels of FVIII inhibitors remained relatively low during the 12-week testing period in mice with hemophilia. In conclusion, the results indicated that prenatal intrahepatic therapy using hAFMSCs has the potential to improve clotting issues in FVIII knockout mice, suggesting it as a potential clinical treatment for individuals with hemophilia A.

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Mechanistic Insights into Factor VIII Immune Tolerance Induction via Prenatal Cell Therapy in Hemophilia A

Cited by 3 publications

References 226 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

In Utero Fetal Therapy: Stem Cells, Cell Transplantation, Gene Therapy, and CRISPR-Cas9

In Utero Cell Treatment of Hemophilia A Mice via Human Amniotic Fluid Mesenchymal Stromal Cell Engraftment

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