In Vivo Hematopoietic Stem Cell Transduction

Richter, Maximilian; Stone, Daniel; Miao, Carol H.; Humbert, Olivier; Kiem, Hans–Peter; Papayannopoulou, Thalia; Lieber, André

doi:10.1016/j.hoc.2017.06.001

Cited by 26 publications

(16 citation statements)

References 74 publications

(80 reference statements)

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“…We developed a minimally invasive and readily translatable approach for in vivo HSPC gene delivery without leukapheresis, myeloablation, and HSPC transplantation (6)(7)(8). It involves injections of G-CSF/AMD3100 to mobilize HSPCs from the bone marrow into the peripheral bloodstream and the intravenous injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system.…”

Section: Introductionmentioning

confidence: 99%

In vivo hematopoietic stem cell gene therapy ameliorates murine thalassemia intermedia

Wang¹,

Georgakopoulou²,

Psatha³

et al. 2018

Journal of Clinical Investigation

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after the second round of selection and reached levels above 80% after the third round. The percentage of γ-globin-expressing cells was approximately 7-to 10-fold higher in erythroid Ter119 + cells versus nonerythroid Ter119cells in peripheral blood and bone marrow (Figure 1D). We used HPLC to measure the level of γ-globin protein in comparison with the adult mouse α-and β-globin chains (Figure 1E and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/ JCI122836DS1). At week 18, these levels reached 10%-15% of adult mouse α-globin and β-major globin and approximately 25% of mouse β-minor globin. This was confirmed on the mRNA level by quantitative reverse transcription PCR (RT-qPCR), where human γ-globin mRNA was approximately 13% of mouse β-major mRNA (Figure 1F). To further demonstrate that primitive, long-term repopulating HSCs were transduced, we transplanted lineagedepleted (Lin-) bone marrow cells from in vivo-transduced/ selected mice into irradiated C57BL/6 mice. Engraftment levels analyzed in peripheral blood, bone marrow, and spleen were greater than 95% and stable over an observation period of 20 weeks (Supplemental Figure 2, A and B). Human γ-globin levels (compared with mouse α-globin) were similar in ("primary") in vivo-transduced mice (analyzed at week 18 after transduction) and secondary recipients analyzed at weeks 14 and 20 after transplantation (Supplemental Figure 2C). The in vivo HSPC transduction/selection approach does not change the SB100X-mediated random transgene integration pattern and does not alter hematopoiesis. We previously showed that in vivo transduction with the hybrid transposon/SB100X HDAd5/35++ system resulted in random transgene integration in HSPCs (6). To evaluate the effect of O 6 BG/BCNU in in vivo selection, we analyzed transgene integration in bone marrow Lincells at the end of the study, i.e., at week 20 in secondary recipients. Linear amplification-mediated PCR (LAM-PCR) followed by deep sequencing showed a random distribution pattern of integration sites in the mouse genome (Figure 2A). Data pooled from 5 mice demonstrated 2.23% integration into exons, 31.58% into introns, 65.17% into intergenic regions, and 1.04% into untranslated regions (Figure 2B). The level of randomness of integration was 99% without preferential integration in any given window of the whole mouse genome (Figure 2C). This indicates that in vivo selection and further expansion of cells in secondary recipients did not result in the emergence of dominant integration sites (Figure 2D). We measured, by qPCR, on average two γ-globin cDNA copies per bone marrow cell in a population containing both transduced and nontransduced cells. We then quantified the integrated transgene copy number on a single-cell level. To do this, we plated bone marrow Lincells from week 18 mice in methylcellulose, isolated individual progenitor colonies, and performed qPCR on genomic DNA. In transgene-positive colonies (n = 113), 86.7% of colonies had 2 or 3 integrated co...

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Section: Introductionmentioning

confidence: 99%

In vivo hematopoietic stem cell gene therapy ameliorates murine thalassemia intermedia

Wang¹,

Georgakopoulou²,

Psatha³

et al. 2018

Journal of Clinical Investigation

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“…In a recent study, we tested SB100x-armed HDAd5/35++ vectors for in vivo HSC transduction. 10 , 19 The approach involved the subcutaneous injection of granulocyte-colony stimulating factor (GCSF)/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of the integrating HDAd5/35++ vector system. We demonstrated in adequate mouse models that our vectors allow for the stable transduction of HSCs, with a preference for transducing primitive HSCs (Lin – /Sca-1 + /c-Kit + [LSK] cells, colony-forming unit [CFU], long-term repopulating cells).…”

Section: Introductionmentioning

confidence: 99%

Integrating HDAd5/35++ Vectors as a New Platform for HSC Gene Therapy of Hemoglobinopathies

Psatha

Wang

et al. 2018

Molecular Therapy - Methods & Clinical Development

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We generated an integrating, CD46-targeted, helper-dependent adenovirus HDAd5/35++ vector system for hematopoietic stem cell (HSC) gene therapy. The ∼12-kb transgene cassette included a β-globin locus control region (LCR)/promoter driven human γ-globin gene and an elongation factor alpha-1 (EF1α)-mgmtP140K expression cassette, which allows for drug-controlled increase of γ-globin-expressing erythrocytes. We transduced bone marrow lineage-depleted cells from human CD46-transgenic mice and transplanted them into lethally irradiated recipients. The percentage of γ-globin-positive cells in peripheral blood erythrocytes in primary and secondary transplant recipients was stable and greater than 90%. The γ-globin level was 10%–20% of adult mouse globin. Transgene integration, mediated by a hyperactive Sleeping Beauty SB100x transposase, was random, without a preference for genes. A second set of studies was performed with peripheral blood CD34+ cells from mobilized donors. 10 weeks after transplantation of transduced cells, human cells were harvested from the bone marrow and differentiated ex vivo into erythroid cells. Erythroid cells expressed γ-globin at a level of 20% of adult α-globin. Our studies suggest that HDAd35++ vectors allow for efficient transduction of long-term repopulating HSCs and high-level, almost pancellular γ-globin expression in erythrocytes. Furthermore, our HDAd5/35++ vectors have a larger insert capacity and a safer integration pattern than currently used lentivirus vectors.

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“…Our platform enables modulation of gene expression in adult mice in an efficient and effective manner. Several different approaches have been previously used to induce genetic alterations in LSK cells, including RNP-based and AAV-based approaches [29][30][31]. Gundry, et al successfully edited genes in both human and murine HSCs with RNP-based approach [30], however, that study were limited to ex vivo analysis with a relatively low efficiency of 60%.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-targeting of CD40 in hematopoietic stem cells limits immune activation mediated by anti-CD40

Wang¹,

Graham²,

Sun³

et al. 2020

PLoS ONE

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Inflammatory bowel diseases (IBD) are complex, multifactorial disorders characterized by chronic relapsing intestinal inflammation. IBD is diagnosed around 1 in 1000 individuals in Western countries with globally increasing incident rates. Association studies have identified hundreds of genes that are linked to IBD and potentially regulate its pathology. The further dissection of the genetic network underlining IBD pathogenesis and pathophysiology is hindered by the limited capacity to functionally characterize each genetic association, including generating knockout animal models for every associated gene. Cutting-edge CRISPR/ Cas9-based technology may transform the field of IBD research by efficiently and effectively introducing genetic alterations. In the present study, we used CRISPR/Cas9-based technologies to genetically modify hematopoietic stem cells. Through cell sorting and bone marrow transplantation, we established a system to knock out target gene expression by over 90% in the immune system of reconstituted animals. Using a CD40-mediated colitis model, we further validated our CRISPR/Cas9-based platform for investigating gene function in experimental IBD. In doing so, we developed a model system that delivers genetically modified mice in a manner much faster than conventional methodology, significantly reducing the time from target identification to in vivo target validation and expediting drug development.

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In Vivo Hematopoietic Stem Cell Transduction

Cited by 26 publications

References 74 publications

In vivo hematopoietic stem cell gene therapy ameliorates murine thalassemia intermedia

In vivo hematopoietic stem cell gene therapy ameliorates murine thalassemia intermedia

Integrating HDAd5/35++ Vectors as a New Platform for HSC Gene Therapy of Hemoglobinopathies

CRISPR/Cas9-targeting of CD40 in hematopoietic stem cells limits immune activation mediated by anti-CD40

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