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Clonal Dominance With Retroviral Vector Insertions Near the ANGPT1 and ANGPT2 Genes in a Human Xenotransplant Mouse Model.

Haemmerle R, Phaltane R, Rothe M, Schröder S, Schambach A, Moritz T, Modlich U - Mol Ther Nucleic Acids (2014)

Bottom Line: Insertional leukemogenesis represents the major risk factor of hematopoietic stem cell (HSC) based gene therapy utilizing integrating viral vectors.Furthermore, we specifically investigated the effect of prolonged in vitro culture in the presence of cytokines recently described to promote HSC expansion or maintenance.No overrepresentation of integrations in the vicinity of cancer-related genes was observed, however, several dominant clones were identified including two clones harboring integrations in the ANGPT1 and near the ANGPT2 genes associated with deregulated ANGPT1- and ANGPT2-mRNA levels.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Research Group Reprogramming and Gene Therapy, Rebirth Cluster-of-Excellence, Hannover Medical School, Hannover, Germany.

ABSTRACT
Insertional leukemogenesis represents the major risk factor of hematopoietic stem cell (HSC) based gene therapy utilizing integrating viral vectors. To develop a pre-clinical model for the evaluation of vector-related genotoxicity directly in the relevant human target cells, cord blood CD34(+) HSCs were transplanted into immunodeficient NOD.SCID.IL2rg(-/-) (NSG) mice after transduction with an LTR-driven gammaretroviral vector (GV). Furthermore, we specifically investigated the effect of prolonged in vitro culture in the presence of cytokines recently described to promote HSC expansion or maintenance. Clonality of human hematopoiesis in NSG mice was assessed by high throughput insertion site analyses and validated by insertion site-specific PCR depicting a GV typical integration profile with insertion sites resembling to 25% those of clinical studies. No overrepresentation of integrations in the vicinity of cancer-related genes was observed, however, several dominant clones were identified including two clones harboring integrations in the ANGPT1 and near the ANGPT2 genes associated with deregulated ANGPT1- and ANGPT2-mRNA levels. While these data underscore the potential value of the NSG model, our studies also identified short-comings such as overall low numbers of engrafted HSCs, limited in vivo observation time, and the challenges of in-depth insertion site analyses by low contribution of gene modified hematopoiesis.

No MeSH data available.


Related in: MedlinePlus

Characterization of the human hematopoiesis in NSG mice. Contribution of human cells to the (a) BM and (b) blood. BM and blood cells were analyzed for the expression of human CD45 (each dot represents a transplanted mouse, filled circle: mice in the first experiment, open circle: mice in the second experiment; mean ± SEM).
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fig2: Characterization of the human hematopoiesis in NSG mice. Contribution of human cells to the (a) BM and (b) blood. BM and blood cells were analyzed for the expression of human CD45 (each dot represents a transplanted mouse, filled circle: mice in the first experiment, open circle: mice in the second experiment; mean ± SEM).

Mentions: Next, we investigated the potential of in vitro transduced and expanded CB-CD34+ cells to engraft and maintain hematopoiesis in NOD.SCID.IL2rg−/− (NSG) mice. Per mouse 5 × 104 cells transduced with the gammaretroviral (GV) vector RSF91.eGFP.pre* and cultured for a total of 4 (STF-ctrl) or 10 days were transplanted (Supplementary Materials and Methods). Two independent experiments were carried out resulting in n = 5–6 mice per experimental group (Table 2). In both experiments, the transduction efficiency was beyond 90%, independent of the cytokine conditions employed during transduction (Supplementary Figure S1). In a control group, NSG mice were transplanted with cells transduced and cultured only for 4 days in STF medium (STF-ctrl; n = 6 mice). In all groups, the contribution of human CD45+ leukocytes in the peripheral blood increased over time indicating effective engraftment (Supplementary Figure S2). After 24 weeks, animals were sacrificed and the human engraftment and lineage contribution in the different hematopoietic tissues were determined by FACS analysis (Figure 2 and Supplementary Figure S3). In all groups, contribution of human cells to overall hematopoiesis varied substantially. There was a tendency towards lower BM engraftment and less human cell contribution to the blood in mice of the GCSF and STF-ctrl groups (Figure 2a,b); however, this was not significant by Kruskal–Wallis analysis across the groups, P = 0.5; (pairwise test in the blood: STF-ctrl versus STF P = 0.08, GCSF versus STF P = 0.1, Mann–Whitney test). In these mice, also human T-cell contribution was low (Supplementary Figure S4) in line with an insufficient thymus repopulation as well as peripheral blood mobilization observed in these groups. In the reconstituted thymi, human T-cell development was normal with human CD4/CD8 double- and single-positive T-cells detectable (Supplementary Figure S4).


Clonal Dominance With Retroviral Vector Insertions Near the ANGPT1 and ANGPT2 Genes in a Human Xenotransplant Mouse Model.

Haemmerle R, Phaltane R, Rothe M, Schröder S, Schambach A, Moritz T, Modlich U - Mol Ther Nucleic Acids (2014)

Characterization of the human hematopoiesis in NSG mice. Contribution of human cells to the (a) BM and (b) blood. BM and blood cells were analyzed for the expression of human CD45 (each dot represents a transplanted mouse, filled circle: mice in the first experiment, open circle: mice in the second experiment; mean ± SEM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4217076&req=5

fig2: Characterization of the human hematopoiesis in NSG mice. Contribution of human cells to the (a) BM and (b) blood. BM and blood cells were analyzed for the expression of human CD45 (each dot represents a transplanted mouse, filled circle: mice in the first experiment, open circle: mice in the second experiment; mean ± SEM).
Mentions: Next, we investigated the potential of in vitro transduced and expanded CB-CD34+ cells to engraft and maintain hematopoiesis in NOD.SCID.IL2rg−/− (NSG) mice. Per mouse 5 × 104 cells transduced with the gammaretroviral (GV) vector RSF91.eGFP.pre* and cultured for a total of 4 (STF-ctrl) or 10 days were transplanted (Supplementary Materials and Methods). Two independent experiments were carried out resulting in n = 5–6 mice per experimental group (Table 2). In both experiments, the transduction efficiency was beyond 90%, independent of the cytokine conditions employed during transduction (Supplementary Figure S1). In a control group, NSG mice were transplanted with cells transduced and cultured only for 4 days in STF medium (STF-ctrl; n = 6 mice). In all groups, the contribution of human CD45+ leukocytes in the peripheral blood increased over time indicating effective engraftment (Supplementary Figure S2). After 24 weeks, animals were sacrificed and the human engraftment and lineage contribution in the different hematopoietic tissues were determined by FACS analysis (Figure 2 and Supplementary Figure S3). In all groups, contribution of human cells to overall hematopoiesis varied substantially. There was a tendency towards lower BM engraftment and less human cell contribution to the blood in mice of the GCSF and STF-ctrl groups (Figure 2a,b); however, this was not significant by Kruskal–Wallis analysis across the groups, P = 0.5; (pairwise test in the blood: STF-ctrl versus STF P = 0.08, GCSF versus STF P = 0.1, Mann–Whitney test). In these mice, also human T-cell contribution was low (Supplementary Figure S4) in line with an insufficient thymus repopulation as well as peripheral blood mobilization observed in these groups. In the reconstituted thymi, human T-cell development was normal with human CD4/CD8 double- and single-positive T-cells detectable (Supplementary Figure S4).

Bottom Line: Insertional leukemogenesis represents the major risk factor of hematopoietic stem cell (HSC) based gene therapy utilizing integrating viral vectors.Furthermore, we specifically investigated the effect of prolonged in vitro culture in the presence of cytokines recently described to promote HSC expansion or maintenance.No overrepresentation of integrations in the vicinity of cancer-related genes was observed, however, several dominant clones were identified including two clones harboring integrations in the ANGPT1 and near the ANGPT2 genes associated with deregulated ANGPT1- and ANGPT2-mRNA levels.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Research Group Reprogramming and Gene Therapy, Rebirth Cluster-of-Excellence, Hannover Medical School, Hannover, Germany.

ABSTRACT
Insertional leukemogenesis represents the major risk factor of hematopoietic stem cell (HSC) based gene therapy utilizing integrating viral vectors. To develop a pre-clinical model for the evaluation of vector-related genotoxicity directly in the relevant human target cells, cord blood CD34(+) HSCs were transplanted into immunodeficient NOD.SCID.IL2rg(-/-) (NSG) mice after transduction with an LTR-driven gammaretroviral vector (GV). Furthermore, we specifically investigated the effect of prolonged in vitro culture in the presence of cytokines recently described to promote HSC expansion or maintenance. Clonality of human hematopoiesis in NSG mice was assessed by high throughput insertion site analyses and validated by insertion site-specific PCR depicting a GV typical integration profile with insertion sites resembling to 25% those of clinical studies. No overrepresentation of integrations in the vicinity of cancer-related genes was observed, however, several dominant clones were identified including two clones harboring integrations in the ANGPT1 and near the ANGPT2 genes associated with deregulated ANGPT1- and ANGPT2-mRNA levels. While these data underscore the potential value of the NSG model, our studies also identified short-comings such as overall low numbers of engrafted HSCs, limited in vivo observation time, and the challenges of in-depth insertion site analyses by low contribution of gene modified hematopoiesis.

No MeSH data available.


Related in: MedlinePlus