Get rid of the toxicity of radiotherapy and chemotherapy via target hematopoietic stem cells in vivo

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Get rid of the toxicity of radiotherapy and chemotherapy via target hematopoietic stem cells in vivo

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Get rid of the toxicity of radiotherapy and chemotherapy via target hematopoietic stem cells in vivo.

Science Breakthrough | Get rid of the toxicity of radiotherapy and chemotherapy by modifying target hematopoietic stem cells in vivo —— CD117/LNP-mRNA targeting system can deplete and transform hematopoietic stem cells in vivo.

Hematopoietic stem cell transplantation ( HSCT ) can replace defective HSCs with genetically engineered or healthy donor hematopoietic stem cells (HSCs) to treat non-malignant hematopoietic diseases such as hemoglobinopathies and immunodeficiencies.

The current gene therapy needs to isolate hematopoietic stem cells from the patient and perform gene editing, and at the same time use chemotherapy or radiotherapy to eliminate the patient’s own HSCs, but in the process, it will cause infertility or secondary disease caused by the accumulation of DNA damage. Side effects such as malignant tumors.

In order to solve this problem, Stefano Rivella ‘s team from Children’s Hospital of Philadelphia and Hamideh Parhiz ‘s team from the University of Pennsylvania published an article entitled In vivo hematopoietic stem cell modification by mRNA delivery in Science magazine , they developed CD117/LNP- mRNA, a lipid nanoparticle that encapsulates mRNA, targets the stem cell factor receptor CD117 on hematopoietic stem cells.

Delivery of an anti-human CD117/LNP editing system almost completely corrected sickle cells, and in vivo delivery of pro-apoptotic factor PUMA mRNA using CD117/LNP depleted HSCs and reduced chemoradiotherapy toxicity in HSCT.

In conclusion, this tool, as an innovative method for targeting hematopoietic stem cells in vivo, eliminates the shortcomings of current hematopoietic stem cell transplantation, such as many side effects, and can be used as the basis for in vivo genome editing to treat genetic diseases.

Although CD45/LNP and CD117/LNP can bind to all hematopoietic derived cells or stem/progenitor cells respectively, CD117/LNP-Cre has a better ability to transform hematopoietic stem cells after testing, so it was selected for subsequent experiments.

The authors transplanted bone marrow cells from Ail4 reporter mice treated with CD117/LNP-Cre and control IgG/LNP-Cre in vitro to lethally irradiated recipient mice (due to the presence of the termination cassette, tdTomato red fluorescence could not be expressed, and after Cre expression Excision of the termination box can express fluorescence) , 4 months after transplantation, almost all lineages of mice receiving CD117/LNP-Cre were edited, and the editing efficiency of long-term hematopoietic stem cells ( LT-HSCs) at 0.1 μg and 1 μg mRNA doses At 95 percent, the efficiency of gene editing in cells within the spleen was comparable to that in the bone marrow.

Figure 1. The editing efficiency of CD117/LNP-Cre on HSCs in vitro.

Given that CD117/LNP-Cre almost completely targets LT-HSCs in vitro, the authors wanted to test whether it is feasible to directly edit LT-HSCs in vivo.

When CD117/LNP-Cre was treated in vivo for 16 weeks, hematopoietic stem cell editing in peripheral blood and bone marrow was dose-dependent, and 5 μg compared with 1 μg increased the percentage of gene-edited LT-HSCs by 5.5 times, and LNP-Cre in vivo editing resulted in Edited red blood cells emerged with kinetics similar to transplanted ex vivo treated bone marrow.

After 4 months of CD117/LNP-Cre treatment, tdTomato+ colony formation could be observed, and Cre-mediated genomic deletion in bone marrow and spleen cells was confirmed by PCR.

To assess the feasibility of using this platform for therapeutic human genome editing, the authors used LNPs containing mRNA encoding an adenine base editor (ABE) fusion protein, and LNPs carrying an sgRNA targeting the β-sickle cell mutation, Treatment of sickle cell samples from different donors with anti-human CD117/LNP resulted in highly efficient editing (up to 88%) when sgRNA was in excess compared with ABE mRNA-containing LNPs, able to convert pathogenic sickle cell hemoglobin (HBBS ) into non-pathogenic HBBG.

The survival of human and mouse hematopoietic stem cells depends on the anti-apoptotic gene Mcl-1 [2] , so the authors wanted to test the ability of CD117/LNP to combine multiple pro-apoptotic mRNA strategies to clear HSCs. After injecting mice with GFP-BM (bone marrow) cells treated with CD117/LNP-PUMA (p53-upregulated apoptosis regulator) , the mice died after 2 weeks, which proved that CD117/LNP-PUMA can make HSCs lose their vitality.

In addition, in C57BL Intravenous injection of 0.05 mg/kg (maximum tolerated dose) of CD117/LNP-PUMA in /6 mice can deplete HSCs in vivo.

Based on this result, the authors further proved that the in vivo targeting of CD117/LNP-PUMA can be used to effectively deplete HSCs, and achieve consistent HSCs transplantation effects without chemoradiotherapy.

In summary, this study shows that the CD117/LNPs system can be delivered to mouse hematopoietic stem cells with only one systemic injection, and then complete functions such as correcting disease mutations and depleting hematopoietic stem cells.

The achievements of biomedical research in the past ten years have been translated into drugs for the treatment of various human diseases.

Original link:
http://doi.org/10.1126/science.ade6967

Get rid of the toxicity of radiotherapy and chemotherapy via target hematopoietic stem cells in vivo

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