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2018 ; 9
(ä): 390-401
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HDAd5/35(++) Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases
CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells
#MMPMID30038942
Li C
; Psatha N
; Gil S
; Wang H
; Papayannopoulou T
; Lieber A
Mol Ther Methods Clin Dev
2018[Jun]; 9
(ä): 390-401
PMID30038942
show ga
We generated helper-dependent HDAd5/35(++) adenovirus vectors expressing
CRISPR/Cas9 for potential hematopoietic stem cells (HSCs) gene therapy of
?-thalassemia and sickle cell disease through re-activation of fetal ?-globin
expression (HDAd-globin-CRISPR). The process of CRISPR/Cas9 gene transfer using
these vectors was not associated with death of human CD34(+) cells and did not
affect their in vitro expansion and erythroid differentiation. However,
functional assays for primitive HSCs, e.g., multi-lineage progenitor colony
formation and engraftment in irradiated NOD/Shi-scid/interleukin-2 receptor ?
(IL-2R?) null (NSG) mice, revealed toxicity of HDAd-globin-CRISPR vectors related
to the prolonged expression and activity of CRISPR/Cas9. To control the duration
of CRISPR/Cas9 activity, we generated an HDAd5/35(++) vector that expressed two
anti-CRISPR (Acr) peptides (AcrII4 and AcrII2) capable of binding to the
CRISPR/Cas9 complex (HDAd-Acr). CD34(+) cells that were sequentially infected
with HDAd-CRISPR and HDAd-Acr engrafted at a significantly higher rate. Target
site disruption frequencies in engrafted human cells were similar to those in
pre-transplantation CD34(+) cells, indicating that genome-edited primitive HSCs
survived. In vitro differentiated HSCs isolated from transplanted mice
demonstrated increased ?-globin expression as a result of genome editing. Our
data indicate that the HDAd-Acr vector can be used as a tool to reduce HSC
cytotoxicity of the CRISPR/Cas9 complex.