Ex vivo CRISPR gene editing in hematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation, sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci, cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. Engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to nontreated cells, with no differences in differentiation. Here we demonstrate nontoxic delivery of the entire CRISPR payload into primary human blood progenitors. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Key Points
The cyclic resveratrol trimer caraphenol A safely enhances lentiviral vector gene delivery to hematopoietic stem and progenitor cells. Caraphenol A decreases interferon-induced transmembrane protein-mediated restriction in an endosomal trafficking-dependent manner.
A hallmark of Fanconi anemia is accelerated decline in hematopoietic stem and
progenitor cells (CD34 +) leading to bone marrow failure. Long-term treatment
requires hematopoietic cell transplantation from an unaffected donor but is
associated with potentially severe side-effects. Gene therapy to correct the
genetic defect in the patient’s own CD34+ cells has been
limited by low CD34+ cell numbers and viability. Here we demonstrate
an altered ratio of CD34Hi to CD34Lo cells in Fanconi
patients relative to healthy donors, with exclusive in vitro
repopulating ability in only CD34Hi cells, underscoring a need for
novel strategies to preserve limited CD34+ cells. To address this
need, we developed a clinical protocol to deplete
lineage+(CD3+, CD14+, CD16+ and
CD19+) cells from blood and marrow products. This process
depletes >90% of lineage+cells while retaining
≥60% of the initial CD34+cell fraction, reduces total
nucleated cells by 1–2 logs, and maintains transduction efficiency and
cell viability following gene transfer. Importantly, transduced
lineage− cell products engrafted equivalently to that of
purified CD34+ cells from the same donor when xenotransplanted at
matched CD34+ cell doses. This novel selection strategy has been
approved by the regulatory agencies in a gene therapy study for Fanconi anemia
patients (NCI Clinical Trial Reporting Program Registry ID
NCI-2011-00202; clinicaltrials.gov identifier:
01331018).
Summary
Myeloid-differentiated hematopoietic stem cells (HSCs) have contributed to a number of novel treatment approaches for lysosomal storage diseases of the central nervous system (CNS), and may also be applied to patients infected with HIV. We quantified hematopoietic stem and progenitor cell (HSPC) trafficking to 20 tissues including lymph nodes, spleen, liver, gastrointestinal tract, CNS, and reproductive tissues. We observed efficient marking of multiple macrophage subsets, including CNS-associated myeloid cells, suggesting that HSPC-derived macrophages are a viable approach to target gene-modified cells to tissues. Gene-marked cells in the CNS were unique from gene-marked cells at any other physiological sites including peripheral blood. This novel finding suggests that these cells were derived from HSPCs, migrated to the brain, were compartmentalized, established myeloid progeny, and could be targeted for lifelong delivery of therapeutic molecules. Our findings have highly relevant implications for the development of novel therapies for genetic and infectious diseases of the CNS.
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