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Hematopoietic reconstitution after bone marrow transplantation is thought to be driven by committed multipotent progenitor cells followed by long-term engrafting hematopoietic stem cells (HSCs). We observed a population of early-engrafting cells displaying HSC-like behavior, which persisted long-term in vivo in an autologous myeloablative transplant model in nonhuman primates. To identify this population, we characterized the phenotype and function of defined nonhuman primate hematopoietic stem and progenitor cell (HSPC) subsets and compared these to human HSPCs. We demonstrated that the CD34CD45RACD90 cell phenotype is highly enriched for HSCs. This population fully supported rapid short-term recovery and robust multilineage hematopoiesis in the nonhuman primate transplant model and quantitatively predicted transplant success and time to neutrophil and platelet recovery. Application of this cell population has potential in the setting of HSC transplantation and gene therapy/editing approaches.

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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:

Alkylating agent chemotherapy for malignant disease results in myelosuppression which significantly limits dose-escalation and potential clinical efficacy. Drug resistance gene therapy with mutant MGMT (P140K) gene-modified hematopoietic stem cells may circumvent this problem but has not been evaluated clinically. Here we show efficient, polyclonal engraftment of P140K-modified hematopoietic cells in patients with unmethylated MGMT promoter glioblastomas with apparently normal BM after well-tolerated, nonmyeloablative conditioning with BCNU. Increases in P140K-modified cells after post-transplant chemotherapy including temozolomide and O6-benzylguanine indicate protection and selection of gene-modified hematopoietic repopulating cells. Longitudinal retroviral integration site (RIS) analysis identified over 12,000 unique RIS in the first three patients with multiple clones present in PB of each patient throughout multiple chemotherapy treatments. To assess safety, RIS distribution was monitored over time and chemotherapy treatments. Two patients exhibited emergence of prominent clones harboring RIS associated with the intronic coding region of PRDM16 or the 3′ UTR of HMGA2 genes with no adverse clinical outcomes. All three patients surpassed the median survival for patients with unmethlyated MGMT promoter glioblastomas, with one patient alive and progression-free more than two years since diagnosis. We conclude that transplantation of P140K-expressing hematopoietic cells mediates chemoprotection and selection, potentially maximizing chemotherapy administration in the treatment of cancer and providing a strategy for increasing gene-corrected/therapeutic cell levels in patients with genetic or infectious diseases.

Key Points Rapamycin significantly enhances lentiviral vector gene delivery to hematopoietic stem cells while preserving engraftment potential. Rapamycin-mediated transduction enhancement is not accompanied by alterations in lentiviral integration profile.

The mammalian non-histone "high mobility group" A (HMGA) proteins are the primary nuclear proteins that bind to the minor groove of AT-rich DNA. They may, therefore, influence the formation and/or repair of DNA lesions that occur in AT-rich DNA, such as cyclobutane pyrimidine dimers (CPDs) induced by UV radiation. Employing both stably transfected lines of human MCF7 cells containing tetracycline-regulated HMGA1 transgenes and primary Hs578T tumor cells, which naturally overexpress HMGA1 proteins, we have shown that cells overexpressing HMGA1a protein exhibit increased UV sensitivity. Moreover, we demonstrated that knockdown of intracellular HMGA1 concentrations via two independent methods abrogated this sensitivity. Most significantly, we observed that HMGA1a overexpression inhibited global genomic nucleotide excision repair of UV-induced CPD lesions in MCF-7 cells. Consistent with these findings in intact cells, DNA repair experiments employing Xenopus oocyte nuclear extracts and lesion-containing DNA substrates demonstrated that binding of HMGA1a markedly inhibits removal of CPDs in vitro. Furthermore, UV "photo-footprinting" demonstrated that CPD formation within a long run of Ts (T 18 -tract) in a DNA substrate changes significantly when HMGA1 is bound prior to UV irradiation. Together, these results suggest that HMGA1 directly influences both the formation and repair of UV-induced DNA lesions in intact cells. These findings have important implications for the role that HMGA protein overexpression might play in the accumulation of mutations and genomic instabilities associated with many types of human cancers.In most organisms, DNA helix-distorting bulky lesions are repaired by nucleotide excision repair (NER) 5 involving the excision and replacement of 24 -32 nt of the damaged DNA strand (1). Many factors, such as (a) the type of DNA damage, (b) the DNA sequence surrounding the lesion, (c) the position in chromatin, and (d) the interactions with DNAbinding proteins, are known to affect the efficiency of NER (2-4). For instance, inhibitory effects of nucleosomes on NER have been observed in vitro and in intact cells (5-9), presumably reflecting the limited access of NER proteins to these lesions (10). In a similar way, certain DNAbinding proteins, such as transcription factor IIIA and HMGB1 proteins, are known to repress NER at their cognate sequences (11-13). Conversely, transcriptional activators and the RNA polymerase II elongation complex are associated with enhanced repair of transcribing genes (14, 15). Furthermore, in contrast to HMGB1, another member of the HMG protein superfamily that specifically binds to nucleosomes, HMGN1, has been shown to likewise enhance NER of UV-damaged DNA in vivo (16).Stable DNA photoproducts produced by UV-induced covalent linkage between adjacent bases are prototypes of helical distorting, bulky lesions (17). Among a variety of possible UV-induced photoproducts, cyclobutane pyrimidine dimers (CPDs) are the most abundant, stable forms (17) and, if unrepaired, are known...