Karin Gaensler

Adenovirus-mediated gene transfer to the peritoneum and hepatic parenchyma of fetal mice in utero ☆ ☆☆

BACKGROUND The development of effective gene transfer in utero will provide alternative approaches to the treatment of genetic disorders. For many disorders, the fetal liver and peritoneum are important target tissues. Our goals were to compare the tissue sites and duration of transferred gene expression after intraperitoneal (i.p.) or intrahepatic adenoviral-mediated gene transfer in utero in the developing murine fetus. METHODS Day 15 CD-1 fetuses were injected intrahepatically or intraperitoneally with recombinant adenoviruses containing the luciferase or beta-galactosidase reporter gene. Tissue levels of luciferase were quantitated, or tissues were examined for X-gal staining. RESULTS Luciferase expression was observed in multiple fetal tissues (including brain, intestine, liver, and lung) and persisted up to 32 days after intrahepatic delivery. Significant hepatic tropism was demonstrated. CONCLUSIONS Intrahepatic and intraperitoneal injection in utero results in transduction of multiple tissues in the developing murine fetus. Transuterine injection of fetal mice via intrahepatic and intraperitoneal routes provides a valuable model for assessing the efficacy of gene delivery vectors in the prenatal treatment of genetic disorders. These studies demonstrate that hepatic and intraperitoneal gene transfer to the developing murine fetus is feasible and may provide therapeutic levels of proteins during fetal development.

Transcription factors KLF1 and KLF2 positively regulate embryonic and fetal β-globin genes through direct promoter binding

Krüppel-like factors (KLFs) control cell differentiation and embryonic development. KLF1 (erythroid Krüppel-like factor) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β-globin genes. KLF1 and KLF2 have highly homologous zinc finger DNA-binding domains. They have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double KO mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic lethality, but double KO embryos are more anemic and die sooner than either single KO. In this work, a dual human β-globin locus transgenic and KLF knockout mouse model was used. The results demonstrate that the human ϵ- (embryonic) and γ-globin (fetal) genes are positively regulated by KLF1 and KLF2 in embryos. Conditional KO mouse experiments indicate that the effect of KLF2 on embryonic globin gene regulation is at least partly erythroid cell-autonomous. KLF1 and KLF2 bind directly to the promoters of the human ϵ- and γ-globin genes, the mouse embryonic Ey- and βh1-globin genes, and also to the β-globin locus control region, as demonstrated by ChIP assays with mouse embryonic blood cells. H3K9Ac and H3K4me3 marks indicate open chromatin and active transcription, respectively. These marks are diminished at the Ey-, βh1-, ϵ- and γ-globin genes and locus control region in KLF1−/− embryos, correlating with reduced gene expression. Therefore, KLF1 and KLF2 positively regulate the embryonic and fetal β-globin genes through direct promoter binding. KLF1 is required for normal histone modifications in the β-globin locus in mouse embryos.

A mechanism for Ikaros regulation of human globin gene switching

The human β globin locus consists of an upstream LCR and functional genes arranged sequentially in the order of their expression during development: 5′‐HBE1, HBG2, HBG1, HBD, HBB‐3′. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Here we show that the transcription factor Ikaros plays a major role in the formation of the β‐globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA‐binding region of Ikaros is disrupted by a point mutation, there is concomitant marked down‐regulation of HBB, and up‐regulation of HBG expression. We show for the first time Ikaros and its family member Eos, bind to critical cis elements implicated in haemoglobin switching and deletional hereditary persistence of fetal haemoglobin (HPFH). Chromatin conformation capture (3C) data demonstrated that Ikaros facilitates long‐distance DNA looping between the LCR and a region upstream of HBD. This study provides new insights into the mechanism of stage‐specific assembly of the β‐globin ACH, and HPFH.

Comparison of Gene Expression after Intraperitoneal Delivery of AAV2 or AAV5 in Utero

Correction of diseases may be achieved by delivery of genes to stem cells and developing organ systems. Our previous studies demonstrated life-long expression after in utero injection of adeno-associated virus (AAV) serotype 2 in mice. In the present studies, we compared levels of expression using the elongation factor 1alpha (EF1alpha) or the CMV promoter in AAV2 and AAV5 linked to luciferase via intraperitoneal injection in day 15 fetuses in utero. An additional AAV construct also contained the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). The level and distribution of luciferase expression were assessed by in vivo bioluminescence and luminometric assays. All mice exhibited luciferase expression for >15 months. In vivo, luciferase expression from AAV5 was greater than that produced from AAV2. Vectors containing the CMV promoter produced higher levels of gene expression in all tissues examined compared to EF1alpha-directed vectors. The WPRE increased expression in vitro fourfold and in vivo eightfold. These studies demonstrate that by modifying the promoter and serotype, increases in the efficiency of AAV-directed expression may be achieved. The efficacy of rAAV-mediated gene delivery in utero supports the potential of these vectors for future therapies.

Physical mapping of yeast artificial chromosomes containing sequences from the human β-globin gene region

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human beta-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult beta-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire beta-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the beta-globin gene cluster.

Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes

In utero injection of cationic liposome–DNA complexes (CLDCs) containing chloramphenicol acetyltransferase, β-galactosidase (β-gal), or human granulocyte colony-stimulating factor (hG-CSF) expression plasmids produced high-level gene expression in fetal rats. Tissues adjacent to the injection site exhibited the highest levels of gene expression. Chloramphenicol acetyltransferase expression persisted for at least 14 days and was reexpressed following postnatal reinjection of CLDCs. Intraperitoneal administration of the hG-CSF gene produced high serum hG-CSF levels. X-gal staining demonstrated widespread β-gal expression in multiple fetal tissues and cell types. No toxic or inflammatory responses were observed, nor was there evidence of fetal–maternal or maternal–fetal gene transfer, suggesting that CLDCs may provide a useful alternative to viral vectors for in utero gene transfer.

An Orthogonal Array Optimization of Lipid-like Nanoparticles for mRNA Delivery in Vivo.

Systemic delivery of mRNA-based therapeutics remains a challenging issue for preclinical and clinical studies. Here, we describe new lipid-like nanoparticles (TT-LLNs) developed through an orthogonal array design, which demonstrates improved delivery efficiency of mRNA encoding luciferase in vitro by over 350-fold with significantly reduced experimental workload. One optimized TT3 LLN, termed O-TT3 LLNs, was able to restore the human factor IX (hFIX) level to normal physiological values in FIX-knockout mice. Consequently, these mRNA based nanomaterials merit further development for therapeutic applications.

Changes in Pyruvate Metabolism Detected by Magnetic Resonance Imaging Are Linked to DNA Damage and Serve as a Sensor of Temozolomide Response in Glioblastoma Cells

Recent findings show that exposure to temozolomide (TMZ), a DNA-damaging drug used to treat glioblastoma (GBM), can suppress the conversion of pyruvate to lactate. To understand the mechanistic basis for this effect and its potential utility as a TMZ response biomarker, we compared the response of isogenic GBM cell populations differing only in expression of the DNA repair protein methyltransferase (MGMT), a TMZ-sensitivity determinant, after exposure to TMZ in vitro and in vivo. Hyperpolarized [1-((13))C]-pyruvate-based MRI was used to monitor temporal effects on pyruvate metabolism in parallel with DNA-damage responses and tumor cell growth. TMZ exposure decreased conversion of pyruvate to lactate only in MGMT-deficient cells. This effect coincided temporally with TMZ-induced increases in levels of the DNA-damage response protein pChk1. Changes in pyruvate to lactate conversion triggered by TMZ preceded tumor growth suppression and were not associated with changes in levels of NADH or lactate dehydrogenase activity in tumors. Instead, they were associated with a TMZ-induced decrease in the expression and activity of pyruvate kinase PKM2, a glycolytic enzyme that indirectly controls pyruvate metabolism. PKM2 silencing decreased PK activity, intracellular lactate levels, and conversion of pyruvate to lactate in the same manner as TMZ, and Chk1 silencing blocked the TMZ-induced decrease in PKM2 expression. Overall, our findings showed how TMZ-induced DNA damage is linked through PKM2 to changes in pyruvate metabolism, and how these changes can be exploited by MRI methods as an early sensor of TMZ therapeutic response.

Sequences in the Aγ–δ intergenic region are not required for stage-specific regulation of the human β-globin gene locus

The human β-globin locus has been extensively studied as a model of tissue and developmental stage-specific gene expression. Structural mapping of naturally occurring mutations, including transfection and transgenic studies, and the recent finding of intergenic transcripts have suggested that there are cis-acting sequence elements in the Aγ–δ intergenic region involved in regulating γ- and β-globin gene expression. To determine whether previously identified sequences in the Aγ–δ intergenic region are required for appropriate developmental expression of the human β-globin gene cluster, transgenic mice were generated by transfer of yeast artificial chromosomes containing the entire human β-globin locus. Three different deletions of the Aγ–δ intergenic region were introduced, including (i) deletion of the 750-bp Aγ 3′ regulatory element (Aγe), (ii) deletion of 3.2 kb upstream of the δ-globin gene encompassing pyrimidine-rich sequences and the recently described intergenic transcript initiation site, and (iii) deletion of a 12.5-kb fragment encompassing most of the Aγ–δ globin intergenic region. Analysis of multiple transgenic lines carrying these deletion constructs demonstrated that the normal stage-specific sequential expression of the ɛ-, γ-, and β-globin genes was preserved, despite deletion of these putative regulatory sequences. These studies suggest that regulatory sequences required for activation and silencing of the human β-globin gene family during ontogeny reside proximally to the genes and immediately 5′ to the human γ- and β-globin genes.

Impact of intensive PBSC mobilization therapy on outcomes following auto-SCT for non-Hodgkin's lymphoma

The best method to mobilize PBSCs in patients with non-Hodgkins Lymphoma (NHL) is uncertain. We hypothesized that PBSC mobilization using an intensive chemotherapy regimen would improve outcomes after autologous hematopoietic stem cell transplantation (ASCT) in NHL patients at high risk for relapse. Fifty NHL patients were prospectively allocated to intense mobilization with high-dose etoposide plus either high-dose cytarabine or CY if they were ‘high risk’ for relapse, whereas 30 patients were allocated to nonintense mobilization with CY if they were ‘standard risk’ (all patients, ±rituximab). All intensely mobilized patients were hospitalized compared with one-third of nonintensely mobilized patients. The EFS after ASCT was the same between the two groups, but overall survival (OS) was better for intensely mobilized patients (<0.01), including the diffuse large B-cell subgroup (P<0.04). We conclude that the intense mobilization of PBSCs in patients with NHL is more efficient than nonintense mobilization, but with greater toxicity. The equalization of EFS and superiority of OS in patients intensely mobilized to those nonintensely mobilized suggests that a treatment strategy using intensive chemotherapy for mobilization may be improving NHL outcomes after ASCT.