Brian R. Davis

Hematopoietic transcriptional regulation by the myeloid zinc finger gene, MZF-1.

Transcriptional regulators control much of hematopoiesis. One such transcriptional regulator is the myeloid zinc finger gene MZF-1. MZF-1 has been localized to the telomere of chromosome 19q, where a large number of related zinc finger genes reside. It has been found to be essential in granulopoiesis. It is a bi-functional transcriptional regulator, repressing transcription in non-hematopoietic cells, and activating transcription in cells of hematopoietic origins. Its consensus DNA binding site has been isolated, and sites in several promoters of myeloid-specific genes, such as CD34, lactoferrin, and myeloperoxidase, have been defined. In co-transfection experiments MZF-1 has been found to regulate transcription from the CD34 promoter.

Targeted Correction and Restored Function of the CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells

Summary Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

Application of SFHR to gene therapy of monogenic disorders

Gene therapy treatment of disease will be greatly facilitated by the identification of genetic mutations through the Human Genome Project. The specific treatment will ultimately depend on the type of mutation as different genetic lesions will require different gene therapies. For example, large rearrangements and translocations may call for complementation with vectors containing the cDNA for the wild-type (wt) gene. On the other hand, smaller lesions, such as the reversion, addition or deletion of only a few base pairs, on single genes, or monogenic disorders, lend themselves to gene targeting. The potential for one gene targeting technique, small fragment homologous replacement (SFHR) to the gene therapy treatment of sickle cell disease (SCD) is presented. Successful conversion of the wt-β-globin locus to a SCD genotype of human lymphocytes (K562) and progenitor/stem hematopoietic cells (CD34+ and lin-CD38−) was achieved by electroporation or microinjection small DNA fragments (SDF).

Liposomes modulate human immunodeficiency virus infectivity

We have investigated the effects of the fusion of liposomes with human immunodeficiency virus type 1 (HIV-1LVA) on the ability of the virus to infect CD4+ and CD4- cells. Fluorescence dequenching measurements indicated that HIV-1 fuses with liposomes composed of either cardiolipin (CL) or N-[2,3-(dioleyloxy) propyl]-N,N,N-trimethyl ammonium chloride (DOTMA) but not appreciably with dioleoylphosphatidylcholine (DOPC) liposomes. Pre-incubation of HIV-1 with DOTMA liposomes enhanced virus production (measured by p24 gag antigen production in the culture medium and in situ) in CD4+ A3.01 and H9 cells in a concentration-dependent manner, but did not mediate the infection of the CD4- cell line, K562. Preincubation of HIV-1 with between 10 and 30 microM-DOTMA liposomes, and subsequent incubation with A3.01 cells, resulted in the production of about 30-fold greater levels of virus than controls. The presence of DOTMA liposomes during the incubation of A3.01 cells with HIV-1 enhanced the infectivity of the virus up to 90-fold compared to controls. Conversely, preincubation of HIV-1 with CL liposomes inhibited infection of A3.01 cells, dependent on the concentration of liposomes; DOPC liposomes did not alter the infectivity of the virus under any of the incubation conditions. Our results thus indicate that fusion of HIV-1 with liposomes alters the ability of the virus to infect its target cells.

5 Effect of human immunodeficiency virus infection on haematopoiesis

The pathogenesis of peripheral blood cytopenias in AIDS patients is clearly multifactorial. Among the various contributing mechanisms, those involving a direct role of HIV-1 have been actively investigated in the past few years. It has now been convincingly demonstrated that HIV can impair the survival/proliferative capacity of purified haematopoietic progenitor cells. Although a subset of haematopoietic progenitor cells are perhaps susceptible to HIV-1 infection, both in vitro and in vivo, the suppressive effect does not require either active or latent infection and is probably mediated by the interaction of viral or virus-associated proteins with the cell membrane of haematopoietic progenitor cells. Both the viral load and the biological characteristics of the virus play an important role in suppression, since different isolates displayed different inhibitory activity. Haematosuppression is not a specific property of monocytotropic versus lymphocytotropic or low-replicating versus high-replicating isolates, and it will be important to exactly establish which viral component is crucial to suppression of haematopoietic progenitor cells. Since the haematopoietic stem cell is the common progenitor to both the myeloid and lymphoid lineages, the capacity of HIV to impair the growth of early haematopoietic progenitor cells could contribute not only to the frequent occurrence of anaemia, granulocytopenia and thrombocytopenia in AIDS patients, but also to the inability of the bone marrow to reconstitute a functional pool of mature CD4+ T-cells. It is also possible that haematopoietic progenitor cells committed to the T-lymphoid lineage are impaired by HIV in their differential pathway within the thymus (Bonyhadi et al, 1993). Infection of megakaryocytes could result in underproduction of platelets and possibly represents a major pathogenetic mechanism of HIV-related thrombocytopenia. Infection of monocytes and T-lymphocytes leads in vitro and probably also in vivo to deranged cytokine production. In the first stages of the disease, increased cytokine production, consequent to a chronic immune activation, is probably responsible for the myelodysplastic/hyperplastic alterations observed at the bone marrow level. In more advanced stages of the disease, the general decline in immune function, the consequent imbalance in cytokine production, and the increase in viral burden, may contribute to dysregulated haematopoiesis and peripheral blood cytopenias.

Enhancement of human immunodeficiency virus type 1 infection by cationic liposomes: the role of CD4, serum and liposome-cell interactions.

We have reported previously the enhancement of the infectivity of human immunodeficiency virus type 1 (HIV-1) by liposomes composed of the cationic lipid N-[2,3-(dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA). To determine the mechanism by which this process occurs, we have investigated the role of CD4, serum concentration and liposome-cell interactions in the DOTMA-mediated stimulation of HIV-1 infection of A3.01 cells. Serum alone significantly inhibited the binding and infectivity of HIV-1, but DOTMA-mediated enhancement of infectivity was more pronounced in the presence of serum than in its absence. HIV-1 binding to cells was increased in the presence of DOTMA liposomes, DEAE-dextran and polybrene, all of which also enhanced infectivity to a similar extent at comparable concentrations. Fluorescence dequenching measurements indicated that DOTMA liposomes fused with HIV-1, but not with cell membranes, in the presence of serum. The enhancing effect of DOTMA liposomes on HIV-1 infectivity was CD4-dependent, and appeared to involve virus-liposome fusion and liposome binding to the cell surface. DOTMA liposomes did not mediate infection of the CD4-K562 and Raji cell lines.

Glass Needle-Mediated Microinjection of Macromolecules and Transgenes into Primary Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into various tissue types, including bone, cartilage, tendon, adipocytes, and marrow stroma, making them potentially useful for human cell and gene therapies. Our objective was to demonstrate the utility of glass needle-mediated microinjection as a method to deliver macromolecules (e.g. dextrans, DNA) to hMSCs for cell and molecular biological studies. hMSCs were isolated and cultured using a specific fetal bovine serum, prescreened for its ability to promote cell adherence, proliferation, and osteogenic differentiation. Successful delivery of Oregon Green-dextran via intranuclear microinjection was achieved, yielding a postinjection viability of 76 ± 13%. Excellent short-term gene expression (63 ± 11%) was achieved following microinjection of GFP-containing vectors into hMSCs. Higher efficiencies of short-term gene expression (∼5-fold) were observed when injecting supercoiled DNA, pYA721, as compared with the same DNA construct in a linearized form, YA721. Approximately 0.05% of hMSCs injected with pYA721 containing both the GFP and neomycin resistance genes formed GFP-positive, drug-resistant colonies that survived >120 days. Injection of linearized YA721 resulted in 3.6% of injected hMSC forming drug-resistant colonies, none of which expressed GFP that survived 60–120 days. These studies demonstrate that glass needle-mediated microinjection can be used as a method of delivering macromolecules to hMSCs and may prove to be a useful technique for molecular and cell biological mechanistic studies and future genetic modification of hMSCs.

New frontier in regenerative medicine: site-specific gene correction in patient-specific induced pluripotent stem cells.

Advances in cell and gene therapy are opening up new avenues for regenerative medicine. Because of their acquired pluripotency, human induced pluripotent stem cells (hiPSCs) are a promising source of autologous cells for regenerative medicine. They show unlimited self-renewal while retaining the ability, in principle, to differentiate into any cell type of the human body. Since Yamanaka and colleagues first reported the generation of hiPSCs in 2007, significant efforts have been made to understand the reprogramming process and to generate hiPSCs with potential for clinical use. On the other hand, the development of gene-editing platforms to increase homologous recombination efficiency, namely DNA nucleases (zinc finger nucleases, TAL effector nucleases, and meganucleases), is making the application of locus-specific gene therapy in human cells an achievable goal. The generation of patient-specific hiPSC, together with gene correction by homologous recombination, will potentially allow for their clinical application in the near future. In fact, reports have shown targeted gene correction through DNA-Nucleases in patient-specific hiPSCs. Various technologies have been described to reprogram patient cells and to correct these patient hiPSCs. However, no approach has been clearly more efficient and safer than the others. In addition, there are still significant challenges for the clinical application of these technologies, such as inefficient differentiation protocols, genetic instability resulting from the reprogramming process and hiPSC culture itself, the efficacy and specificity of the engineered DNA nucleases, and the overall homologous recombination efficiency. To summarize advances in the generation of gene corrected patient-specific hiPSCs, this review focuses on the available technological platforms, including their strengths and limitations regarding future therapeutic use of gene-corrected hiPSCs.

Somatic mosaicism in the Wiskott–Aldrich syndrome: Molecular and functional characterization of genotypic revertants

The reasons underlying the occurrence of multiple revertant genotypes in Wiskott-Aldrich syndrome (WAS) patients remain unclear. We have identified more than 30 revertant genotypes in a C995T WAS patient having 10-15% revertant, WAS protein (WASp)-expressing circulating lymphocytes. Of 497 allospecific T-cell clones generated from the peripheral blood, 47.1% carried a revertant sequence. All revertant T-cell clones exhibited restoration of WASp expression. However, anti-CD3-induced proliferative responses varied greatly amongst revertants. Several revertant T-cell clones expressed an internally deleted WASp mutant lacking much of the proline-rich region. This potentially accounts for the reduced anti-CD3 proliferative responses of these T-cell clones. We found no evidence for an increased DNA mutation rate in this patient. We conclude that the diversity of revertant genotypes in our patient does not result from an extraordinary mutation rate and that the amino acid sequence space explored by WASp in revertant T-cells is significantly smaller than might have been predicted from the diversity of revertant genotypes.

Unprecedented diversity of genotypic revertants in lymphocytes of a patient with Wiskott-Aldrich syndrome

Spontaneous somatic reversions of inherited mutations are poorly understood phenomena that are thought to occur uncommonly in a variety of genetic disorders. When molecularly characterized, revertant cells have rarely exhibited more than one revertant genotype per patient. We analyzed individual allospecific T-cell clones derived from a Wiskott-Aldrich syndrome (WAS) patient identified by flow cytometry to have 10% to 15% revertant, WAS protein-expressing lymphocytes in his blood. Genotypic analysis of the clones revealed a remarkable diversity of deletions and base substitutions resulting in at least 34 different revertant genotypes that restored expression of WASp. A large fraction of these revertant genotypes were also identified in primary T cells purified from peripheral blood. These data suggest that the use of sensitive methods may reveal the presence of wide arrays of individual genotypic revertants in WAS patients and offer opportunities for further understanding of their occurrence.