Paula M. Cannon

Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo

CCR5 is the major HIV-1 co-receptor, and individuals homozygous for a 32-bp deletion in CCR5 are resistant to infection by CCR5-tropic HIV-1. Using engineered zinc-finger nucleases (ZFNs), we disrupted CCR5 in human CD34+ hematopoietic stem/progenitor cells (HSPCs) at a mean frequency of 17% of the total alleles in a population. This procedure produces both mono- and bi-allelically disrupted cells. ZFN-treated HSPCs retained the ability to engraft NOD/SCID/IL2rγnull mice and gave rise to polyclonal multi-lineage progeny in which CCR5 was permanently disrupted. Control mice receiving untreated HSPCs and challenged with CCR5-tropic HIV-1 showed profound CD4+ T-cell loss. In contrast, mice transplanted with ZFN-modified HSPCs underwent rapid selection for CCR5−/− cells, had significantly lower HIV-1 levels and preserved human cells throughout their tissues. The demonstration that a minority of CCR5−/− HSPCs can populate an infected animal with HIV-1-resistant, CCR5−/− progeny supports the use of ZFN-modified autologous hematopoietic stem cells as a clinical approach to treating HIV-1.

Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders caused by mutations in the human type VII collagen gene (COL7A1). Individuals with DEB lack type VII collagen and anchoring fibrils, structures that attach epidermis and dermis. The current lack of treatment for DEB is an impetus to develop gene therapy strategies that efficiently transfer and stably express genes delivered to skin cells in vivo. In this study, we delivered and expressed full-length type VII collagen using a self-inactivating minimal lentivirus-based vector. Transduction of lentiviral vectors containing the COL7A1 transgene into recessive DEB (RDEB) keratinocytes and fibroblasts (in which type VII collagen was absent) resulted in persistent synthesis and secretion of type VII collagen. Unlike RDEB parent cells, the gene-corrected cells had normal morphology, proliferative potential, matrix attachment and motility. We used these gene-corrected cells to regenerate human skin on immune-deficient mice. Human skin regenerated by gene-corrected RDEB cells had restored expression of type VII collagen and formation of anchoring fibrils at the dermal–epidermal junction in vivo. These studies demonstrate that it is possible to restore type VII collagen gene expression in RDEB skin in vivo.

HIV-1 Vpu and HIV-2 Env counteract BST-2/tetherin by sequestration in a perinuclear compartment

BackgroundIn the absence of the Vpu protein, newly formed HIV-1 particles can remain attached to the surface of human cells due to the action of an interferon-inducible cellular restriction factor, BST-2/tetherin. Tetherin also restricts the release of other enveloped viral particles and is counteracted by a several viral anti-tetherin factors including the HIV-2 Env, SIV Nef and KSHV K5 proteins.ResultsWe observed that a fraction of tetherin is located at the surface of restricting cells, and that co-expression of both HIV-1 Vpu and HIV-2 Env reduced this population. In addition, Vpu, but not the HIV-2 Env, reduced total cellular levels of tetherin. An additional effect observed for both Vpu and the HIV-2 Env was to redirect tetherin to an intracellular perinuclear compartment that overlapped with markers for the TGN (trans-Golgi network). Sequestration of tetherin in this compartment was independent of tetherins normal endocytosis trafficking pathway.ConclusionsBoth HIV-1 Vpu and HIV-2 Env redirect tetherin away from the cell surface and sequester the protein in a perinuclear compartment, which likely blocks the action of this cellular restriction factor. Vpu also promotes the degradation of tetherin, suggesting that it uses more than one mechanism to counteract tetherin restriction.

Genomic Editing of the HIV-1 Coreceptor CCR5 in Adult Hematopoietic Stem and Progenitor Cells Using Zinc Finger Nucleases

The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5Δ32/Δ32) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5Δ32/Δ32 donors, we reasoned that engineered autologous CD34+ hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.

International AIDS Society global scientific strategy: towards an HIV cure 2016

Antiretroviral therapy is not curative. Given the challenges in providing lifelong therapy to a global population of more than 35 million people living with HIV, there is intense interest in developing a cure for HIV infection. The International AIDS Society convened a group of international experts to develop a scientific strategy for research towards an HIV cure. This Perspective summarizes the groups strategy.

Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors.

Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by adeno-associated virus (AAV) serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune-deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome-editing technologies in HSPCs.

Sequences in the cytoplasmic tail of the gibbon ape leukemia virus envelope protein that prevent its incorporation into lentivirus vectors.

ABSTRACT Pseudotyping retrovirus and lentivirus vectors with different viral fusion proteins is a useful strategy to alter the host range of the vectors. Although lentivirus vectors are efficiently pseudotyped by Env proteins from several different subtypes of murine leukemia virus (MuLV), the related protein from gibbon ape leukemia virus (GaLV) does not form functional pseudotypes. We have determined that this arises because of an inability of GaLV Env to be incorporated into lentivirus vector particles. By exploiting the homology between the GaLV and MuLV Env proteins, we have mapped the determinants of incompatibility in the GaLV Env. Three modifications that allowed GaLV Env to pseudotype human immunodeficiency virus type 1 particles were identified: removal of the R peptide (C-terminal half of the cytoplasmic domain), replacement of the whole cytoplasmic tail with the corresponding MuLV region, and mutation of two residues upstream of the R peptide cleavage site. In addition, we have previously proposed that removal of the R peptide from MuLV Env proteins enhances their fusogenicity by transmitting a conformational change to the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392–5398, 1998). Our analysis of chimeric MuLV/GaLV Env proteins provides further evidence in support of this model and suggests that proper Env function involves both interactions within the cytoplasmic tail and more long-range interactions between the cytoplasmic tail, the membrane-spanning region, and the ectodomain of the protein.

Ebola Virus Glycoprotein Counteracts BST-2/Tetherin Restriction in a Sequence-Independent Manner That Does Not Require Tetherin Surface Removal

ABSTRACT BST-2/tetherin is an interferon-inducible protein that restricts the release of enveloped viruses from the surface of infected cells by physically linking viral and cellular membranes. It is present at both the cell surface and in a perinuclear region, and viral anti-tetherin factors including HIV-1 Vpu and HIV-2 Env have been shown to decrease the cell surface population. To map the domains of human tetherin necessary for both virus restriction and sensitivity to viral anti-tetherin factors, we constructed a series of tetherin derivatives and assayed their activity. We found that the cytoplasmic tail (CT) and transmembrane (TM) domains of tetherin alone produced its characteristic cellular distribution, while the ectodomain of the protein, which includes a glycosylphosphatidylinositol (GPI) anchor, was sufficient to restrict virus release when presented by the CT/TM regions of a different type II membrane protein. To counteract tetherin restriction and remove it from the cell surface, HIV-1 Vpu required the specific sequence present in the TM domain of human tetherin. In contrast, the HIV-2 Env required only the ectodomain of the protein and was sensitive to a point mutation in this region. Strikingly, the anti-tetherin factor, Ebola virus GP, was able to overcome restriction conferred by both tetherin and a series of functional tetherin derivatives, including a wholly artificial tetherin molecule. Moreover, GP overcame restriction without significantly removing tetherin from the cell surface. These findings suggest that Ebola virus GP uses a novel mechanism to circumvent tetherin restriction.

New World Clade B Arenaviruses Can Use Transferrin Receptor 1 (TfR1)-Dependent and -Independent Entry Pathways, and Glycoproteins from Human Pathogenic Strains Are Associated with the Use of TfR1

ABSTRACT Arenaviruses are rodent-borne viruses, with five members of the family capable of causing severe hemorrhagic fevers if transmitted to humans. To date, two distinct cellular receptors have been identified that are used by different pathogenic viruses, α-dystroglycan by Lassa fever virus and transferrin receptor 1 (TfR1) by certain New World clade B viruses. Our previous studies have suggested that other, as-yet-unknown receptors are involved in arenavirus entry. In the present study, we examined the use of TfR1 by the glycoproteins (GPs) from a panel of New World clade B arenaviruses comprising three pathogenic and two nonpathogenic strains. Interestingly, we found that TfR1 was only used by the GPs from the pathogenic viruses, with entry of the nonpathogenic strains being TfR1 independent. The pathogenic GPs could also direct entry into cells by TfR1-independent pathways, albeit less efficiently. A comparison of the abilities of TfR1 orthologs from different species to support arenavirus entry found that the human and feline receptors were able to enhance entry of the pathogenic strains, but that neither the murine or canine forms were functional. Since the ability to use TfR1 is a characteristic feature of the human pathogens, this interaction may represent an important target in the treatment of New World hemorrhagic fevers. In addition, the ability to use TfR1 may be a useful tool to predict the likelihood that any existing or newly discovered viruses in this family could infect humans.

Chemokine receptor 5 knockout strategies.

Purpose of reviewIndividuals homozygous for a deletion in the chemokine receptor 5 (CCR5) gene (CCR5Δ32) are almost completely resistant to HIV-1 infection. A recent report that transplantation of hematopoietic stem or progenitor cells (HSCs) from a CCR5Δ32 homozygous donor effectively cured an HIV patient has increased interest in the development of strategies that could be used to recreate this phenotype using a patients own cells. This review will focus on recent developments to disrupt CCR5 expression in both autologous T cells and HSCs. Recent findingsCCR5 expression in HIV-1 target cells can be suppressed by RNA-based gene suppression technologies such as RNA interference, or completely eliminated by zinc finger nuclease (ZFN)-mediated gene disruption. ZFNs bind specifically to a DNA sequence and generate a double-stranded DNA break, whose subsequent repair by the cells error-prone nonhomologous end-joining pathway can lead to permanent disruption of the genes open reading frame. Recent developments in humanized mouse models have facilitated preclinical studies that have demonstrated the ability of CCR5-targeted ZFNs to suppress HIV-1 in vivo, when used to modify human T cells or HSCs. The same CCR5 ZFNs are now being evaluated in a phase I clinical trial of ex vivo expanded autologous T cells. SummaryCCR5 gene knockout in T cells or HSCs by ZFNs effectively suppresses the replication of CCR5-tropic strains of HIV-1 in animal models. ZFNs are currently being evaluated in a phase I clinical trials using ex vivo expanded T cells and HSCs targeted therapies are under development.