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1.
N Engl J Med ; 371(15): 1407-17, 2014 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-25295500

RESUMO

BACKGROUND: In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus-based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancer-mediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS: We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS: All patients received bone marrow-derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS: This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.).


Assuntos
Gammaretrovirus/genética , Terapia Genética , Vetores Genéticos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Animais , Antígenos CD34 , DNA Complementar/uso terapêutico , Expressão Gênica , Inativação Gênica , Terapia Genética/efeitos adversos , Humanos , Lactente , Subunidade gama Comum de Receptores de Interleucina/genética , Masculino , Camundongos , Mutação , Linfócitos T/imunologia , Transdução Genética , Transgenes/fisiologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia
2.
Nature ; 475(7355): 217-21, 2011 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-21706032

RESUMO

Editing of the human genome to correct disease-causing mutations is a promising approach for the treatment of genetic disorders. Genome editing improves on simple gene-replacement strategies by effecting in situ correction of a mutant gene, thus restoring normal gene function under the control of endogenous regulatory elements and reducing risks associated with random insertion into the genome. Gene-specific targeting has historically been limited to mouse embryonic stem cells. The development of zinc finger nucleases (ZFNs) has permitted efficient genome editing in transformed and primary cells that were previously thought to be intractable to such genetic manipulation. In vitro, ZFNs have been shown to promote efficient genome editing via homology-directed repair by inducing a site-specific double-strand break (DSB) at a target locus, but it is unclear whether ZFNs can induce DSBs and stimulate genome editing at a clinically meaningful level in vivo. Here we show that ZFNs are able to induce DSBs efficiently when delivered directly to mouse liver and that, when co-delivered with an appropriately designed gene-targeting vector, they can stimulate gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to correct the prolonged clotting times in a mouse model of haemophilia B, and remained persistent after induced liver regeneration. Thus, ZFN-driven gene correction can be achieved in vivo, raising the possibility of genome editing as a viable strategy for the treatment of genetic disease.


Assuntos
Reparo do DNA/genética , Modelos Animais de Doenças , Marcação de Genes/métodos , Terapia Genética/métodos , Genoma/genética , Hemofilia B/genética , Hemostasia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , Éxons/genética , Fator IX/análise , Fator IX/genética , Vetores Genéticos/genética , Células HEK293 , Hemofilia B/fisiopatologia , Humanos , Íntrons/genética , Fígado/metabolismo , Regeneração Hepática , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Fenótipo , Homologia de Sequência , Dedos de Zinco
3.
Nucleic Acids Res ; 43(11): 5647-63, 2015 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-25969444

RESUMO

Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD ß1-ß2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD ß1-ß2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD ß1-ß2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection.


Assuntos
Integrases/química , Vírus da Leucemia Murina de Moloney/enzimologia , Proteínas Virais/química , Domínio Catalítico , DNA/química , DNA/metabolismo , Integrases/genética , Integrases/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Proteínas Virais/metabolismo
4.
Genes Dev ; 23(5): 633-42, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19270161

RESUMO

At least 8% of the human genome was formed by integration of retroviral DNA sequences. Here we analyze the forces directing the accumulation of human endogenous retroviruses (HERVs) by comparing de novo HERV integration targeting with the distribution of fixed HERV elements in the human genome. All known genomic HERVs are inactive due to mutation, but we were able to study integration targeting using a reconstituted consensus HERV-K (designated HERV-K(Con)). We found that HERV-K(Con) integrated preferentially in transcription units, in gene-rich regions, and near features associated with active transcription units and associated regulatory regions. In contrast, genomic HERV-K proviruses are found preferentially outside transcription units. The minority of genomic HERVKs present inside transcription units are in opposite transcriptional orientation relative to the host gene, the orientation predicted to be minimally disruptive to host mRNA synthesis, but de novo HERV-K(Con) integration within transcription units showed no orientation bias. We also found that the youngest HERV-K elements in the human genome showed a distribution intermediate between de novo HERV-K(Con) integration sites and older fixed HERV-Ks. These findings indicate that accumulation of HERVs in the human germline is a two-step process: integration targeting biases direct initial accumulation, then purifying selection leads to loss of proviruses disrupting gene function.


Assuntos
Retrovirus Endógenos/metabolismo , Genoma Humano/genética , Integração Viral/fisiologia , Células/virologia , Cromatina/metabolismo , Mapeamento Cromossômico , Retrovirus Endógenos/genética , Histonas/metabolismo , Humanos , Metilação , Integração Viral/genética
5.
PLoS Pathog ; 10(2): e1003911, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24516383

RESUMO

The Bunyaviridae comprise a large family of RNA viruses with worldwide distribution and includes the pathogenic New World hantavirus, Andes virus (ANDV). Host factors needed for hantavirus entry remain largely enigmatic and therapeutics are unavailable. To identify cellular requirements for ANDV infection, we performed two parallel genetic screens. Analysis of a large library of insertionally mutagenized human haploid cells and a siRNA genomic screen converged on components (SREBP-2, SCAP, S1P and S2P) of the sterol regulatory pathway as critically important for infection by ANDV. The significance of this pathway was confirmed using functionally deficient cells, TALEN-mediated gene disruption, RNA interference and pharmacologic inhibition. Disruption of sterol regulatory complex function impaired ANDV internalization without affecting virus binding. Pharmacologic manipulation of cholesterol levels demonstrated that ANDV entry is sensitive to changes in cellular cholesterol and raises the possibility that clinically approved regulators of sterol synthesis may prove useful for combating ANDV infection.


Assuntos
Colesterol/metabolismo , Infecções por Hantavirus/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Orthohantavírus/patogenicidade , Internalização do Vírus , Linhagem Celular , Citometria de Fluxo , Humanos , Microscopia Confocal , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Transdução de Sinais/fisiologia , Esteróis/metabolismo , Transdução Genética , Replicação Viral/fisiologia
6.
PLoS Pathog ; 10(5): e1004171, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24874515

RESUMO

The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies in vitro. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC50 of 0.024 µM) blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology.


Assuntos
Antivirais/farmacologia , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Multimerização Proteica/efeitos dos fármacos , Integração Viral/efeitos dos fármacos , Linhagem Celular , HIV-1/efeitos dos fármacos , Humanos , Ligação Proteica/efeitos dos fármacos , Quinolinas/farmacologia , Replicação Viral/efeitos dos fármacos
7.
Nucleic Acids Res ; 42(9): 5917-28, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24623816

RESUMO

We report alterations to the murine leukemia virus (MLV) integrase (IN) protein that successfully result in decreasing its integration frequency at transcription start sites and CpG islands, thereby reducing the potential for insertional activation. The host bromo and extraterminal (BET) proteins Brd2, 3 and 4 interact with the MLV IN protein primarily through the BET protein ET domain. Using solution NMR, protein interaction studies, and next generation sequencing, we show that the C-terminal tail peptide region of MLV IN is important for the interaction with BET proteins and that disruption of this interaction through truncation mutations affects the global targeting profile of MLV vectors. The use of the unstructured tails of gammaretroviral INs to direct association with complexes at active promoters parallels that used by histones and RNA polymerase II. Viruses bearing MLV IN C-terminal truncations can provide new avenues to improve the safety profile of gammaretroviral vectors for human gene therapy.


Assuntos
Integrases/química , Vírus da Leucemia Murina/genética , Proteínas de Ligação a RNA/química , Proteínas Virais/química , Integração Viral , Sequência de Aminoácidos , Sítios de Ligação , Ilhas de CpG , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNA , Deleção de Sequência , Fatores de Transcrição , Sítio de Iniciação de Transcrição
8.
Nucleic Acids Res ; 42(8): 4868-81, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24520112

RESUMO

The importance of understanding the molecular mechanisms of murine leukemia virus (MLV) integration into host chromatin is highlighted by the development of MLV-based vectors for human gene-therapy. We have recently identified BET proteins (Brd2, 3 and 4) as the main cellular binding partners of MLV integrase (IN) and demonstrated their significance for effective MLV integration at transcription start sites. Here we show that recombinant Brd4, a representative of the three BET proteins, establishes complementary high-affinity interactions with MLV IN and mononucleosomes (MNs). Brd4(1-720) but not its N- or C-terminal fragments effectively stimulate MLV IN strand transfer activities in vitro. Mass spectrometry- and NMR-based approaches have enabled us to map key interacting interfaces between the C-terminal domain of BRD4 and the C-terminal tail of MLV IN. Additionally, the N-terminal fragment of Brd4 binds to both DNA and acetylated histone peptides, allowing it to bind tightly to MNs. Comparative analyses of the distributions of various histone marks along chromatin revealed significant positive correlations between H3- and H4-acetylated histones, BET protein-binding sites and MLV-integration sites. Our findings reveal a bimodal mechanism for BET protein-mediated MLV integration into select chromatin locations.


Assuntos
Integrases/metabolismo , Vírus da Leucemia Murina/enzimologia , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ciclo Celular , DNA/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Integrases/química , Vírus da Leucemia Murina/fisiologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Integração Viral
9.
Proc Natl Acad Sci U S A ; 110(23): 9487-92, 2013 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-23661057

RESUMO

The p12 protein of murine leukemia virus (MuLV) group-specific antigen (Gag) is associated with the preintegration complex, and mutants of p12 (PM14) show defects in nuclear entry or retention. Here we show that p12 proteins engineered to encode peptide sequences derived from known viral tethering proteins can direct chromatin binding during the early phase of viral replication and rescue a lethal p12-PM14 mutant. Peptides studied included segments of Kaposi sarcoma herpesvirus latency-associated nuclear antigen (LANA)(1-23), human papillomavirus 8 E2, and prototype foamy virus chromatin-binding sequences. Amino acid substitutions in Kaposi sarcoma herpesvirus LANA and prototype foamy virus chromatin-binding sequences that blocked nucleosome association failed to rescue MuLV p12-PM14. Rescue by a larger LANA peptide, LANA(1-32), required second-site mutations that are predicted to reduce peptide binding affinity to chromosomes, suggesting that excessively high binding affinity interfered with Gag/p12 function. This is supported by confocal microscopy of chimeric p12-GFP fusion constructs showing the reverted proteins had weaker association to condensed mitotic chromosomes. Analysis of the integration-site selection of these chimeric viruses showed no significant change in integration profile compared with wild-type MuLV, suggesting release of the tethered p12 post mitosis, before viral integration.


Assuntos
DNA Viral/genética , Produtos do Gene gag/genética , Vírus da Leucemia Murina/genética , Integração Viral/genética , Replicação Viral/genética , Animais , Antígenos Virais/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular , Cromatina/metabolismo , Clonagem Molecular , Cães , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Microscopia Confocal , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Engenharia de Proteínas , Análise de Sequência de DNA , Transativadores/metabolismo
10.
Proc Natl Acad Sci U S A ; 110(6): E478-87, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23091042

RESUMO

Chromosome structure and function are influenced by transposable elements, which are mobile DNA segments that can move from place to place. hAT elements are a superfamily of DNA cut and paste elements that move by excision and integration. We have characterized two hAT elements, TcBuster and Space Invaders (SPIN), that are members of a recently described subfamily of hAT elements called Buster elements. We show that TcBuster, from the red flour beetle Tribolium castaneum, is highly active in human cells. SPIN elements are currently inactive elements that were recently highly active in multiple vertebrate genomes, and the high level of sequence similarity across widely diverged species and patchy phylogenetic distribution suggest that they may have moved between genomes by horizontal transfer. We have generated an intact version of this element, SPIN(ON), which is highly active in human cells. In vitro analysis of TcBuster and SPIN(ON) shows that no proteins other than transposase are essential for recombination, a property that may contribute to the ability of SPIN to successfully invade multiple organisms. We also analyze the target site preferences of de novo insertions in the human genome of TcBuster and SPIN(ON) and compare them with the preferences of Sleeping Beauty and piggyBac, showing that each superfamily has a distinctive pattern of insertion. The high-frequency transposition of both TcBuster and SPIN(ON) suggests that these transposon systems offer powerful tools for genome engineering. Finally, we describe a Saccharomyces cerevisiae assay for TcBuster that will provide a means for isolation of hyperactive and other interesting classes of transposase mutants.


Assuntos
Elementos de DNA Transponíveis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Transferência Genética Horizontal , Genes de Insetos , Engenharia Genética , Células HeLa , Humanos , Dados de Sequência Molecular , Filogenia , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Transposases/metabolismo , Tribolium/genética
11.
Proc Natl Acad Sci U S A ; 110(29): 12036-41, 2013 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-23818621

RESUMO

The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy.


Assuntos
Integrases/metabolismo , Vírus da Leucemia Murina/enzimologia , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição/fisiologia , Integração Viral/fisiologia , Animais , Azepinas , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Humanos , Espectrometria de Massas , Camundongos , Células NIH 3T3 , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteômica/métodos , Interferência de RNA , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Triazóis , Integração Viral/genética
12.
Proc Natl Acad Sci U S A ; 110(25): E2279-87, 2013 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-23723351

RESUMO

The transposon piggyBac is being used increasingly for genetic studies. Here, we describe modified versions of piggyBac transposase that have potentially wide-ranging applications, such as reversible transgenesis and modified targeting of insertions. piggyBac is distinguished by its ability to excise precisely, restoring the donor site to its pretransposon state. This characteristic makes piggyBac useful for reversible transgenesis, a potentially valuable feature when generating induced pluripotent stem cells without permanent alterations to genomic sequence. To avoid further genome modification following piggyBac excision by reintegration, we generated an excision competent/integration defective (Exc(+)Int(-)) transposase. Our findings also suggest the position of a target DNA-transposase interaction. Another goal of genome engineering is to develop reagents that can guide transgenes to preferred genomic regions. Others have shown that piggyBac transposase can be active when fused to a heterologous DNA-binding domain. An Exc(+)Int(-) transposase, the intrinsic targeting of which is defective, might also be a useful intermediate in generating a transposase whose integration activity could be rescued and redirected by fusion to a site-specific DNA-binding domain. We show that fusion to two designed zinc finger proteins rescued the Int(-) phenotype. Successful guided transgene integration into genomic DNA would have broad applications to gene therapy and molecular genetics. Thus, an Exc(+)Int(-) transposase is a potentially useful reagent for genome engineering and provides insight into the mechanism of transposase-target DNA interaction.


Assuntos
Elementos de DNA Transponíveis/genética , Engenharia Genética/métodos , Proteínas do Tecido Nervoso/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Técnicas de Transferência de Genes , Genoma Humano/genética , Células HEK293 , Células HeLa , Humanos , Mamíferos , Dados de Sequência Molecular , Mutagênese Insercional/métodos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Dedos de Zinco/genética
13.
J Biol Chem ; 289(30): 20477-88, 2014 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-24904063

RESUMO

HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75-IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75-IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Integrase de HIV/metabolismo , HIV-1/fisiologia , Multimerização Proteica , Fatores de Transcrição/metabolismo , Replicação Viral/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Linhagem Celular , Cristalografia por Raios X , Integrase de HIV/química , Integrase de HIV/genética , Inibidores de Integrase de HIV/farmacologia , Humanos , Estrutura Terciária de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , Replicação Viral/fisiologia
14.
Bioinformatics ; 30(11): 1493-500, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24489369

RESUMO

MOTIVATION: Gene therapy with retroviral vectors can induce adverse effects when those vectors integrate in sensitive genomic regions. Retroviral vectors are preferred that target sensitive regions less frequently, motivating the search for localized clusters of integration sites and comparison of the clusters formed by integration of different vectors. Scan statistics allow the discovery of spatial differences in clustering and calculation of false discovery rates providing statistical methods for comparing retroviral vectors. RESULTS: A scan statistic for comparing two vectors using multiple window widths is proposed with software to detect clustering differentials and compute false discovery rates. Application to several sets of experimentally determined HIV integration sites demonstrates the software. Simulated datasets of various sizes and signal strengths are used to determine the power to discover clusters and evaluate a convenient lower bound. This provides a toolkit for planning evaluations of new gene therapy vectors. AVAILABILITY AND IMPLEMENTATION: The geneRxCluster R package containing a simple tutorial and usage hints is available from http://www.bioconductor.org.


Assuntos
Vetores Genéticos , HIV/genética , Integração Viral , Algoritmos , Inteligência Artificial , DNA/química , Interpretação Estatística de Dados , Genômica , Humanos , Células Jurkat , Software
15.
JAMA ; 313(15): 1550-63, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25898053

RESUMO

IMPORTANCE: Wiskott-Aldrich syndrome is a rare primary immunodeficiency associated with severe microthrombocytopenia. Partially HLA antigen-matched allogeneic hematopoietic stem cell (HSC) transplantation is often curative but is associated with significant comorbidity. OBJECTIVE: To assess the outcomes and safety of autologous HSC gene therapy in Wiskott-Aldrich syndrome. DESIGN, SETTING, AND PARTICIPANTS: Gene-corrected autologous HSCs were infused in 7 consecutive patients with severe Wiskott-Aldrich syndrome lacking HLA antigen-matched related or unrelated HSC donors (age range, 0.8-15.5 years; mean, 7 years) following myeloablative conditioning. Patients were enrolled in France and England and treated between December 2010 and January 2014. Follow-up of patients in this intermediate analysis ranged from 9 to 42 months. INTERVENTION: A single infusion of gene-modified CD34+ cells with an advanced lentiviral vector. MAIN OUTCOMES AND MEASURES: Primary outcomes were improvement at 24 months in eczema, frequency and severity of infections, bleeding tendency, and autoimmunity and reduction in disease-related days of hospitalization. Secondary outcomes were improvement in immunological and hematological characteristics and evidence of safety through vector integration analysis. RESULTS: Six of the 7 patients were alive at the time of last follow-up (mean and median follow-up, 28 months and 27 months, respectively) and showed sustained clinical benefit. One patient died 7 months after treatment of preexisting drug-resistant herpes virus infection. Eczema and susceptibility to infections resolved in all 6 patients. Autoimmunity improved in 5 of 5 patients. No severe bleeding episodes were recorded after treatment, and at last follow-up, all 6 surviving patients were free of blood product support and thrombopoietic agonists. Hospitalization days were reduced from a median of 25 days during the 2 years before treatment to a median of 0 days during the 2 years after treatment. All 6 surviving patients exhibited high-level, stable engraftment of functionally corrected lymphoid cells. The degree of myeloid cell engraftment and of platelet reconstitution correlated with the dose of gene-corrected cells administered. No evidence of vector-related toxicity was observed clinically or by molecular analysis. CONCLUSIONS AND RELEVANCE: This study demonstrated the feasibility of the use of gene therapy in patients with Wiskott-Aldrich syndrome. Controlled trials with larger numbers of patients are necessary to assess long-term outcomes and safety.


Assuntos
Terapia Genética , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas , Lentivirus , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Adolescente , Criança , Pré-Escolar , Estudos de Viabilidade , Expressão Gênica , Terapia Genética/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Lactente , Recém-Nascido , Masculino , Índice de Gravidade de Doença , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia
17.
Retrovirology ; 10: 90, 2013 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-23953889

RESUMO

BACKGROUND: HIV infection can be treated effectively with antiretroviral agents, but the persistence of a latent reservoir of integrated proviruses prevents eradication of HIV from infected individuals. The chromosomal environment of integrated proviruses has been proposed to influence HIV latency, but the determinants of transcriptional repression have not been fully clarified, and it is unclear whether the same molecular mechanisms drive latency in different cell culture models. RESULTS: Here we compare data from five different in vitro models of latency based on primary human T cells or a T cell line. Cells were infected in vitro and separated into fractions containing proviruses that were either expressed or silent/inducible, and integration site populations sequenced from each. We compared the locations of 6,252 expressed proviruses to those of 6,184 silent/inducible proviruses with respect to 140 forms of genomic annotation, many analyzed over chromosomal intervals of multiple lengths. A regularized logistic regression model linking proviral expression status to genomic features revealed no predictors of latency that performed better than chance, though several genomic features were significantly associated with proviral expression in individual models. Proviruses in the same chromosomal region did tend to share the same expressed or silent/inducible status if they were from the same cell culture model, but not if they were from different models. CONCLUSIONS: The silent/inducible phenotype appears to be associated with chromosomal position, but the molecular basis is not fully clarified and may differ among in vitro models of latency.


Assuntos
Linfócitos T CD4-Positivos/virologia , HIV/fisiologia , Integração Viral , Latência Viral , Células Cultivadas , HIV/genética , Humanos , Provírus/genética , Provírus/fisiologia
18.
PLoS Pathog ; 7(3): e1001313, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21423673

RESUMO

Genome-wide siRNA screens have identified host cell factors important for efficient HIV infection, among which are nuclear pore proteins such as RanBP2/Nup358 and the karyopherin Transportin-3/TNPO3. Analysis of the roles of these proteins in the HIV replication cycle suggested that correct trafficking through the pore may facilitate the subsequent integration step. Here we present data for coupling between these steps by demonstrating that depletion of Transportin-3 or RanBP2 altered the terminal step in early HIV replication, the selection of chromosomal sites for integration. We found that depletion of Transportin-3 and RanBP2 altered integration targeting for HIV. These knockdowns reduced HIV integration frequency in gene-dense regions and near gene-associated features, a pattern that differed from that reported for depletion of the HIV integrase binding cofactor Psip1/Ledgf/p75. MLV integration was not affected by the Transportin-3 knockdown. Using siRNA knockdowns and integration targeting analysis, we also implicated several additional nuclear proteins in proper target site selection. To map viral determinants of integration targeting, we analyzed a chimeric HIV derivative containing MLV gag, and found that the gag replacement phenocopied the Transportin-3 and RanBP2 knockdowns. Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration.


Assuntos
HIV/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , beta Carioferinas/metabolismo , Regulação Viral da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , RNA Interferente Pequeno/genética , Replicação Viral , beta Carioferinas/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
19.
Blood ; 117(12): 3311-9, 2011 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-21106988

RESUMO

Gene transfer using adeno-associated virus (AAV) vectors has great potential for treating human disease. Recently, questions have arisen about the safety of AAV vectors, specifically, whether integration of vector DNA in transduced cell genomes promotes tumor formation. This study addresses these questions with high-dose liver-directed AAV-mediated gene transfer in the adult mouse as a model (80 AAV-injected mice and 52 controls). After 18 months of follow-up, AAV-injected mice did not show a significantly higher rate of hepatocellular carcinoma compared with controls. Tumors in mice treated with AAV vectors did not have significantly different amounts of vector DNA compared with adjacent normal tissue. A novel high-throughput method for identifying AAV vector integration sites was developed and used to clone 1029 integrants. Integration patterns in tumor tissue and adjacent normal tissue were similar to each other, showing preferences for active genes, cytosine-phosphate-guanosine islands, and guanosine/cytosine-rich regions. [corrected] Gene expression data showed that genes near integration sites did not show significant changes in expression patterns compared with genes more distal to integration sites. No integration events were identified as causing increased oncogene expression. Thus, we did not find evidence that AAV vectors cause insertional activation of oncogenes and subsequent tumor formation.


Assuntos
Dependovirus/genética , Terapia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Dependovirus/fisiologia , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/fisiologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional/fisiologia , Testes de Mutagenicidade
20.
Blood ; 117(11): 3113-22, 2011 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-21228324

RESUMO

Human T-lymphotropic virus type 1 (HTLV-1) persists by driving clonal proliferation of infected T lymphocytes. A high proviral load predisposes to HTLV-1-associated diseases. Yet the reasons for the variation within and between persons in the abundance of HTLV-1-infected clones remain unknown. We devised a high-throughput protocol to map the genomic location and quantify the abundance of > 91,000 unique insertion sites of the provirus from 61 HTLV-1(+) persons and > 2100 sites from in vitro infection. We show that a typical HTLV-1-infected host carries between 500 and 5000 unique insertion sites. We demonstrate that negative selection dominates during chronic infection, favoring establishment of proviruses integrated in transcriptionally silenced DNA: this selection is significantly stronger in asymptomatic carriers. We define a parameter, the oligoclonality index, to quantify clonality. The high proviral load characteristic of HTLV-1-associated inflammatory disease results from a larger number of unique insertion sites than in asymptomatic carriers and not, as previously thought, from a difference in clonality. The abundance of established HTLV-1 clones is determined by genomic features of the host DNA flanking the provirus. HTLV-1 clonal expansion in vivo is favored by orientation of the provirus in the same sense as the nearest host gene.


Assuntos
Genoma Humano/genética , Infecções por HTLV-I/genética , Infecções por HTLV-I/virologia , Interações Hospedeiro-Patógeno/genética , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Provírus/genética , Linfócitos T/virologia , Proliferação de Células , Células Clonais , Epigênese Genética , Infecções por HTLV-I/imunologia , Humanos , Pessoa de Meia-Idade , Mutagênese Insercional/genética , Reação em Cadeia da Polimerase , Linfócitos T/patologia , Fatores de Tempo , Transcrição Gênica , Integração Viral/genética
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