Although the risk of developing lymphoma has decreased in the highly active antiretroviral therapy era, this cancer remains the major cause of mortality in HIV-infected patients. Autologous hematopoietic stem cell transplantation (ASCT) outcome does not differ for HIV-infected versus HIV-uninfected patients. We propose to develop a new treatment for HIV-associated high-risk lymphoma based on autologous transplantation of two genetically modified products: CD4(+) T lymphocytes and CD34(+) hematopoietic stem cells (HSPCs). The cells will be transduced ex vivo with the Cal-1 lentiviral vector encoding for both a short hairpin RNA (shRNA) against CCR5 (sh5) and the HIV-1 fusion inhibitor C46. The transduced cells will be resistant to HIV infection by two complementary mechanisms: impaired binding of the virus to the cellular CCR5 co-receptor and decreased fusion of the virus as C46 interacts with gp41 and inhibits HIV infection. This phase I/II pilot study, also entitled GENHIV, will involve two French participating centers: Saint Louis Hospital and Necker Hospital in Paris. We plan to enroll five HIV-1-infected patients presenting with high-risk lymphoma and require a treatment with ASCT. The primary objective of this study is to evaluate the safety, feasibility, and success of engraftment of Cal-1 gene-transduced CD4(+) T lymphocytes and CD34(+) HSPCs.

BackgroundThe gene therapeutic Cal-1 comprises the anti-HIV agents: (i) sh5, a short hairpin RNA to CCR5 that down-regulates CCR5 expression and (ii) maC46 (C46), a peptide that inhibits viral fusion with the cell membrane. These constructs were assessed for inhibition of viral replication and selective cell expansion in a number of settings. MethodsHIV replication, selective outgrowth and cell surface viral binding were analysed with a single cycle infection assay of six pseudotyped HIV strains and a static and longitudinal passaging of MOLT4/CCR5 cells with HIV. Pronase digestion of surface virus and fluorescence microscopy assessed interactions between HIV virions and transduced cells. ResultsCal-1 reduced CCR5 expression in peripheral blood mononuclear cells to CCR532 heterozygote levels. Even low level transduction resulted in significant preferential expansion in MOLT4/CCR5 gene-containing cells over a 3-week HIV challenge regardless of viral suppression [12.5% to 47.0% (C46), 46.7% (sh5), 62.2% (Dual), respectively]. The sh5 and Dual constructs at > 95% transduction also significantly suppressed virus to day 12 in the passage assay and all constructs, at varying percentage transduction inhibited virus in static culture. No escape mutations were present through 9weeks of challenge. The Dual construct significantly suppressed infection by a panel of CCR5-using viruses, with its efficacy being independently determined from the single constructs. Dual and sh5 inhibited virion internalisation, as determined via pronase digestion of surface bound virus, by 70% compared to 13% for C46. ConclusionsThe use of two anti-HIV genes allows optimal preferential survival and inhibition of HIV replication, with the impact on viral load being dependent on the percentage of gene marked cells.

The Runx3 transcription factor is essential for development and diversification of the dorsal root ganglia (DRGs) TrkC sensory neurons. In Runx3-deficient mice, developing TrkC neurons fail to extend central and peripheral afferents, leading to cell death and disruption of the stretch reflex circuit, resulting in severe limb ataxia. Despite its central role, the mechanisms underlying the spatiotemporal expression specificities of Runx3 in TrkC neurons were largely unknown. Here we first defined the genomic transcription unit encompassing regulatory elements (REs) that mediate the tissue-specific expression of Runx3. Using transgenic mice expressing BAC reporters spanning the Runx3 locus, we discovered three REs—dubbed R1, R2, and R3—that cross-talk with promoter-2 (P2) to drive TrkC neuron-specific Runx3 transcription. Deletion of single or multiple elements either in the BAC transgenics or by CRISPR/Cas9-mediated endogenous ablation established the REs’ ability to promote and/or repress Runx3 expression in developing sensory neurons. Our analysis reveals that an intricate combinatorial interplay among the three REs governs Runx3 expression in distinct subtypes of TrkC neurons while concomitantly extinguishing its expression in non-TrkC neurons. These findings provide insights into the mechanism regulating cell type-specific expression and subtype diversification of TrkC neurons in developing DRGs.

Background: The transcription factor NKX2-5 is crucial for heart development and mutations in this gene have been implicated in diverse congenital heart diseases (CHD) and conduction defects (CD) in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown. Methods and Results: Mutation screening was performed in 220 probands with adult-onset dilated cardiomypathy (DCM). Six NKX2-5 coding sequence variants were identified, including 3 non-synonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain (HD), was identified in one family. A subset of family members had CHD, but there was an unexpectedly high prevalence of DCM. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes, due to reduced degradation by the ubiquitinproteasome system (UPS). In functional assays, DNA binding activity of I184M was reduced, resulting in impaired activation of target genes, despite increased expression levels of mutant protein. Conclusions: Certain NKX2-5 HD mutations show abnormal protein degradation via the UPS and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function, and contribute to NKX2-5-related cardiomyopathies with graded severity.

Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA) for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4(+) T lymphocytes, and CD34(+) hematopoietic stem/progenitor cells (HSPC). CCR5-targeted shRNA (sh5) and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.

BACKGROUND:The transcription factor NKX2-5 is crucial for heart development, and mutations in this gene have been implicated in diverse congenital heart diseases and conduction defects in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown. METHODS AND RESULTS:Mutation screening was performed in 220 probands with adult-onset dilated cardiomyopathy. Six NKX2-5 coding sequence variants were identified, including 3 nonsynonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain, was identified in 1 family. A subset of family members had congenital heart disease, but there was an unexpectedly high prevalence of dilated cardiomyopathy. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes because of reduced degradation by the Ubiquitin-proteasome system. In functional assays, DNA-binding activity of I184M was reduced, resulting in impaired activation of target genes despite increased expression levels of mutant protein. CONCLUSIONS:Certain NKX2-5 homeodomain mutations show abnormal protein degradation via the Ubiquitin-proteasome system and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function and contribute to NKX2-5-related cardiomyopathies with graded severity.

Rationale: The cardiac gene regulatory network (GRN) is controlled by transcription factors and signaling inputs, but network logic in development and it unraveling in disease is poorly understood. In development, the membrane-tethered signaling ligand Neuregulin (Nrg)1, expressed in endocardium, is essential for ventricular morphogenesis. In adults, Nrg1 protects against heart failure and can induce cardiomyocytes to divide. Objective: To understand the role of Nrg1 in heart development through analysis of null and hypomorphic Nrg1 mutant mice. Methods and Results: Chamber domains were correctly specified in Nrg1 mutants, although chamber-restricted genes Hand1 and Cited1 failed to be activated. The chamber GRN subsequently decayed with individual genes exhibiting decay patterns unrelated to known patterning boundaries. Both trabecular and nontrabecular myocardium were affected. Network demise was spatiotemporally dynamic, the most sensitive region being the central part of the left ventricle, in which the GRN underwent complete collapse. Other regions were partially affected with graded sensitivity. In vitro, Nrg1 promoted phospho-Erk1/2–dependent transcription factor expression, cardiomyocyte maturation and cell cycle inhibition. We monitored cardiac pErk1/2 in embryos and found that expression was Nrg1-dependent and levels correlated with cardiac GRN sensitivity in mutants. Conclusions: The chamber GRN is fundamentally labile and dependent on signaling from extracardiac sources. Nrg1–ErbB1/4–Erk1/2 signaling critically sustains elements of the GRN in trabecular and nontrabecular myocardium, challenging our understanding of Nrg1 function. Transcriptional decay patterns induced by reduced Nrg1 suggest a novel mechanism for cardiac transcriptional regulation and dysfunction in disease, potentially linking biomechanical feedback to molecular pathways for growth and differentiation.

Development of the mammalian heart is mediated by complex interactions between myocardial, endocardial, and neural crest‐derived cells. Studies in Drosophila have shown that the Slit‐Robo signaling pathway controls cardiac cell shape changes and lumen formation of the heart tube. Here, we demonstrate by in situ hybridization that multiple Slit ligands and Robo receptors are expressed in the developing mouse heart. Slit3 is the predominant ligand transcribed in the early mouse heart and is expressed in the ventral wall of the linear heart tube and subsequently in chamber but not in atrioventricular canal myocardium. Furthermore, we identify that the homeobox gene Nkx2‐5 is required for early ventral restriction of Slit3 and that the T‐box transcription factor Tbx2 mediates repression of Slit3 in nonchamber myocardium. Our results suggest that patterned Slit‐Robo signaling may contribute to the control of oriented cell growth during chamber morphogenesis of the mammalian heart. Developmental Dynamics 239:3303–3311, 2010. © 2010 Wiley‐Liss, Inc.

The homeodomain factor Nkx2-5 is expressed in cardiac progenitor cells of the first and second heart fields, and in differentiating cardiomyocytes and conduction cells. NKX2-5 mutations cause congenital heart disease (CHD) and conduction disease (CD) in humans. Nkx2-5 acts as both a repressor and an activator, and one of its early roles is to repress, through a negative feedback loop, the expression of the Bmp2 gene, which encodes one of the upstream inducers of cardiogenesis and is itself a repressor of cardiac progenitor cell proliferation. In this way Nkx2-5 orchestrates a transition between a period of cardiac induction and one of heart progenitor cell expansion and deployment. We have also studied the Nkx2-5 transcriptional cofactor, Tbx20, a member of the T-box transcription factor family, and shown that Tbx20 mutations also cause CHD as well as adult-onset dilated cardiomyopathy (DCM). The T-box DNA binding domain of Tbx20 has unique biophysical properties, with its tertiary hydrophobic interactions existing in a state of dynamic equilibrium akin to a molten globule, a state that may facilitate formation and/or stability of transcriptional complexes. Gain-of-function mutations can exacerbate this state. These studies address CHD at the molecular, cellular and organ level and contribute to an understanding of CHD origins and patient management.

Bistability in developmental pathways refers to the generation of binary outputs from graded or noisy inputs. Signaling thresholds are critical for bistability. Specification of the left/right (LR) axis in vertebrate embryos involves bistable expression of transforming growth factor beta (TGFbeta) member NODAL in the left lateral plate mesoderm (LPM) controlled by feed-forward and feedback loops. Here we provide evidence that bone morphogenetic protein (BMP)/SMAD1 signaling sets a repressive threshold in the LPM essential for the integrity of LR signaling. Conditional deletion of Smad1 in the LPM led to precocious and bilateral pathway activation. NODAL expression from both the left and right sides of the node contributed to bilateral activation, indicating sensitivity of mutant LPM to noisy input from the LR system. In vitro, BMP signaling inhibited NODAL pathway activation and formation of its downstream SMAD2/4-FOXH1 transcriptional complex. Activity was restored by overexpression of SMAD4 and in embryos, elevated SMAD4 in the right LPM robustly activated LR gene expression, an effect reversed by superactivated BMP signaling. We conclude that BMP/SMAD1 signaling sets a bilateral, repressive threshold for NODAL-dependent Nodal activation in LPM, limiting availability of SMAD4. This repressive threshold is essential for bistable output of the LR system.

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.

The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up.

The Rel/NF-κB transcription factors have been shown to regulate apoptosis in different cell types, acting as inducers or blockers in a stimuli- and cell type-dependent fashion. One of the Rel/NF-κB subunits, RelA, has been shown to be crucial for normal embryonic development, in which it functions in the embryonic liver as a protector against TNFα-induced physiological apoptosis. This study assesses whether NF-κB may be involved in the embryo's response to teratogens. Fot this, we evaluated how NF-KappaB DNA binding activity in embryonic organs demonstraiting differential sensitivity to a reference teratogen, cyclophosphamide, correlates with dysmorphic events induced by the teratogen at the cellular level (excessive apoptosis) and at the organ level (structural anomalies).

The general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 12-14 TBP-associated factors (TAF(II)s). Some TAF(II)s act as bridges between transcription activators and the general transcription machinery through direct interaction with activation domains. Although TAF-mediated transcription activation has been established, there is little genetic evidence connecting it to binding of an activator. TAF(II)105 is a substoichiometric subunit of transcription factor IID highly expressed in B lymphocytes. In this study, we examined the physiological role of TAF(II)105 and its mechanism of action in vivo by expressing two forms of dominant-negative mutant TAF(II)105 in mice. We show that TAF(II)105 has a pro-survival role in B and T lymphocytes, where the native protein is expressed. In addition, TAF(II)105 is important for T cell maturation and for production of certain antibody isotypes. These phenotypic alterations were absent in mice expressing a dominant-negative mutant that lacks one of the domains mediating p65/RelA binding in vitro. These findings provide support to the notion that interaction between the activator and TAF is important for their function in vivo.

ABSTRACT NF-κB induces the expression of genes involved in immune response, apoptosis, inflammation, and the cell cycle. Certain NF-κB-responsive genes are activated rapidly after the cell is stimulated by cytokines and other extracellular signals. However, the mechanism by which these genes are activated is not entirely understood. Here we report that even though NF-κB interacts directly with TAFIIs, induction of NF-κB by tumor necrosis factor alpha (TNF-α) does not enhance TFIID recruitment and preinitiation complex formation on some NF-κB-responsive promoters. These promoters are bound by the transcription apparatus prior to TNF-α stimulus. Using the immediate-early TNF-α-responsive gene A20 as a prototype promoter, we found that the constitutive association of the general transcription apparatus is mediated by Sp1 and that this is crucial for rapid transcriptional induction by NF-κB. In vitro transcription assays confirmed that NF-κB plays a postinitiation role since it enhances the transcription reinitiation rate whereas Sp1 is required for the initiation step. Thus, the consecutive effects of Sp1 and NF-κB on the transcription process underlie the mechanism of their synergy and allow rapid transcriptional induction in response to cytokines.

Early stages of B cell development occur in the bone marrow, resulting in formation of immature B cells. From there these immature cells migrate to the spleen where they differentiate to mature cells. This final maturation step is crucial for the B cells to become responsive to antigens and to participate in the immune response. Recently, invariant chain (Ii), a major histocompatibility complex class II chaperone, as well as the transcription factors c-Rel and p65/RelA, were found to play a role in the final antigen-independent differentiation stage of B cells in the spleen. In this study, we investigated a possible link between Ii-dependent B cell maturation and the NF-kappaB pathway. Our studies indicate that Ii-induced B cell maturation involves activation of transcription mediated by the NF-kappaB p65/RelA homodimer and requires the B cell-enriched coactivator TBP-associated factor (II)105.