Genetics
Abstract
SCID patients have been successfully treated by administration of ex vivo gene-corrected stem cells. However, despite its proven efficacy, such treatment carries specific risks and difficulties. We hypothesized that some of these drawbacks may be overcome by in situ gene correction of T lymphoid progenitors in the thymus. Indeed, in vivo intrathymic transfer of a gene that provides a selective advantage for transduced prothymocytes should result in the generation of functional T lymphocyte progeny, allowing long-term immune reconstitution. We assessed the feasibility of this approach in a murine model of ZAP-70–deficient SCID. A T cell–specific ZAP-70–expressing lentiviral vector was injected into thymi of adult ZAP-70–/– mice without prior conditioning. This resulted in the long-term differentiation of mature TCR-αβ+ thymocytes, indicating that the vector had integrated into progenitor cells. Moreover, peripheral ZAP-70–expressing T cells demonstrated a partially diversified receptor repertoire and were responsive to alloantigens in vitro and in vivo. Improved treatment efficacy was achieved in infant ZAP-70–/– mice, in which the thymus is proportionately larger and a higher percentage of prothymocytes are in cycle. Thus, intrathymic injection of a lentiviral vector could represent a simplified and potentially safer alternative to ex vivo gene-modified hematopoietic stem cell transplantation for gene therapy of T cell immunodeficiencies.
Authors
Oumeya Adjali, Gilles Marodon, Marcos Steinberg, Cédric Mongellaz, Véronique Thomas-Vaslin, Chantal Jacquet, Naomi Taylor, David Klatzmann
The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth
Abstract
The long-term integrity of an articulating joint is dependent upon the nourishment of its cartilage component and the protection of the cartilage surface from friction-induced wear. Loss-of-function mutations in lubricin (a secreted glycoprotein encoded by the gene PRG4) cause the human autosomal recessive disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP). A major feature of CACP is precocious joint failure. In order to delineate the mechanism by which lubricin protects joints, we studied the expression of Prg4 mRNA during mouse joint development, and we created lubricin-mutant mice. Prg4 began to be expressed in surface chondrocytes and synoviocytes after joint cavitation had occurred and remained strongly expressed by these cells postnatally. Mice lacking lubricin were viable and fertile. In the newborn period, their joints appeared normal. As the mice aged, we observed abnormal protein deposits on the cartilage surface and disappearance of underlying superficial zone chondrocytes. In addition to cartilage surface changes and subsequent cartilage deterioration, intimal cells in the synovium surrounding the joint space became hyperplastic, which further contributed to joint failure. Purified or recombinant lubricin inhibited the growth of these synoviocytes in vitro. Tendon and tendon sheath involvement was present in the ankle joints, where morphologic changes and abnormal calcification of these structures were observed. We conclude that lubricin has multiple functions in articulating joints and tendons that include the protection of surfaces and the control of synovial cell growth.
Authors
David K. Rhee, Jose Marcelino, MacArthur Baker, Yaoqin Gong, Patrick Smits, Véronique Lefebvre, Gregory D. Jay, Matthew Stewart, Hongwei Wang, Matthew L. Warman, John D. Carpten
Abstract
CC(A/T)6GG–dependent (CArG-dependent) and serum response factor–dependent (SRF-dependent) mechanisms are required for gene expression in smooth muscle cells (SMCs). However, an unusual feature of many SMC-selective promoter CArG elements is that they contain a conserved single G or C substitution in their central A/T-rich region, which reduces binding affinity for ubiquitously expressed SRF. We hypothesized that this CArG degeneracy contributes to cell-specific expression of smooth muscle α-actin in vivo, since substitution of c-fos consensus CArGs for the degenerate CArGs resulted in relaxed specificity in cultured cells. Surprisingly, our present results show that these substitutions have no effect on smooth muscle–specific transgene expression during normal development and maturation in transgenic mice. However, these substitutions significantly attenuated injury-induced downregulation of the mutant transgene under conditions where SRF expression was increased but expression of myocardin, a smooth muscle–selective SRF coactivator, was decreased. Finally, chromatin immunoprecipitation analyses, together with cell culture studies, suggested that myocardin selectively enhanced SRF binding to degenerate versus consensus CArG elements. Our results indicate that reductions in myocardin expression and the degeneracy of CArG elements within smooth muscle promoters play a key role in phenotypic switching of smooth muscle cells in vivo, as well as in mediating responses of CArG-dependent smooth muscle genes and growth regulatory genes under conditions in which these 2 classes of genes are differentially expressed.
Authors
Jennifer A. Hendrix, Brian R. Wamhoff, Oliver G. McDonald, Sanjay Sinha, Tadashi Yoshida, Gary K. Owens
Abstract
Spinal muscular atrophy (SMA) is a frequent recessive autosomal disorder. It is caused by mutations or deletion of the telomeric copy of the survival motor neuron (SMN) gene, leading to depletion in SMN protein levels. The treatment rationale for SMA is to halt or delay the degeneration of motor neurons, but to date there are no effective drug treatments for this disease. We have previously demonstrated that pseudotyping of the nonprimate equine infectious anemia virus (using the lentivector gene transfer system) with the glycoprotein of the Evelyn-Rokitnicki-Abelseth strain of the rabies virus confers retrograde axonal transport on these vectors. Here, we report that lentivector expressing human SMN was successfully used to restore SMN protein levels in SMA type 1 fibroblasts. Multiple single injections of a lentiviral vector expressing SMN in various muscles of SMA mice restored SMN to motor neurons, reduced motor neuron death, and increased the life expectancy by an average of 3 and 5 days (20% and 38%) compared with LacZ and untreated animals, respectively. Further extension of survival by SMN expression constructs will likely require a knowledge of when and/or where high levels of SMN are needed.
Authors
Mimoun Azzouz, Thanh Le, G. Scott Ralph, Lucy Walmsley, Umrao R. Monani, Debbie C.P. Lee, Fraser Wilkes, Kyriacos A. Mitrophanous, Susan M. Kingsman, Arthur H.M. Burghes, Nicholas D. Mazarakis
Abstract
Hailey-Hailey disease (HHD) is an autosomal dominant trait characterized by erythematous and oozing skin lesions preponderantly involving the body folds. In the present unusual case, however, unilateral segmental areas along the lines of Blaschko showing a rather severe involvement were superimposed on the ordinary symmetrical phenotype. Based on this observation and similar forms of mosaicism as reported in other autosomal dominant skin disorders, we postulated that in such cases, 2 different types of segmental involvement can be distinguished. Accordingly, the linear lesions as noted in the present case would exemplify type 2 segmental HHD. In the heterozygous embryo, loss of heterozygosity occurring at an early developmental stage would have given rise to pronounced linear lesions reflecting homozygosity or hemizygosity for the mutation. By analyzing DNA and RNA derived from blood and skin samples as well as keratinocytes of the index patient with various molecular techniques including RT-PCR, real-time PCR, and microsatellite analysis, we found a consistent loss of the paternal wild-type allele in more severely affected segmental skin regions, confirming this hypothesis for the first time, to our knowledge, at the molecular and cellular level.
Authors
Pamela Poblete-Gutiérrez, Tonio Wiederholt, Arne König, Frank K. Jugert, Yvonne Marquardt, Albert Rübben, Hans F. Merk, Rudolf Happle, Jorge Frank
Abstract
Although inflammation and protease/antiprotease imbalance have been postulated to be critical in cigarette smoke–induced (CS-induced) emphysema, oxidative stress has been suspected to play an important role in chronic obstructive pulmonary diseases. Susceptibility of the lung to oxidative injury, such as that originating from inhalation of CS, depends largely on its upregulation of antioxidant systems. Nuclear factor, erythroid-derived 2, like 2 (Nrf2) is a redox-sensitive basic leucine zipper protein transcription factor that is involved in the regulation of many detoxification and antioxidant genes. Disruption of the Nrf2 gene in mice led to earlier-onset and more extensive CS-induced emphysema than was found in wild-type littermates. Emphysema in Nrf2-deficient mice exposed to CS for 6 months was associated with more pronounced bronchoalveolar inflammation; with enhanced alveolar expression of 8-oxo-7,8-dihydro-2′-deoxyguanosine, a marker of oxidative stress; and with an increased number of apoptotic alveolar septal cells — predominantly endothelial and type II epithelial cells — as compared with wild-type mice. Microarray analysis identified the expression of nearly 50 Nrf2-dependent antioxidant and cytoprotective genes in the lung that may work in concert to counteract CS-induced oxidative stress and inflammation. The responsiveness of the Nrf2 pathway may act as a major determinant of susceptibility to tobacco smoke–induced emphysema by upregulating antioxidant defenses and decreasing lung inflammation and alveolar cell apoptosis.
Authors
Tirumalai Rangasamy, Chung Y. Cho, Rajesh K. Thimmulappa, Lijie Zhen, Sorachai S. Srisuma, Thomas W. Kensler, Masayuki Yamamoto, Irina Petrache, Rubin M. Tuder, Shyam Biswal
Abstract
Poly(ADP-ribosyl)ation is rapidly formed in cells following DNA damage and is regulated by poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is known to be involved in various cellular processes, such as DNA repair, genomic stability, transcription, and cell death. During apoptosis, PARP-1 is cleaved by caspases to generate 89-kDa and 24-kDa fragments, a hallmark of apoptosis. This cleavage is thought to be a regulatory event for cellular death. In order to understand the biological significance of PARP-1 cleavage, we generated a PARP-1 knockin (PARP-1KI/KI) mouse model, in which the caspase cleavage site of PARP-1, DEVD214, was mutated to render the protein resistant to caspases during apoptosis. While PARP-1KI/KI mice developed normally, they were highly resistant to endotoxic shock and to intestinal and renal ischemia-reperfusions, which were associated with reduced inflammatory responses in the target tissues and cells due to the compromised production of specific inflammatory mediators. Despite normal binding of NF-κB to DNA, NF-κB–mediated transcription activity was impaired in the presence of caspase-resistant PARP-1. This study provides a novel insight into the function of PARP-1 in inflammation and ischemia-related pathophysiologies.
Authors
Virginie Pétrilli, Zdenko Herceg, Paul O. Hassa, Nimesh S.A. Patel, Rosanna Di Paola, Ulrich Cortes, Laura Dugo, Helder-Mota Filipe, Christoph Thiemermann, Michael O. Hottiger, Salvatore Cuzzocrea, Zhao-Qi Wang
Abstract
Complex I deficiency, the most common respiratory chain defect, is genetically heterogeneous: mutations in 8 nuclear and 7 mitochondrial DNA genes encoding complex I subunits have been described. However, these genes account for disease in only a minority of complex I–deficient patients. We investigated whether there may be an unknown common gene by performing functional complementation analysis of cell lines from 10 unrelated patients. Two of the patients were found to have mitochondrial DNA mutations. The other 8 represented 7 different (nuclear) complementation groups, all but 1 of which showed abnormalities of complex I assembly. It is thus unlikely that any one unknown gene accounts for a large proportion of complex I cases. The 2 patients sharing a nuclear complementation group had a similar abnormal complex I assembly profile and were studied further by homozygosity mapping, chromosome transfers, and microarray expression analysis. NDUFS6, a complex I subunit gene not previously associated with complex I deficiency, was grossly underexpressed in the 2 patient cell lines. Both patients had homozygous mutations in this gene, one causing a splicing abnormality and the other a large deletion. This integrated approach to gene identification offers promise for identifying other unknown causes of respiratory chain disorders.
Authors
Denise M. Kirby, Renato Salemi, Canny Sugiana, Akira Ohtake, Lee Parry, Katrina M. Bell, Edwin P. Kirk, Avihu Boneh, Robert W. Taylor, Hans-Henrik M. Dahl, Michael T. Ryan, David R. Thorburn
Abstract
Gene-based delivery can establish a sustained supply of therapeutic proteins within the nervous system. For diseases characterized by extensive CNS and peripheral nervous system (PNS) involvement, widespread distribution of the exogenous gene may be required, a challenge to in vivo gene transfer strategies. Here, using lentiviral vectors (LVs), we efficiently transduced hematopoietic stem cells (HSCs) ex vivo and evaluated the potential of their progeny to target therapeutic genes to the CNS and PNS of transplanted mice and correct a neurodegenerative disorder, metachromatic leukodystrophy (MLD). We proved extensive repopulation of CNS microglia and PNS endoneurial macrophages by transgene-expressing cells. Intriguingly, recruitment of these HSC-derived cells was faster and more robust in MLD mice. By transplanting HSCs transduced with the arylsulfatase A gene, we fully reconstituted enzyme activity in the hematopoietic system of MLD mice and prevented the development of motor conduction impairment, learning and coordination deficits, and neuropathological abnormalities typical of the disease. Remarkably, ex vivo gene therapy had a significantly higher therapeutic impact than WT HSC transplantation, indicating a critical role for enzyme overexpression in the HSC progeny. These results indicate that transplantation of LV-transduced autologous HSCs represents a potentially efficacious therapeutic strategy for MLD and possibly other neurodegenerative disorders.
Authors
Alessandra Biffi, Michele De Palma, Angelo Quattrini, Ubaldo Del Carro, Stefano Amadio, Ilaria Visigalli, Maria Sessa, Stefania Fasano, Riccardo Brambilla, Sergio Marchesini, Claudio Bordignon, Luigi Naldini
Abstract
Excessive inflammatory responses can emerge as a potential danger for organisms’ health. Physiological balance between pro- and anti-inflammatory processes constitutes an important feature of responses against harmful events. Here, we show that cannabinoid receptors type 1 (CB1) mediate intrinsic protective signals that counteract proinflammatory responses. Both intrarectal infusion of 2,4-dinitrobenzene sulfonic acid (DNBS) and oral administration of dextrane sulfate sodium induced stronger inflammation in CB1-deficient mice (CB1–/–) than in wild-type littermates (CB1+/+). Treatment of wild-type mice with the specific CB1 antagonist N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716A) mimicked the phenotype of CB1–/– mice, showing an acute requirement of CB1 receptors for protection from inflammation. Consistently, treatment with the cannabinoid receptor agonist R(-)-7-hydroxy-Δ6-tetra-hydrocannabinol-dimethylheptyl (HU210) or genetic ablation of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) resulted in protection against DNBS-induced colitis. Electrophysiological recordings from circular smooth muscle cells, performed 8 hours after DNBS treatment, revealed spontaneous oscillatory action potentials in CB1–/– but not in CB1+/+ colons, indicating an early CB1-mediated control of inflammation-induced irritation of smooth muscle cells. DNBS treatment increased the percentage of myenteric neurons expressing CB1 receptors, suggesting an enhancement of cannabinoid signaling during colitis. Our results indicate that the endogenous cannabinoid system represents a promising therapeutic target for the treatment of intestinal disease conditions characterized by excessive inflammatory responses.
Authors
Federico Massa, Giovanni Marsicano, Heike Hermann, Astrid Cannich, Krisztina Monory, Benjamin F. Cravatt, Gian-Luca Ferri, Andrei Sibaev, Martin Storr, Beat Lutz
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)